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ISBN 1-904761-16-X
Disclaimer
Every reasonable effort has been made to ensure that the material in this book is true, correct, complete
and appropriate at the time of writing. Nevertheless, the publishers and authors do not accept
responsibility for any omission or error, or for any injury, damage, loss or financial consequences arising
from the use of the book.
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CONTENTS
Preface
xix
1
1
7
25
32
34
45
45
51
51
56
57
58
58
59
60
66
67
69
73
74
74
76
76
79
82
89
92
92
95
96
97
97
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Contents
Mitochondrial capsular selenoprotein
Selenoprotein N
Selenoprotein R
Selenoprotein S
Selenoprotein U
Selenoprotein T and selenoprotein X
Selenium-vitamin E interactions: antioxidant properties and beyond
Prooxidant properties of selenite and possible antioxidant protection
by selenomethionine
References
116
151
151
156
161
171
181
184
184
191
198
213
214
224
228
233
240
243
251
252
257
260
262
279
279
281
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99
100
100
101
101
103
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Contents
Selenoproteins and their role in antioxidant protection in semen
Conclusions
References
286
304
305
317
318
324
326
330
342
347
347
397
400
405
411
415
418
422
424
445
446
454
459
459
469
473
474
475
476
479
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363
364
374
385
Contents
9. Selenium in ruminant nutrition
Introduction
Selenium deficiency in ruminants
Selenium deficiency in dairy and beef industries
Important features of Se metabolism in ruminants
Meeting selenium requirements
Methods for Se status assessment
Routes of selenium supplementation
Practical applications of Se nutrition of ruminants
Selenized yeast vs selenite
Conclusions
References
487
488
518
521
528
531
537
538
546
559
561
10. Selenium in nutrition of other animals: horses, dogs, cats and fish
Selenium in horse nutrition
Introduction
Se deficiency
Se excess
Se requirement
Selenium and GSH-Px
Se metabolism and effective sources
Antioxidant defences and exercise
Se and maternal effect
Se and immunity
Conclusions
589
589
589
590
592
592
593
595
597
599
599
601
601
601
602
603
604
605
607
608
612
615
616
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Contents
Se deficiency
Se toxicity
Se requirement
Health and immunity
Selenium and fish quality
Effective forms of Se in fish diets
Se-fish as human food
Conclusions
References
616
618
620
621
622
624
626
627
628
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643
643
644
651
654
657
658
662
664
665
666
671
672
682
687
694
705
708
720
729
729
731
732
733
735
736
738
741
746
751
Contents
Conclusions
References
754
755
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809
810
814
817
821
827
837
842
844
857
857
859
859
861
862
864
865
867
868
870
871
873
874
874
876
880
882
882
883
883
885
885
Contents
Selenomethionine
Synthetic antioxidants
Specific place for Se-dependent enzymes in antioxidant defence of GIT
Other antioxidant mechanisms
What should be changed to have a healthy diet?
Antioxidant-prooxidant balance in the intestine and animal health
Conclusions
References
14. Conclusions: Looking ahead
References
923
949
Index
955
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886
886
887
892
895
896
897
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ABBREVIATIONS
AA
Ab
ACS
AD
AFB1
ALA
Apo-AI
ApoB
ApoII
AR
BDI scores
BHT
BHA
CAT
CE
CH
CHD
CIND
CoQ
Con A
CPK
CRP
CSF
CVD
DAA
DAT
DHA
DM
DMBA
DON
DPA
DTA
DTH
ECs
Ascorbic acid
Antibody
Acute coronary syndromes
Alzheimers disease
Aflatoxin B1
Alpha-linolenic acid
Apoprotein AI
Apoprotein B
Apoprotein II
Androgen receptor
Beck Depression Inventory scores
Butylated hydroxytoluene
Butylated hydroxyanisole
Catalase
Cholesteryl esters
Cholesterol
Coronary heart disease
Cognitive impairment non-dementia
Coenzyme Q
Concanavalin A
Creatine phosphokinase
C-reactive protein
Cerebrospinal fluid
Cardiovascular diseases
Dehydroascorbic acid
Dementia of the Alzheimer type
Docosahexaenoic acid
Dry matter
7,12-Dimethylbenz[a]anthracene
Deoxynivalenol
Docosapentaenoic acid
Docosatetraenoic acid
Delayed-type hypersensitivity
Endothelial cells
ED
EPA
FB1
FCR
Exudative diathesis
Eicosapentaenoic acid
Fumonisin B1
Feed conversion ratio
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Abbreviations
FFA
GC-MS
GI-GSH-Px
GIT
GOT
GR
GSH
GSSG
GSH-Px
GST
HA
Hb
HCC
5-HETE
15-HPET
HI
HDL
HNE
HO-1
HPLC
HSP
ID
IFN
IL-1
IL-6
IL-2R
IMI
KD
LA
LE
LNA
LDL
LDH
5- LO
LOOH
LP
LPL
LPO
LPS
LTA
LTB
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Abbreviations
MAP
MCS
MDA
MD
Met
MODA
NDV
MIF
MIP-2
MCP
MSA
MsrA and MsrB
NE
NK cells
NMD
NPO
NRC
NRP
OA
OPN
OVA
PBMC
PC
PCA
PFC test
PGE2
pGSH-Px
PAM
PL
PLC
PMN
PG
PHA
PH-GSH-Px
PIH
PLA2
POPs
PRECISE
PSA
PUFA
PWM
Microangiopathy
Mitochondrial capsular selenoprotein
Malondialdehyde
Mareks disease
Methionine
Milan overall dementia assessment
Newcastle disease virus
Migration inhibitory factor
Macrophage inflammatory protein 2
Mitochondrial capsule protein
Methylseleninic acid
Methionine sulfoxide reductases
Nutritional encephalomalacia
Natural killer cells
Nutritional muscular distrophy
2'-(4-nitrophenoxy)oxirane
National Research Council
Nutrient Requirements of Poultry
Ochratoxin A
Osteopontin
Ovalbumin
Peripheral blood mononuclear cells
Phosphatidylcholine
Prostate cancer
Plaque-forming cell test
Prostaglandin E2
Plasma glutathione peroxidase
Prematurely aging mice
Phospholipid
Primary liver cancer
Polymorphonuclea cells, neutrophils
Prostaglandin
Phytohemagglutin
Phospholipid glutathione peroxidase
Pregnancy induced hypertension
Phospholipase A2
Persistent organic pollutants
Prevention of Cancer by Intervention with Se
Prostate-specific antigen
Polyunsaturated fatty acid
Pokeweed mitogen
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Abbreviations
RA
RBC
RDA
ROS
RNS
RP
RR
RSV
SCC
SCCs
SDG
SeCys
Sel-Plex
SECIS
SELECT
SeN
SeMet
SeP
SeR
SeS
SeW
SeRU
SFA
sn-GSH-Px
SOD
SPS
SRBC
SST
Sptrx-1
STZ
T3
T4
TBA
TBARS
Th
TG
TNF-a
TPA
Trx
TPx
TR
Rheumatoid arthritis
Red blood cells
Recommended Dietary Allowances
Reactive oxygen species
Reactive nitrogen species
Retained placenta
Relative risk
Respiratory syncytial virus
Somatic cell count
Ssquamous carcinoma cells
Selenodiglutathione
Selenocysteine
Selenium enriched yeast
Selenocysteine insertion sequence
The Selenium and Vitamin E Cancer Prevention Trial
Selenoprotein N
Selenomethionine
Selenoprotein P
Selenoprotein R
Selenoprotein S
Selenoprotein W
Se-responsive unthriftiness
Saturated fatty acids
Sperm nuclei glutathione peroxidase
Superoxide dismutase
Selenophosphate synthetase-2
Sheep red blood cells
Sperm storage tubules
Sperm-specific thioredoxin
Streptozotocin
Triiodthyronine
Thyroxine
Thiobarbituric acid
Thiobarbituric acid reactive substances
T helper
Triacylglycerol
Tumour necrosis factor a
12-O-tetradecanoylphorbol-13-acetate
Thioredoxin
Thioredoxin peroxidase
Thioredoxin reductase
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Abbreviations
TRAIL
Txl-2
VEGF
VLDL
vMDV
WMD
p-XSC
YSM
Zea
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PREFACE
Among many minerals selenium has a special place being the most controversial trace
element. Indeed a narrow gap between essentiality and toxicity and environmental issues
from the one hand and global selenium deficiency from the other hand, fuel research in
this field. There were several breakthroughs in selenium research. The first one was a
discovery of Se essentiality in early 1960th. The second one was a discovery in 1973
that glutathione peroxidase is a selenoprotein. The third one came almost 30 years later
with characterisation of main selenoproteins in human and animal body and further
understanding the role of selenium in nutrition and health. Indeed, this third breakthrough
is really a selenium revolution creating many hypotheses, stimulating new research
and providing practical applications in medicine and agriculture. New insight in the
role of free radicals as signalling molecules, understanding the role of nutrients in gene
expression and maternal programming, tremendous progress in human and animal
genome work created new demands for further research related to biological roles of
selenium. For the last few years several comprehensive monographs and reviews have
been published addressing various Se-related issues. However, most of them were dealing
with Se roles in human health. However, animal food-producing industry is developing
very quickly and a great body of information was accumulated indicating importance
of Se in maintenance of animal health, productive and reproductive performance.
Unfortunately up to now there was no comprehensive review combining all major aspects
of selenium in relating to animal and human health. Indeed a chain: Se in soil - selenium
in plants-selenium in animals- selenium in human was not well characterised.
Therefore the goal of this volume is to provide up to date information about the
roles of Se in nutrition and health of human and farm animals, including poultry, pigs,
ruminants, horses, cats, dogs and fish. In the first chapter a special emphasis is given to
the role of selenium as an essential part of the integrated antioxidant system of the
body with regulatory functions providing necessary connections between different
antioxidants. In fact selenium is called the chief executive of the antioxidant defence.
The second chapter is addressing molecular mechanisms of Se action describing major
functions of the selenoproteins. Indeed, the family of selenoproteins includes 25
members and functions of many of them are still not well understood. Selenium in
food and feed is described in the third chapter. The main idea of this chapter is to
characterise main Se sources in relation to new knowledge about Se speciation in various
food and feed ingredients. It seems likely, that in grains and some other important food
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Preface
ingredients selenomethionine is the main Se form. The idea was put forward that during
evolution the digestive system of human and animals was adapted to natural form of
selenium consisting of SeMet and other organic selenocompounds. Therefore this form
of Se is more efficiently assimilated in the body than inorganic forms of selenium. In
fact SeMet is considered to be the storage form of selenium in the body. Accumulation
of the Se reserves in the body as a result of organic selenium consumption is considered
as an adaptive mechanism providing additional antioxidant defences in stress conditions.
The fourth chapter is devoted to the role of selenium in immunity. It is difficult to
overestimated immunomodulating properties of selenium and increased resistance to
various diseases of human and animals is a result of optimal Se status. The possibility
of virus mutation in the body of animals or human deficient in selenium is of great
importance for understanding mechanisms of spreading such diseases as AIDS, chicken
influenza, etc. New findings in the field of male reproduction in relation to Se status are
described in the chapter 5. From the data presented it is clear that Se has special important
roles in maintaining animal reproduction. It is well known that antioxidants play
important roles in preventing various stresses. Mycotoxins are considered to be the
biggest feed-related stress in farm animals. Therefore, possible protective role of Se in
mycotoxicosis is described in the chapter 6. Next four chapters are devoted to practical
applications of Se in animal nutrition. The data presented indicated importance of Se in
growth, development and reproduction of poultry, pigs, ruminants, horses, cats, dogs
and fish. The main idea of the chapters is to show possibilities of improvement of
productive and reproductive performance of animals and animal produce quality by
optimal Se supplementation. Indeed, organic selenium in the form of selenized yeast is
proven to be the most effective form of Se supplementation for poultry, farm animals,
companion animals and fish. Role of selenium in human health is described in the
chapter 11. Specific emphasis is given to anticancer properties of Se with detailed
analysis of possible molecular mechanisms of such Se action. Beneficial role of Se in
other diseases, including cardio-vascular diseases, diabetes, arthritis, pancreatitis,
reproductive disorders as well as protective role of selenium against radiation are also
described. The link between animal industry and human health is described in the
chapter 12 devoted to production of Se-enriched eggs, meat and milk. Indeed, production
of a range of Se-enriched products is considered as an important solution for global Se
deficiency. Se-enriched eggs are already on supermarket shelves in more than 25
countries worldwide with millions such eggs sold daily. The technologies of producing
Se-meat and milk are also developed and await practical applications. An important
issue of antioxidant-prooxidant balance in the digestive tract is considered in the chapter
13. It seems likely that this balance has been overlooked by scientists. However, from
the one hand this balance could explain a beneficial effect of fruit and vegetables in
human diet. On the other hand, this balance is a key for general health. In the conclusive
chapter of the book the general trends for future research in the field of the Se biology
are shown. Information provided in this book will be of practical importance to medical
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Preface
and pharmaceutical industry professionals, dieticians, egg, meat and milk producers,
animal scientists, feed formulators as well as for students of biological, medical,
veterinary and other related sciences at universities and colleges.
Much of the material in this book is based on ideas and concepts that in many
instances have been developed from work carried out in the Department of Biochemistry
and Nutrition and further in the Avian Science Research Centre at the Scottish
Agricultural College. I am, therefore, grateful to the SAC for providing an environment
to develop these ideas, and to the Scottish Executive Environment and Rural Affairs
Department for its generous support of this research. I understand that my views on
the role of selenium in nutrition and health are sometime different from those of other
scientists and therefore I would appreciate very much receiving any comments from
readers which will help me in my future research.
I would like to thank my colleagues with whom I have had a pleasure to collaborate
and share my ideas related to natural antioxidants and selenium in particular, who
helped me at various stages of this research by providing reprints of their recent
publications. I am also indebted to the Alltech Inc. for providing organic selenium for
experiments and some important information and ideas and to the Worlds Poultry
Science Association for the Research Award.
P.F. Surai
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Introduction
For the majority of organisms on Earth, life without oxygen is impossible. Animals,
plants and many microorganisms rely on oxygen for the efficient production of
energy. However, the high oxygen concentration in the atmosphere is potentially
toxic for living organisms. It is interesting that oxygen toxicity was first described
in laboratory animals in 1878 (see Knight, 1998). For the last few years free
radical research has generated valuable information for further understanding not
only detrimental, but also beneficial role of free radicals in cell signaling and
other physiological processes. The benefit or harm of free radicals ultimately
depend on the level of their production and efficiency of antioxidant defence.
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Table 1.1 Internal and external sources of free radicals (Adapted from Furst, 1996 and Halliwell, 1996).
Internally generated
External sources
Mitochondria (ETC)
Phagocytes
Xanthine oxidase
Reactions with Fe2+ or Cu+
Arachidonate pathways
Peroxisomes
Inflammation
Biomolecule oxidation (adrenaline, dopamine,
tetrahydrofolates, ect.)
Cigarette smoke
Radiation
UV light
Pollution
Certain drugs
Chemical reagents
Industrial solvents
Table 1.2 Reactive oxygen and nitrogen species (Adapted from Halliwell and Gutteridge, 1999).
Radicals
Non-radicals
Alkoxyl, RO*
Hydroperoxyl, HOO*
Hydroxyl, *OH
Peroxyl, ROO*
Superoxide, O2*
Nitric oxide, NO*
Nitrogen dioxide, NO2*
Superoxide (O2*-) is the main free radical produced in biological systems during
normal respiration in mitochondria and by autoxidation reactions with half-life at
37C in the range of 1 x 10-6 second. Superoxide can inactivate some enzymes
due to formation of unstable complexes with transition metals of enzyme prosthetic
groups, followed by oxidative self-destruction of the active site (Chaudiere and
Ferrari-Iliou, 1999). Depending on condition, superoxide can act as oxidizing or
a reducing agent. It is necessary to mention that superoxide, by itself, is not
extremely dangerous and does not rapidly cross lipid membrane bilayer (Kruidenier
and Verspaget, 2002). However, superoxide is a precursor of other, more powerful
ROS. For example it reacts with nitric oxide with a formation of peroxynitrite
(ONOO-), a strong oxidant, which lead to formation of reactive intermediates due
to spontaneous decomposition (Kontos, 2001; Mruk et al., 2002). In fact ONOOwas shown to damage a wide variety of biomolecules, including proteins (via
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Fe2+/Cu+ + O2
Further reactions of Fe2+ and Cu+ with H2O2 are a source of the hydroxyl radical
(*OH) in the Fenton reaction:
H2O2 + Fe2+/Cu+
OH + OH- + Fe3+/Cu2+
The sum of reaction of superoxide radical with transition metals and transition
metals with hydrogen peroxide is known as the Haber-Weiss reaction. It is
necessary to underline that superoxide radical is a double-edged sword. It is
beneficial when produced by activated polymorphonuclea leukocytes and other
phagocytes as an essential component of their bactericidal activities but in excess
it may result in tissue damage associated with inflammation.
Did you know that superoxide radical is the major radical
produced in biological systems?
Hydroxyl radical is the most reactive species with an estimated half-life of only
about 10-9 second. It can damage any biological molecule it touches, however, its
diffusion capability is restricted to only about two molecular diameters before
reacting (Yu, 1994). Therefore, in most cases damaging effect of hydroxyl radical
is restricted to the site of its formation. In general, hydroxyl radical can be generated
in human/animal body as a result of radiation exposure from natural sources (radon
gas, cosmic radiation) and from man-made sources (electromagnetic radiation
and radionuclide contamination). In fact in many cases hydroxyl radical is a trigger
of chain reaction in lipid peroxidation.
Did you know that hydroxyl radical is short-lived but most
powerful radical in biological systems?
Therefore, ROS/RNS (Table 1.3) are constantly produced in vivo in the course of
the physiological metabolism in tissues. It is generally accepted that the electron-
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Table 1.3 Effect of free radicals on DNA damages (Adapted from Diplock, 1998).
Radical
Effect
ROO*
OH*
O2*-, H2O2
ONOO-
Guanine oxidized
All four bases are affected
No base changes
Xanthine, hypoxanthine, 8-nitroguanine are affected
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Initiator
L*
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The initiator in this reaction could by the hydroxyl radical, radiation or some
other events or compounds. In presence of oxygen these radicals (L*) react with
oxygen producing peroxyl radicals starting the next stage of lipid peroxidation
called the propagation phase:
LOO*
L* +O2
LOOH + L*
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oxidatively altered DNA molecules have been identified. The chemistry of attack
by ROS on DNA is very complex and lesions in chromatin include damage to
bases, sugar lesions, single strand-breaks, basic lesions and DNA-nucleoprotein
cross-links (Diplock, 1994).
Did you know that an old rat has accumulated about 66,000
oxidative DNA lesions per cell?
The complex structure of proteins and a variety of oxidizable functional groups
of the amino acids make them susceptible to oxidative damage. In fact, the
accumulation of oxidized proteins has been implicated in the aging process and
in other age-related pathologies. A range of oxidized proteins and amino acids
has been characterised in biological systems (Table 1.4; Kehrer, 2000; Dean et
al., 1997).
Table 1.4 Oxidized protein and amino acids found in biologic systems (Adapted from Kehrer, 2000;
Dean et al., 1997).
2-Oxohistidine
3-chlorotyrosine
3-Nitrotyrosine
5-Hydroxy-2-aminovaleric acid
Aminomalonic acid
Dimers of hydroxylated aromatic amino acids
Dopa
Hydro(pero)xyleucine
Hydro(pero)xyvaline
N-Formylkynureinine
Kynurenine
o- and m-tyrosine
p-Hydroxyphenylacetaldehyde
Protein carbonyls
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the nature and relative location of the oxidant or free radical source;
nature and structure of protein;
the proximity of ROS to protein target ;
the nature and concentrations of available antioxidants.
Free radicals are implicated in the initiation or progression phase of various diseases,
including cardiovascular disease, some forms of cancer, cataracts, age-related
macular degeneration, rheumatoid arthritis and a variety of neuro-degenerative
diseases (Hogg, 1998; McCord, 2000; Table 1.5). In general, it is widely believed
that most human diseases at different stages of their development are associated
with free radical production and metabolism. Normally, there is a delicate balance
between the amount of free radicals generated in the body and the antioxidants to
protect against them. For the majority of organisms on Earth, life without oxygen
is impossible, animals, plants and many micro-organisms relying on oxygen for
the efficient production of energy. However, they pay a high price for pleasure of
living in an oxygenated atmosphere since high oxygen concentration in the
atmosphere is potentially toxic for living organisms.
Did you know that free radicals damage not only lipids
but also DNA and proteins?
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Table 1.5 Free radical involvement in the development of human diseases (adapted from Surai and
Sparks, 2001; McCord, 2000; Hogg, 1998 and references there).
Liver
Reperfusion
Toxic effects of chemicals: halogenated
hydrocarbons, quinones, iron,
acetaminophen, ethanol
Endotoxin
Eye
Retionopathy of prematurity
Photic retinopathy
Macular degeneration
Ocular hemorrhage
Cataracts
Kidney
Autoimmune nephrosis:inflammation
Toxic effects of chemicals: aminoglycosides, heavy metals
Muscle
Muscular dystrophy
Over-exercising
Lung
Normobaric hyperoxic injury
Bronchopulmonary displasia
Toxic effects of chemicals: paraquat,
bleomicin
Emphysema
Asbestosis
Idiopathic pulmonary fibrosis
Skin
Radiation (UV or ionising)
Thermal injury
Toxic effects of chemicals:
tetracyclines stimulating
photosensitization
Contact dermatitis
Porphyria
Gastrointestinal tract
Inflammatory-immune system
Reperfusion
Glomerulonephritis
Toxic effects of chemicals: nonsteroidal
Vasculitis
and anti-inflammatory agents, alloxan, iron
Autoimmune disease
Pancreatitis, Colitis, Intestinal ischemia,
Lupus erythermatosus
Gastric ulcers
Reumatroid arthritis
Blood
Malaria
Various anemias
Protoporphyrin photooxidation
Toxic effects of chemicals: phenylhydrazine, primaquine and related drugs,
sulfonamides, lead etc.
Favism
Fanconis anemia
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Miscellaneous/general
Aging
AIDS, Cancer, Diabetes
Inflammation
Trauma
Ischemia/reperfusion
Radiation injury
Rheumatoid arthritis and lupus
Toxic effects of chemicals: alloxan
(diabetes), iron overload
Acute pancreatitis, Amyloidosis
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2O2* +2H+
H2O2+O2
First level of defence:
Prevention of radical formation
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N Mn-SOD manipulationm
Effects
H2O2 + 2GSH
2H2O2
Catalase
GSSG+2H2O
2H2O + O2
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13
copper ions per molecule preventing their participation in free radical generation.
About 5% of human plasma copper is bound to albumin or to amino acids and the
rest is bound to ceruloplasmin. Furthermore ceruloplasmin possesses antioxidant
properties itself being able to scavenge superoxide radical (Yu, 1994). Therefore,
it is now quite clear that metal sequestration is an important part of extracellular
antioxidant defence.
Unfortunately this first level of antioxidant defence in the cell is not sufficient
to completely prevent free radical formation and some radicals do escape through
the preventive first level of antioxidant safety screen initiating lipid peroxidation
and causing damage to DNA and proteins. Therefore the second level of defence
consists of chain-breaking antioxidants - vitamin E, ubiquinol, carotenoids, vitamin
A, ascorbic acid, uric acid and some other antioxidants. Glutathione and
thioredoxin systems also have a substantial role in the second level of antioxidant
defence (for details see Chapter 2). Chain-breaking antioxidants inhibit
peroxidation by keeping the chain length of the propagation reaction as small as
possible. Therefore, they prevent the propagation step of lipid peroxidation by
scavenging peroxyl radical intermediates in the chain reaction:
Toc* + LOOH
LOO* + Toc
ROOH + 2GSH
Thus, vitamin E performs only half the job in preventing lipid peroxidation by
scavenging free radicals and forming hydroperoxides. The second part of this
important process of antioxidant defence is due to Se-GSH-Px. It is necessary to
underline, that vitamin E and selenium work in a tandem; and even very high
doses of dietary vitamin E cannot replace Se which is needed (in the form of
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Both ascorbate and ascorbyl radical have low reduction potentials and can
react with most other biologically relevant radicals and oxidants;
Ascorbyl radical has a low reactivity as a result of resonance stabilisation of
unpaired electron and readily dismutates to ascorbate and dehydroascorbic
acid (DAA);
Ascorbyl radical and DAA can be converted into active ascorbate form by
enzyme-dependent or independent pathways. In particular, ascorbyl radical
can be reduced by NADH-dependent semidehydroascorbate reductase or
by thioredoxin reductase. At the same time DAA can be reduced to AA by
GSH, lipoic acid or glutaredoxin.
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Table 1.7 Antioxidant properties of ascorbate (Adapted from Halliwell, 1996; 1999a).
Glutathione (GSH) is the most abundant non-protein thiol in avian and mammalian
cells, and considered to be an active antioxidant in biological systems providing
cells with their reducing milieu (Meister, 1992). Cellular GSH plays a key role in
many biological processes (Sen and Packer, 2000):
Furthermore, GSH thiolic group can react directly with (Lenzi et al., 2000; Meister
and Anderson, 1983):
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H 2O 2;
superoxide anion;
hydroxyl radicals;
alkoxyl radicals;
hydroperoxides
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Therefore, a crucial role for GSH is as free radical scavenger, particularly effective
against the hydroxyl radical (Bains and Shaw, 1997), since there are no enzymatic
defences against this species of radical. Usually decreased GSH concentration in
tissues is associated with increased lipid peroxidation (Thompson et al., 1992).
Furthermore in stress conditions GSH prevents the loss of protein thiols and vitamin
E (Palamanda and Kehrer, 1993) and plays an important role as a key modulator
of cell signaling (Elliott and Koliwad, 1997). Animals and human are able to
synthesise glutathione.
Taurine, a sulphur containing amino acid (2-aminoethane sulfonic acid) is
synthesised from methionine and cysteine in the presence of vitamin B6 and
found in almost all tissues in mammals. It is the most abundant intracellular amino
acid in humans which is not incorporated into proteins. Antioxidant properties of
taurine were shown in various model systems in vivo and in vitro (Cozzi et al.,
1995; Haber et al., 2003; Sethupathy et al., 2002). However, antioxidant effect of
taurine is not restricted to PUFAs. For example, Ogasawara et al. (1993; 1994)
showed an inhibiting effect of taurine against the modification of protein, as well
as an antioxidative effect through the reactions of taurine with aldehydes in vivo.
It seems likely that in many cases in biological systems taurine could interact with
other antioxidants being an important part of an integrated antioxidant system.
For example, taurine supplementation of rats restored kidney GSH content and
GSH-Px activity and reduced MDA production levels in the kidney tissue following
cisplatin treatment (Saad and Al-Rikabi, 2002). In streptozotocin-diabetic rats,
dietary taurine supplementation ameliorates MDA levels, GSSG/GSH, and NAD+/
NADH (Obrosova and Stevens, 1999). Taurine reduced significantly a decrease
of glutathione antioxidant system activity protecting tissues against GSH pool
depletion and preventing a decrease of glutathione reductase and glutathione
peroxidase activities in acute severe hypoxia (Mankovska et al., 1998).
Uric acid is traditionally considered to be a metabolically inert end-product of
purine metabolism in man, without any physiological value. However, this
ubiquitous compound has proven to be a selective antioxidant (Becker, 1993;
Maples and Mason, 1988) which can:
react with hydroxyl radicals and hypochlorous acid, itself being converted
to innocuous products
serve as an oxidisable cosubstrate for the enzyme cyclooxygenase
protect against reperfusion damage induced by activated granulocytes
prevents oxidative inactivation of endothelial enzymes in stress conditions
chelate transition metal ions and scavenging ROS
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antioxidant defence (Slupphaug et al., 2003). The role of ubiquinones and uric
acid in the farm animal and poultry development has not yet been studied.
However, even the second level of antioxidant defence in the cell is not able to
prevent damaging effects of ROS and RNS on lipids, proteins and DNA. In this
case, the third level of defence is based on systems that eliminate damaged
molecules or repair them. This level of antioxidant defence includes lipolytic
(lipases), proteolytic (peptidases or proteases) and other enzymes (DNA repair
enzymes, ligases, nucleases, polymerases, proteinases, phospholipases and various
transferases).
Since maintaining the integrity of the genome is of the vital importance, living
organisms have evolved a range of systems that can recognise, signal the presence
of, and repair the various forms of DNA damage. In fact, DNA repair is one of
the fundamental processes of life (130 human DNA repair genes have been
identified; Wood et al., 2001) and if the systems are compromised devastating
consequences would be expected. In order to deal with the deleterious effects of
such lesions, leading to genomic instability, cells have evolved a number of DNA
repair mechanisms. They include the direct reversal of the lesion, mismatch repair,
the base excision repair, nucleotide excision repair, nucleotide incision repair,
transcription-coupled repair, global genome repair, translesion synthesis,
homologous recombination and non-homologous end-joining (Gros et al., 2002;
Slupphaug et al., 2003). These repair pathways are universally present in living
cells and extremely well conserved.
Did you know that 130 human DNA repair genes
have been identified?
Therefore, DNA repair systems include a number of enzymatic processes ranging
from base recognition and excision to ligation of DNA strands. In particular, the
DNA glycosylases recognise damaged purines and pyrimidines and hydrolyse
the bond linking the abnormal base to the sugar-phosphate backbone (Halliwell
and Gutteridge, 1999); the 5I-apurinic endonucleases process strand breaks, sites
of base loss, and the products of DNA glycosylase/apurinic lyase action. DNA
polymerase fills in the one-nucleotide gap with the correct base. DNA ligases
complete the repair process by sealing the 3I end of the newly synthesised stretch
of DNA to the original portion of the DNA chain (Cardozo-Pelaez et al., 2000;
Wallace et al., 1997; Croteau and Bohr, 1997).
It is believed that most damaged or inappropriate bases in DNA are removed
by excision repair, while a minority are repaired by direct damage reversal (Krokan
et al., 2000). The importance of these DNA repair systems is confirmed by the
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fact that defects in these can result in cell death and hypersensitivity to endogenous
or environmental mutagens (Jackson, 1998). Therefore removing mutagenic
lesions in DNA is a vital task for repair systems. In general, the repair DNA damage
mechanisms in bacteria are well defined, whereas in higher eukaryotes the genes
and proteins responsible for repair await further investigation (Croteau and Bohr,
1997). It seems likely that DNA repair is integrated with cell cycle regulation,
transcription and replication and use some common factors (Slupphaug et al.,
2003). However, the enzymes of the third level of antioxidant defence do not
achieve complete repair or removal of damaged DNA molecules and this could
lead to arrest of cell cycle and cell death. In fact, programmed cell death (apoptosis)
is involved in maintenance of the genetic integrity by removing genetically altered
cells.
Selenium in an organic form can directly or indirectly effect DNA integrity
and repair. For example, elderly male dogs were fed on the diet supplemented
with selenium in the form of SeMet or high-selenium yeast at 3 g/kg or 6 g/kg
body weight per day for 7 months and a comparison was made with
unsupplemented control dogs. The extent of DNA damage in prostate cells and in
peripheral blood lymphocytes, as determined by the alkaline comet assay, was
lower among the selenium-supplemented dogs than among the control dogs
(Waters et al., 2003). Furthermore, Seo et al. (2002a) showed that selenium in the
form of SeMet induces a DNA repair response in normal human fibroblasts in
vitro, and protects cells from DNA damage. A possible mechanism for the inducible
DNA repair response was shown to be enhanced repair complex formation in
selenomethionine-treated cells. Similarly, treatment with SeMet either on initiation
or on selection/promotion, or during the entire experiment showed that SeMet
was most effective in regulating the cellular antioxidant defence systems, DNA
chain break control and reducing aberrant crypt foci in the colorectal tissues of
rats (Mukherjee et al., 2001).
Did you that SeMet can affect DNA-repair enzyme systems?
Protective effect of Se against DNA damage depends on the dose used. For
example, measurements in acinar cells in Syrian golden hamsters suggested a
more rapid repair of single-strand breaks DNA in hamsters prefed 2.5 ppm Se
than in those prefed 0.1 ppm Se (Birt et al., 1988). It has also been shown that
selenium in the form of SeMet can activate the p53 tumor suppressor protein by a
redox mechanism that requires the redox factor Ref1 (Seo et al., 2002). Specifically,
SeMet induced sequence-specific DNA binding and transactivation by p53 and
therefore the DNA repair branch of the p53 pathway was activated. Recognition
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and signalling of DNA damage is an important event for the induction of subsequent
cellular responses such as increased repair, cell cycle arrest and apoptosis.
Recognition of DNA breaks is accomplished by a group of phosphatidylinositol3-kinases. These kinases are ATM (ataxia telangiectasia mutated), ATR (ataxia
telangiectasia related) and the catalytic subunit of DNAPK (Christmann et al.,
2003). ATR and ATM can bind to DNA ends of damaged DNA, which results in
activation of the kinase activity. It is interesting that treatment with SeMet was
shown to enhance ATR and CHK2 gene expression in cultured human thyroid
epithelial cells (Kennedy et al., 2004). The authors suggested that SeMet may
prevent radiation-induced adverse biological effects by enhancing the DNA repair
machinery in irradiated cells. Therefore it is clear that SeMet has a unique specific
effect on the DNA repair system as well as provides a protection against DNA
damage and this effect is not the case or even can be opposite when selenite is
used.
In spite of important roles of protein oxidation in pathogenesis of the
development of various diseases, mechanisms for the control of protein oxidation
and their repair have not been well studied and this has been a topic of great
interest for the last few years. The oxidative damage to proteins is associated with
alteration of transport proteins and ion dis-balance, disruption to the receptors
and impair signal transduction, enzyme inactivation etc. It is believed that
conversion of SH groups into disulphides and other oxidized species (e.g.
oxyradicals) is one of the earliest events during the radical-mediated oxidation of
proteins. Therefore, thioredoxin plus thioredoxin reductase deal with these changes
by reducing protein disulphides to thiols and regulating redox-sensitive
transcription factors (Dean et al., 1997).
It is interesting that reversible oxidation of cysteine could be an important
cellular redox sensor in some proteins (Finkel, 2000). Methionine residues in
proteins are also very susceptible to oxidation with methionine sulfoxide formation,
which was detected in native proteins (Gao et al., 1998). This could affect activity
of various proteins (Table 1.8). In fact, almost all forms of ROS oxidize methionine
residues of proteins to a mixture of the R- and S-isomers of methionine sulfoxide
(Stadtman et al., 2002). Methionine sulfoxide reductase (Msr) can reduce either
the free or the protein-bound methionine sulfoxide back to methionine. Therefore
Msr is considered a repair mechanism for dealing with the product of reaction of
oxidants with methionine residues (Levine et al., 1996). The authors hypothesized
that methionine residues function as a last chance antioxidant defence system
for proteins. It was shown that in bacterial glutamine synthetase surface-exposed
methionine residues surrounding the entrance to the active site are preferentially
oxidised and other residues (e.g. cystein) within the critical regions of the protein
are protected without loss of catalytic activity of the protein (Levine et al., 1996).
Indeed, due to Msr activity the methionine-methionine sulfoxide pair can function
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Table 1.8 Proteins and peptides with activity altered by methionine oxidation (adapted from Levine et al.,
2000).
Adrenocorticotropic hormone
-1-antitrypsin
-2-antitrypsin
-2-macroglobulin
Amyloid beta peptide
Antiflammin
Antitrombin
Apolipoprotein
Bombesin
Bugarotoxin
Calcitonin
Calmodulin
Chemotactic peptide f-Met-Leu-Phe
Cholecystokinin
Chorionic somatomammotropin
Chymotrypsin
Complement C5
Cytochrom c peroxidase
Cytochrome c
Echistatin
Enkephalin
Factor VII
Fibronectin
Glucagon
Glutamine synthetase
Growth hormone
Hemoglobin
HIV-2 protease
Interferon -2b
Interferon
Interleukin 6
Keratinocyte growth hormone
Lypoxygenase
Lutropin
Lysozyme
Mucus proteinase inhibitor
Neuropeptide Y
Ovoinhibitor
Parathyroid hormone
Pepsin
Phosphoglucomutase
Plasminogen activator inhibitor
Potassium channel
Prolactin
Ribonuclease
Ribosomal protein L12
Secretory leukocyte proteinase inhibitor
Small heat shork protein
Snake venon cardiotoxin
Stem cell factor
Subtilisin
Thrombomodulin
Tissue plasminogen activator
Triptophanase
Vasoactive intestinal peptide
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On the other hand, overexpretion of the MsrA gene in the nervous system markedly
extends the lifespan of the fruit fly Drosophila (Ruan et al., 2002), human T-cells
(Moscovitz et al., 1998) and mouse (Moscovitz et al., 2001). Furthermore, the
authors showed that MsrA transgenic flies are more resistant to paraquat-induced
oxidative stress. In fact Msr is also considered to be a potential missing link in the
post-translational modification cycle involved in the specific oxidation and
reduction of methionine residues in cellular signalling proteins which can change
cellular excitability (Hoshi and Heinemann, 2001). This could well be the regulatory
mechanism similar to protein phosphorylation. The general scheme of antioxidant
function of Msr is shown in Figure 1.2.
CO2+
Pentose
G-6-P
Thioredoxin
NADPH
-ox
NADP+
Thioredoxinred
Msr -red
Msr -ox
ProteinMetSO
RSox
ProteinMet
RSred
Figure 1.2 Function of methionine sulfoxide reductase (adapted from Levine et al., 2000).
RSox represents a reactive species which is reduced with concomitant oxidation of methionine residue in
the protein. Thioredoxin-red and ox- are reduced and oxidised thioredoxin. Msr-ox and red- are
methionine sulfoxide reductase.
MsrA has been known for a long time, and its repairing function is well
characterised, however, recently, a new methionine sulfoxide reductase was
characterised (Grimaud et al., 2001). It was referred to as MsrB and it was shown
that the gene of MsrB is present in genomes of eubacteria, archaebacteria, and
eucaryotes. Therefore, in mammals two methionine sulfoxide reductases, MsrA
and MsrB, are expressed with different substrate specificity (Grimaud et al., 2001).
They catalyze the thioredoxin-dependent reduction of the S-isomer and R-isomer
of methionine sulfoxide to methionine, respectively.
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Niacin
Membrane Se
Transport
Riboflavin
Vit .E quinone
Loss
Loss
CO2+
Pentose NADPH GSSH AA
2
Glucose
Figure 1.2
Loss
Diketo-L- gulonic
acid
Redox cycle of vitamin E (Adapted from Winkler et al., 1994 ; Surai, 1999)
As a result of antioxidant action of vitamin E, tocopheroxyl radical is formed. This radical can be reduced
back to an active form of -tocopherol by coupling with ascorbic acid oxidation. Ascorbic acid can be
regenerated back from the oxidised form by recycling with glutathione which can receive a reducing
potential from NADPH, synthesised in the pentose phosphate cycle of carbohydrate metabolism. Enzymes
involved in vitamin E recycling are as follows: 1. Thioredoxin reductase; 2. Glutathione reductase; 3.
Glucose-6-phosphate dehydrogenase. Due to incomplete regeneration (the efficiency of recycling is usually
less than 100%) in biological systems, the antioxidants have to be obtained from the diet (vitamin E and
carotenoids) or synthesised in the tissues (ascorbic acid and glutathione).
Therefore the antioxidant protection in the cell depends not only on vitamin E
concentration and location, but also relies on the effective recycling. Indeed, if
the recycling is effective then even low vitamin E concentrations are able to
maintain high antioxidant protection in physiological conditions. For example,
this could be demonstrated using chicken brain as a model system. Indeed, our
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25
data (Surai, 2002) indicate that the brain is characterised by extremely high
concentrations of long chain polyunsaturated fatty acids predisposing this tissue
to lipid peroxidation. Furthermore, brain contains much lower levels of vitamin E
than other body tissues. However, in fresh chicken brain, levels of products of
lipid peroxidation are very low, which could be a reflection of an effective vitamin
E recycling by ascorbic acid which is present in this tissue in comparatively high
concentrations. Antioxidant recycling is the most important element in
understanding mechanisms involved in antioxidant protection against oxidative
stress. The rate of regeneration, or recycling, of the vitamin E radicals may affect
both its antioxidant efficiency and its lifetime in biological systems. As can be
seen from data presented above the antioxidant defence includes several options
(Surai, 2002):
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Stress conditions
Se,Mn
Zn, Cu
Nutritional
Diet optimization
Environmental condition
optimization
Disease prevention and
treatment by antibiotics and
other drugs
Environmental
Internal
Temperature, humidity,
hyperoxia,
radiation, UV,
microwave etc.
Diseases: bacterial, viral,
allergy etc.
Fre e ra d ic a l g e n e ra tio n
Electron-transport chain,
Phagocytes,
Xanthine oxidase etc.
Lipid p eroxidation,
d amag es to lipids,
p rotein s, DN A
Membrane damage
Nutrient absorption
decrease
Nutrient imbalance
Decrease of productive
and reproductive
performances
Figure 1.4 Antioxidant-prooxidant balance in the organism (adapted from Surai, 1999).
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Did you know that all antioxidants in the body are working as a
team responsible for antioxidant defence and we call this
team the antioxidant system?
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Table 1.9 Some regulatory functions of free radicals (adapted from McCord, 2000; Droge, 2002; Yoon
et al., 2002; Thundathil et al., 2003; de Lamirande and Gagnon, 2003).
Type of radical
Source of radical
Superoxide and
related ROS
Any source
ROS
Any source
Physiological process
Smooth muscle relaxation (control of
vascular tone) and various other cGMPdependent functions; specific role in signal
transduction events leading to sperm
capacitation
Control of ventilation; Control of
erythropoitenin production and other
hypoxia-inductible functions; Smooth
muscle relaxation; Signal transduction from
various membrane receptors/enhancement of
immunological functions
Oxidative stress responses and the
maintenance of redox homeostasis; An
increased intracellular ROS level stimulates
cell proliferation, apoptosis and
differentiation depending on the relative
concentration of oxidants in the cell;
Promotes physiological capacitation in
sperm allowing the acquisition of fertilizing
capacity
Activation of a variety of kinases including
Src kinase family, preotein kinase C,
mitogen-activated protein kinase (MAPK),
receptor tyrosine kinases and transriptional
factors such as AP-1 and NF-kB;
Modification of redox-sensitive proteins
(e.g. thioredoxin) affecting stress kinases
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Increased interval between an egg being laid and its cooling down for
storage. Eggs should be collected more frequently on warm days. In such
conditions free radical damages to lipids and proteins could occur and
antioxidant protection would be beneficial.
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Delay in collecting chicks from incubator. Since not all chicks are hatched
at the same time because of heterogeneity of the starting material (eggs
from older breeders hatch earlier than those from young flocks and chicks
from smaller eggs hatch earlier than those from large eggs; Decuypere et
al., 2001), some would be in the incubator for 2-12 hours longer than others.
This puts pressure on antioxidant defence capacity. Furthermore, any delay
in food and/or water intake after hatching usually negatively affect a number
of performance parameters and a delay occurs in the maturation of the
enzymatic systems that control metabolism (Decuypere et al., 2001) and
free radical production and protection against them.
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cell death (Cereser et al., 2001). Similarly, in a recent study forty-one male,
healthy agricultural sprayers, exposed to pesticides for 5 years, were
compared with twenty one controls matched for age and economic status
with respect to free radical generation, lipid peroxidation, antioxidant status
and concentration of cellular enzymes were determined (Prakasam et al.,
2001). Significantly increased TBARS levels and decreased concentrations
of antioxidants such as GSH, alpha-tocopherol, ascorbic acid and
ceruloplasmin were observed in sprayer populations, when compared to
controls. When Mallard eggs were exposed to diquat dibromide, a commonly
used aquatic herbicide, significant manifestations of oxidative stress were
apparent in hatchlings and included increased hepatic lipid peroxidation
and decreased brain reduced glutathione concentration (Sewalk et al., 2001).
Another herbicide ANITEN I caused an oxidative stress in pheasant kidney
and liver by decreasing activities of antioxidant enzymes (Holovska et al.,
1998).
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Oxidized fat in the diet can cause oxidative stress in the intestine increasing
antioxidant requirement to prevent damages. For example the intake of
oxidized oil caused a growth depression and increased TBARS concentrations
in plasma and decreased concentrations of tocopherols, lutein, beta-carotene
and retinol in plasma and tissues of broilers (Enberg et al., 1996).
Furthermore, toxic products of lipid peroxidation may damage the brush
border membrane in the intestine (Kimura et al., 1984) decreasing absorption
of antioxidants. When a chicken diet includes spent fat after its high
temperature treatment, the fat usually contains peroxides and hydroperoxides
which can contribute substantially to oxidative stress. Therefore it is
necessary to evaluate a benefit and disadvantages of using such fat sources.
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Vitamin A excess in the diet is shown (Surai et al., 1998; 2000) to cause an
oxidative stress decreasing vitamin E and carotenoid concentrations in
tissues and increasing tissue susceptibility to lipid peroxidation.
The list of potential stresses can vary from one poultry farm to another, but
overproduction of free radicals and the critical need for antioxidant protection are
common factors. It is interesting, that in wild, birds are often exposed to
electromagnetic fields, which cause oxidative stress and suppressed plasma total
protein, hematocrit and carotenoids in kestrels (Fernie and Bird, 2001). Similar
stresses can be attributed to farm animal production. Some potential stressconditions, related to the production of free radicals in the digestive tract of human
and animals are presented in Chapter 10.
Did you know that antioxidant-prooxidant balance in the cell
is an important determinant of various physiological functions?
Conclusions
Antioxidant-prooxidant balance in the cell is an important determinant of various
physiological functions. Indeed, oxidative stress occurs when this balance is
disturbed due to overproduction of free radicals or compromised antioxidant
defences. Free radical overproduction and oxidative stress are considered as a
pathobiochemical mechanism involved in the initiation or progression phase of
various diseases.. In animal production free radial generation and lipid peroxidation
are responsible for the development of various diseases as well as for the decrease
of animal productivity and product quality (Hurley and Doane, 1989; McDowell,
2000). Dietary antioxidants may be especially important in protecting against the
development of the oxidative stress.
In general, ingestion of excessive amounts of antioxidants is presumed to
shift the oxidant-antioxidant balance toward the antioxidant side. This is supposed
to be beneficial; however, this may also adversely affect key physiological
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Introduction
It is generally accepted that in biological systems Se participates in various
physiological functions as an integral part of a range selenoproteins. In fact, it is
well known that sulphur and selenium occur in proteins as constituents of the
amino acids cysteine, methionine and selenocysteine, and selenomethionine,
respectively. The redox activity of those amino acids under physiological conditions
allows a wide variety of posttranslational protein modifications, metal free redox
pathways, and unusual chalcogen redox states (Jacob et al., 2003). For example,
unlike any other amino acid, cysteine and SeCys can participate in several distinct
redox pathways, including exchange and radical reactions, as well as atom-,
electron-, and hydride-transfer reactions. Furthermore, the position of selenium
in the periodic table between the metals and the non-metals makes selenoproteins
effective catalysts for many biological redox transformations (Jacob et al., 2003).
Selenoprotein family
The human selenoproteome consists of 25 selenoproteins (Kryukov et al., 2003;
Table 2.1) and about 35 Se-containing proteins or protein subunits can be distinguished
by two-dimensional electrophoresis after in vivo labelling with 75Se (Behne at al.,
2000; Table 2.2). Recently, 24 Se-containing proteins have been found in subcellular
fractions of normal human liver (Chen et al., 2002). The molecular weights of the
subunits were mostly in the range 20-30 kDa and 50-80 kDa. In accordance with
another estimation the number of selenoproteins in mammals could reach 30-50
(Kohrle et al, 2000). There is also a range of prokaryotic selenoproteins (Table 2.3).
Did you know that there are at least 25 selenoproteins
in the human body?
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Protein
Protein length
SeCys position
201
190
226
197
221
499
656
523
249
265
278
122
397
94
145
556
669
381
Selenoprotein R (SelR)
Selenoprotein S (SelS)
SPS2
Selenoprotein T (SelT)
Selenoprotein V (SelT)
Selenoprotein W (SelW)
15-kDa Selenoprotein (Sel15)
116
189
448
182
346
87
162
47
40
73
73
73
498
655
522
126
133
144
44
387
92
48
428
667
59, 300,318,330,345,
352,367,369,376,378
95
188
60
36
273
13
93
Table 2.2 Other eukaryotic selenoproteins (adapted from Kryukov and Gladyshev, 2002; Behne and
Kyriakopoulos, 2001)
Protein
Selenoprotein X3 (SelX)
Selenoprotein Z3 (SelZf1 and SelZf2)
Selenoprotein Pb1 (SelPb)
Selenoprotein W21 ) (SelW2)
Selenoprotein U4(SelU)
Selenoprotein T21 (SelT2)
Selenoprotein G-rich2 (SelG-rich)
Selenoprotein BthD2 (SelBthD)
18-kDa Selenoprotein5 (Sel18)
Protein length
SeCys position
265
94
165
109
249
162
64
13
19
110
37
93
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Selenoprotein
Function
Glycine reductase
Glycine/sarcosine/betaine reductase
Selenoprotein A
Glycine reductase selenoprotein B
Sarcosine reductase selenoprotein B
Betaine reductase selenoprotein B
Proline reductase
Heterodisulfide reductase
Seleno-peroxiredoxin
Putative redox active selenoprotein
Formate dehydrogenase
Formylmethanofuran dehydrogenase
NiFeSe-hydrogenase
F420 non-reducing hydrogenase
F420 reducing hydrogenase
Selenophosphate synthetase
Formation of a selenoether
Redox function
Transfer of selenoether
Formation of selenoether
Formation of selenoether
Formation of selenoether
Redox function, formation of selenoether
Redox function
Redox function (peroxidase)
Redox function
Hydrogen donor
Redox function
Hydrogen donor
Redox function
Redox function
Formation of key metabolite for selenoprotein
synthesis
Formation of a carbon oxide selenide
Unknown
Unknown
CO dehydrogenase
Nicotinic acid hydroxylase
Xanthine dehydrogenase
The mammalian Se-containing selenoproteins are divided into three groups (Figure
2.1; Behne and Kyriakopoulos, 2001).
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1.
2.
3.
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48
Se-Met
Se-Cys
Intermediary
Se pool
Specific
incorporation
(Se-Cys)
Selenate
Selenite
Specific
binding
Figure 2.1 Incorporation of Se into proteins (Adapted from Behne and Kyriakopoulos, 2001).
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50
Table 2.4 Selenoprotein genes in completely sequenced eukaryotic genomes (Adapted from Kryukov et
al., 2003)
Organism
Genome size
Homo sapiens
Mus musculus
Drosophila melangaster
Caenorhabditis elegans
Arabidopsis thaliana
Saccharomyces cerevisiae
3,400,000,000
3,454,200,000
137,000,000
97,000,000
100,000,000
12,067,280
Estimated number
Number of
of genes
selenoprotein genes
40,000
40,000
14,331
20,206
25,000
6,312
25
24
3
1
0
0
Glutathione peroxidase (GSH-Px) and thioredoxin reductase (TR) are the most
abundant antioxidant Se-containing proteins in mammals (Gladyshev et al., 1998).
Major characteristics of GSH-Px and TR are shown in Table 2.5 and Figure 2.2.
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Oxidixed ascorbate
Thioredoxin reductase
NADP+
Reduced Thioredoxin
Reduced ascorbate
Oxidized Thioredoxin
Cell growth
Inhibited apoptosis
Thioredoxin
peroxidase
Ribonucleotide
reductase
Transcription
factors
Cellular sensitivity to
glucocorticoids
Immunomodulation
Pregnancy and birth
Antioxidant
DNA synthesis
Gene transcription
Neuronal survival
Figure 2.2 Thioredoxin reductase functions (Adapted from Mastacich and Powis, 2000; Arner and
Holmgren. 2000;Makino et al., 1996)
Glutathione peroxidase
Glutathione peroxidase family includes several members from which there are at
least 6 different Se-dependent enzymes. They differ in molecular weight, substrate
specificity, cell distribution and perform a variety of functions.
Did you know that there are six Se-dependent glutathione
peroxidases?
CYTOSOLIC GLUTATHIONE PEROXIDASE (cGSH-PX OR GSH-Px1)
Glutathione peroxidase (glutathione H 2O 2 oxidoreductase E.C. 1.11.1.9) was
discovered by Mills in 1957, who showed that this enzyme had a protective effect
in erythrocytes against H2O2- or ascorbate-induced hemolysis. Sixteen years later
it became clear that GSH-Px was a selenoenzyme (Rotruck et al., 1973; Flohe et
al., 1973). In fact, Rotruck et al. (1973) were the first to show that in rat red cells
Se was tightly bound to the enzyme and demonstrated the uptake of 75Se by GSHPx (Ahrens, 1996). In the same year Flohe et al. (1973) reported that bovine
GSH-Px contained one Se atom per subunit. It is interesting that 1973 was a year
of Se discoveries, since in the same year selenoproteins have been described in
micro-organisms.
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GSH-Px5
Epididymal
GSH-Px
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Epididymal fluid,
epididymis
25.0
25.2
22.1
25.5
21.9
21.9
H2O2, organic
hydroperoxides,
Phospholipid
Hydroperoxides
Low activity
towards H2O2 and
organic hydroperoxides
GSH
GSH
Caput epididymis
Expressed in kidney
GSH,
thioredoxin,
glutaredoxin
H2O2, t-BHP,
phospholipid
hydroperoxides
Mucosal epithelial
cells in GIT
Erythrocytes,
kidney and liver
GSH
GSH
H2O2, t-BHP
H2O2, t-BHP
NonSeGSH-Px
GSH-Px4
Phospholipid
hydroperoxide
GSH-Px
Non-selenium
GSH-Px
Plasma
GSH-Px3
Extracellular
(plasma)
GSH-Px
Intracellular,
partly cytosolic,
partly mitochondrial, partly
membrane-bound
Intracellular,
cytosolic
Gastrointestinal GSH-Px2
GSH-Px
Intracellular,
cytosolic, partly
mitochondria
Other
characteristics
GSH-Px1
Electron
donors
Cytosolic
GSH-Px
Substrates
Nomenclature
Glutathione
peroxidase
GSH-Px
Se
Table 2.5 Glutathione peroxidase characteristics (Adapted from Hayes and McLellan, 1999; Singh and Shichi, 1998; Schwaab et al., 1998)
Subunit
size (kDa)
Localization
52
ROOH + 2GSH
H2O2 + 2GSH
ROH +GSSG+H2O
GSSG + 2H2O
GSH-Px
ROOH
ROH
ONOE-Se-OH
E-Se-H
GSSG
GSH
GSH
H 2O
E-Se-SG
Figure 2.3 Catalytic mechanisms of GSH-Px (Adapted from Mugesah and Singh, 2000; Sies et al., 1997)
GSH-Px
ONO- +GSSG+H2O
GR
2GSH + NADP+
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Substrate
Substrate
H2O2
Allopregnanolone 17-hydroperoxide
Cholesterol 7-hydroperoxide
Cholesteryl hydroperoxyoctadecadienoates
Cumene hydroperoxide
Ethyl hydroperoxide
Ethyl hydroperoxyacetadecatrienoate
Glyceryl 1-hydroxyoctadecadianoates
15-Hydroperoxyprostaglandine E1
1-linoleoyl lysophosphatidylcholine
hydroperoxide2
Morgan et al., 2004; 2Marinho et al., 1997; 3Kaplan and Ansari, 1984; 4Chaudiere and
Tappel, 1983; 5Gebicki et al., 2002; 6Sies et al., 1997
Table 2.7 Specificity of the antioxidant enzymes to peroxide substrates (as initial rate of reaction), mol/
min/mg protein (Adapted from Brigelius-Flohe et al., 2002)
Enzyme
cGSH-Px
eGSH-Px
PH-GSH-Px
SeP
t-BuOOH
H2O2
PC-OOH1
PC-OOH2
PC-OOH3
552
199
9.56
<0.02
793
373
34.8
<0.02
<0.02
54.1
2.65
<0.02
<0.02
0.03
64.8
1.11
<0.02
0.18
28.8
1.15
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Species
Tissue
Rat*
Trout*
Japanese sea bass1
Black sea bass2
Liver
Lung
Erythrocytes
Lens
Erythrocytes
Liver
Aorta
Erythrocytes
Placenta
Liver
Liver
Liver
76,000
84,000
83,800-85,000
96.600
88,000-89,000
77,000-80,000
84,000
95,000-100,000
85,500
95,000
70,000
72,000
Human3
Placenta
85,000
Human4
Hamster5
Rabbit6
Human7
Human8
Rat9
Bovine10
Human11
Plasma
Liver
Lung macrophages
Platelet
Milk
Lung
Erythrocytes
Erythrocytes
92,000
92,000
80,000
92,000
92,000
80,000
83,800
95,000
Cow*
Sheep*
Pig*
Human*
*Adapted from Combs and Combs, 1986; 1Nagai et al., 2002; 2Braddon-Galloway and
Balthrop, 1985;3Awasthi et al., 1979; 4Broderick et al., 1987; 5Chaudiere and Tappel, 1983;
6
Chiba et al., 1999; 7Rey et al., 1994; 8Bhattacharya et al, 1988; 9Chiu et al., 1976; 11Flohe
et al., 1971;12 Awasthi et al., 1975;
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GSH-Px activity was shown to have species- and tissue-specificity. For example,
Chiaradia et al. (2002) determined lymphocyte GSH-Px activity, plasmatic GSH
levels and the effect of H2O2 on the responsiveness of lymphocytes to proliferative
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Table 2.9 Comparison of GSH-Px knockout animals with wild type animals
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Effect
System
References
Mice fibroblasts
Mice
Galactosamine/
Li et al., 2003
endotoxin induced
toxicity in mice
Hyperhomocysteinemic mice
Mice
Ho, 2002
Mice
Mice
Ischemia/
reperfusion mouse
model
Cold-induced
injury in mice
Mice
Fu et al., 2001a
Mice exposed to
Ohlemiller et al.,
2000
broadband noise
Mice
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Effect
System
References
Mice lenses
Mice
Mice
Mice
Mice
Fu et al., 1999
Mice
Mice
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Effect
System
References
Transgenic mice
Rats
Endothelial cells
Human cells
Kretz-Remy and
Arrigo, 2003
Human breast
cancer T47D cells
Mice
Mice
Lei, 2001
Human breast
cancer cells
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Effect
System
References
MCF-7 cells
Li et al., 2001
Mice
Insulin-producing
N-acetyl-D,L-penicillamine and
sodium nitroprusside, which generate both
NO and reactive oxygen species
RINm5F cells
Mice
Mirochnitchenko et
al., 1999
Mice
Bensadoun et al.,
1998
Insulin-producing
RINm5F cells
Mice
Mice
Weisbrot-Lefkowitz et
al., 1998
Human MCF-7
breast cancer cells
Doroshow, 1995
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al., 2000
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System
References
Breast carcinoma
cells
Mice
Mice
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when organic Se is used in the diet Se reserves in the form of SeMet nonspecifically incorporated in various proteins, for-example in muscles, are
built
in stress conditions requirement for selenoproteins to prevent free-radical
related damages is increased, but selenium bioavailability decreased due to
decreased feed consumption
redox status of muscle cells decreased due to depletion of antioxidants and
probably some proteins are oxidised
proteosomes are activated and protein degradation is increased
SeMet is released from proteins and used for additional synthesis of
selenoproteins
Antioxidant defences are improved and redox status of the cells changed
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transcription using different first exons Ia and Ib, respectively (Imai et al., 2003).
Recent data (Ufer et al., 2003) indicate that the basic PH-GSH-Px promoter
constitutes a 200 bp oligonucleotide, which is localized immediately upstream of
the 3'-ATG and involves functional stimulating proteins (SP1/SP3), nuclear factor
Y (NF-Y) and SMAD binding sites. The authors suggested that the corresponding
trans-regulatory proteins may contribute to differential expression regulation of
the mitochondrial and cytosolic PH-GSH-Px isoforms. Indeed, the genomic
sequence of rat PH-GSH-Px4 was established and investigated in respect to
expression into the cytosolic, mitochondrial, and nuclear forms of PH-GSH-Px.
In silico analysis suggested the presence of two distinct promoter regions, the
upstream one leading to transcripts translating into cPH-GSH-Px or mPH-GSHPx and the downstream one yielding nPH-GSH-Px (Maiorino et al., 2003). The
data suggested that the formation of nPH-GSH-Px is due to alternative transcription
and not to alternative splicing. In particular, transcripts encoding nPH-GSH-Px
were most abundant in testis although not restricted to this organ. This observation
points to a general role of the nuclear PH-GSH-Px variant in regulating cell division.
It is well known that PH-GSH-Px is widely expressed in normal tissue, and
especially high in testis (Imai et al., 1995), where it has an important role in
spermatogenesis and sperm function and is under gonadotropin control. In this
organ a relevant PH-GSH-Px activity is strongly linked to mitochondria of cells
undergoing differentiation to spermatozoa. In tested mitochondrial for of PHGSH-Px is electrostatically bound to the inner surfaces of the organelle. (Roveri
et al.,1994). In fact, there was no difference between the soluble and the
mitochondrial enzyme in terms of chromatographic properties, the electrophoretic
mobility, the reactivity to antibodies and the fragmentation patterns and substrate
specificity. However, two-dimensional electrophoresis followed by
immunostaining with monoclonal antibodies, showed the presence of multiple
isoforms with a different pattern between the soluble and the mitochondrial enzyme.
(Roveri et al., 1994)
The gene encoding for the porcine PH-GSH-Px was cloned (Brigelius-Flohe et
al., 1994) and the complete genomic sequences of the human (Kelner and Montoya,
1998) and the murine (Borchert et al., 1999) orthologs were reported. A full-length
cDNA for PH-GSH-Px including exon Ib from rat and mouse testis was also cloned
(Nakamura et al., 2003). In Western blot analysis of proteins extracted from testes of
mutant mice and from developing mouse testes, two signals at 19- and 22-kDa were
observed which confirm the existence of two PH-GSH-Px forms in testicular cells
(Nayernia et al., 2004). It is interesting that PH-GSH-Px is found to be localised in the
midpiece of spermatozoa in various species including Drosophila melanogaster, frog,
fish, cock, mouse, rat, pig, bull, and human (Nayernia et al., 2004). It is also important
to mentioned that PH-GSH-Px mRNA expression in the male reproductive organs is
under estrogen control (Nam et al., 2003).
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This process coincides with a complete loss of GSH in late sperm maturation
The aggregates could only be dissolved by drastic treatment with low nuclear
weight thiols
In vitro aggregation of solubilised capsular material by H2O2 depends on
the absence of GSH
It was postulated that an oxidised form of PH-GSH-Px reacts with surface
SH groups of itself and other proteins to create dead-end intermediates (Mauri
et al., 2003) building up mitochondrial capsule.
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Effect
System
References
MCF-7 cells
Rabbit aortic
smooth muscle
cells
Neonatal rat
cardiac myocytes
RBL2H3 cells
Human dermal
fibroblasts
Dissemond et al.,
2003
RBL2H3 cells
Stimulated
RBL-2H3 cells
A431 cells
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Effect
System
References
Tumor epithelial
cell line
Human breast
cancer MCF-7
cells
Brigelius-Flohe et al.,
2000
rat basophile
leukaemia (RBL)
2H3 cells
RBL-2H3 cells
RBL2H3 cells
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the rate constants describing the reactivity of human p-GSHPx and human
GSHPx-1 with LinOOH and H2O2 are in the same range;
pGSH-Px is more reactive with LinOOH and GSHPx-1 is more reactive
with H2O2.
pGSHPx has a low level of reducing activity toward cholesterol 7 alphaOOH and no detectable activity with the 5 alpha-OOH isomer in contrast to
PH-GSPx which readily reduced both isomers. However, it catalyzed the
reduction of phosphatidylcholine hydroperoxides to the corresponding
hydroxy derivatives (Yamamoto et al., 1993). In fact, the reductions of
aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide,
t-amyl hydroperoxide, t-butyl hydroperoxide, paramenthane hydroperoxide)
were better catalyzed by pGSH-Px than were reductions of the more
physiological substrates (linoleic acid hydroperoxide, hydrogen peroxide,
peroxidized plasma lipids, and oxidized cholesterol) (Howard and Hawkes,
1998);
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and spermatid (Nakamura et al., 2003). It seems likely that regulation of expression
of the PH/snGSH-Px gene involves transcriptional and post-transcriptional events.
Therefore, snGSH-Px has cytosolic localisation and has negative regulatyory
elements in the first intron of the PH/snGSH-Px gene with several protein-binding
sites (Borchert et al., 2003). In Se-depleted rats the concentration of sn-GSH-Px
decreased to a third of the normal level and chromatin condensation was severely
disturbed (Pfeifer et al., 2001). The authors suggested that sn-GSH-Px is a
protamine thiol peroxidase responsible for disulfide cross-linking by reduction of
reactive oxygen species. It seems likely that antioxidant protection of the DNA
could be another important role for this enzyme.
GSH-Px6
It is a close homolog to plasma GSH-Px3 and the gene encoding human and
porcine GSH-Px6 were cloned (Kryukov et al., 2003). However, in rodents SeCys
was replaced by Cys and therefore this enzyme is not a selenoprotein. The authors
suggested that in rats GSH-Px6 was previously described as odorant-metabolizing
protein. In fact, GSH-Px6 was only detected in embryos and olfactory epithelium
(Kryukov et al., 2003).
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There are several specific non-Se GSH-Px which have some unique tissue-specific
features of their expression. For example, a secreted 24-kDa sperm-bound
selenium-independent GSH-Px protein was characterized and cloned and called
GSH-Px5. It contains a 663 bp open-reading frame that, after conceptual translation,
shows extensive identity with proteins belonging to the GSH-Px family (Vernet et
al., 1996). The authors showed that GSH-Px5-expressing cells can metabolize
hydrogen peroxide in a manner that is consistent with a peroxidase activity.
However, GSH-Px5 is insensitive to a regular inhibitor of GSH-Px enzymes. The
expression of GSH-Px5 was found to be restricted to the mouse caput epididymidis
(Schwaab et al., 1998), but low levels of expression were shown in the kidney
and liver (Dufaure et al., 1996) and in other mouse tissues (Lahti et al., 2001). It
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An apparent MW of 112 kDa by gel filtration and 29 kDa by SDSpolyacrylamide gel electrophoresis.
The enzyme consisted of four identical subunits.
The enzyme was active with a variety of peroxides including H2O2, cumene
hydroperoxide, t-butyl hydroperoxide, triphenylcarbinyl hydroperoxide,
linoleic hydroperoxide and 5-phenylpentenyl hydroperoxide
Glutathione was essential for the reaction, however, L-cysteine, dithiothreitol
and 2-mercaptoethanol were inactive.
No selenium was found in the enzyme by the fluorometric assay with 2.3diaminonaphthalene.
The enzyme demonstrated no glutathione S-transferase activity when tested
with 1-chloro-2,4-dinitrobenzene, and several other compounds.
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It seems likely that non-Se GSH-Px family is much bigger that it had been suggested
before. Recently a novel non-selenium GSH-Px has been identified from rat lung
(Power and Nicholas, 1999). In fact the same enzyme is known as 1-Cys
peroxiredoxin (a bifunctional enzyme with two distinct active sites) called
nonselenium GSH-Px (NSGP) and is characterised by Ca 2+ -independent
phospholipase A2 and GSH-Px activity and has a specific role in the regulation of
phospholipid turnover as well as in protection against oxidative injury (Chen et
al., 2000). Recombinant human NSGPx expressed in Escherichia coli from a
human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2linolenoyl- or sn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate;
NADPH or thioredoxin could not substitute for GSH (Fisher et al., 1999). It was
shown that the enzyme catabolised H 2O 2, as well as organic hydroperoxides
including phospholipid hydroperoxides. It is interesting that the enzyme had no
GSH S-transferase activity. Bovine cDNA encoding NSGPx showed >95%
similarity to previously published human, rat, and mouse sequences and does not
contain the TGA stop codon. The molecular mass of bovine NSGPx deduced
from the cDNA is 25,047 Da (Fisher et al., 1999). In normal human brain there
was a very low level of NSGP in astrocytes and this enzyme was upregulated in
pathological conditions including Parkinsons disease and demnia (Power et al.,
2002).
Clearly, the GSH-Px family is an important part of antioxidant defences in
human and animal bodies and specific roles of the enzymes in regulation of other
important functions warrant further investigation.
Did you know that first selenoprotein called cytosolic GSH-Px
was described in 1973 and the last member of this family
(GSH-Px6) was discovered 30 years later in 2003?
Thioredoxin reductase
Redox status of the cell is a major determinant of many different pathways
including gene regulation (Morel and Barouki, 1999). A thiol redox system
consisting of the glutathione system (glutathione/glutathione reductase/
glutaredoxin/glutathione peroxidase, Holmgren, 1989; Cotgreave and Gerdes,
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Table 2.12 Classification of human thioredoxins and thioredoxin reductases (Adapted from MirandaVizuete et al., 2004)
Thioredoxins
Size, kDa
Name
Chromosomal
localization
Trx-1
9q31
11.71
Trx-2
Txl-1/Trp32
Erdj5/JDPI
22q13.1
18q21.2
2p22.1-23.1
11.87
32.25
91.08
Sptrx-1
18p11.2-11.31
53.27
Sptrx-2
7p14.1
67.27
Sptrx-3
Txl-2
Not dtermined
3q22.3-23
14.57
36.85
TrxR1
TrxR2
TGR
Thioredoxin
12q23-24.1
54.71
22q11.21
53.06
3p13-q13.33
63.63
Tissue
specificity
Subcellular
localization
Ubiquitous
Mainly cytosolic,
nuclear upon
certain stimuli
Ubiquitous
Mitochondrial
Ubiquitous
Cytosolic
Ubiquitous
Endoplasmic
reticulum
Testis/spermatid Sperm fibrous
sheath
Testis/spermatid Sperm fibrous
sheath
Testis/spermatid Golgi
Ubiquitous,
Associated with
especially in
microtubules in
testis and lung cilia and flagella
reductases
Ubiquitous
Cytosolic
Ubiquitous
Mitochondrial
Ubiquitous, but Cytosolic
highly expressed
in testis
10
8
6
4
2
0
Liver
Kidney
Thymus
Spleen
Lung
Brain
Heart
Erythrocytes
Figure 2.4 Concentrations of thioredoxins in tissues of 1-week-old calf (Adapted from Gromer et al.,
2004)
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Biological roles of the thioredoxin system are diverse and include (Gromer et al.,
2004; Das, 2004; Rundolf and Arner, 2004):
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There are at least three forms of this enzyme (Table 13). TrxR1 is located
predominantly in the cytosol; TrxR2 is found in mitochondria (Miranda-Vizuete
et al., 1999; Powis et al., 2000). In fact, human mitochondrial TrxR consists of
521 amino acid residues with a calculated molecular mass of 56.2 Kda. It is also
highly homologous to the previously described cytosolic TrxR1. It is interesting
that TrxR2 has extra 33 amino acids in its molecule at the N-termins. It was
shown that mRNA for TrxR2 is highly expressed in prostate, testis and liver. TrxR2
gene consists of 18 exons spanning about 67 kb with a chromosomal localization
at position 22q11.2 (Miranda-Vizuele et al., 1999). One more TrxR3 is located in
the testes (Sun et al., 1999). Recently, Sun et al. (2001) demonstrated that testes
TR has broad substrate specificity and can reduce several components of the
thioredoxin and glutathione systems. It has been predicted that other members of
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Reactions
Reactions
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Tissue
Liver
Lung
Heart
Kidney
Brain
Breast muscle
Bursa
Thymus
Spleen
RBC
Plasma
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Control,
(0 Se)
Selenite
(0.3 ppm)
Sel-Plex
(0.3 ppm)
Sel-Plex +Selenite
(0.3 ppm)
0.023
0.047
0.024
0.061
0.060
0.028
0.026
0.038
0.025
0.030
0.034
0.084
0.082
0.062
0.087
0.076
0.069
0.070
0.092
0.045
0.066
0.031
0.071
0.085
0.114
0.079
0.149
0.083
0.098
0.140
0.065
0.040
0.025
0.053
0.034
0.056
0.051
0.129
0.066
0.058
0.076
0.076
0.021
0.039
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Cellular distribution
Liver homogenate
Nuclear pellet
Post nuclear supernatant
Mitochondrial pellet
Post-mitochondrial supernatant
Mitochondrial pellet
Mitochondrial membranes
0.107
0.129
0.087
0.065
0.107
0.133
0.093
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RT3>>T4>T3
Homodimer of a 27-kDa subunit
Substrate specificity
Structure
Decreased
Cloned in species
Se deficiency
29,000
Molecular mass of
monomer, Da
Subcellular location
Induction
Expression
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T4>rT3
Decreased
Selenocysteine?
30,500
Systemic>local T3 production,
Local>systemic T3 production;
degradation of rT3 and sulfated
provides intracellular T3 in specific
iodothyronines; source of plasma T3 tissues; souce of plasma T3 (50%)
Function
Type II
5I-deiodinase
Type I
5I-deiodinase
Property
T3>T4
Unknown
Selenocysteine
31,500
Endoplasmic reticulum;
Inactivation of T3 and T4
Type III
5I-deiodinase
Table 2.16 Main mammalian deiodinases (Adapted from Kohrle, 2000; Arthur and Beckett, 1994; Germain, 2001:Bianco et al., 2002 ).
90
Selenium in Nutrition and Health
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Table 2.17 Effect of Se deficiency on thyroid hormone metabolism (Adapted from Germain, 2001)
Increased
Unchanged
Decreased
Serum T4
Serum free T4
Serum T4 Sulfate
Serum T3 Sulfate
D1 (liver, kidney)
D2 (brain, pituitary, brown adipose
tissue)
D3 (brain, skin, uterus)
Tissue T3 levels (brain, liver)
cGSH-Px (liver, thyroid)
PH-GSH-Px (liver, thyroid)
Table 2.18 Downregulation of selenoproteins in selenium-deficient rats (adapted from Sunde, 2001)
Selenoprotein
99
59
95
90
90
90
<10
50
<10-33
<30
GSH-Px1
GSH-Px4
DI
SeP
TrxR1
Other selenoproteins
In addition to the well-characterised selenoproteins mentioned above, there other
selenoproteins that are less well characterised and their functions are less obvious.
They include:
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Selenoprotein W is found in muscle, spleen, testis and brain, but was not
detected in liver, kidney, intestinal mucosa, lungs, heart, plasma and
erythrocytes. It is necessary to take into account that SeW concentration in
tissues depends on Se dietary provision. For example, Se W was undetectable
in skeletal muscle of rats fed the basal diet, detectable in those fed 0.1 ppm
selenium in the diet, and much higher in muscle from rats fed 4 ppm
selenium diet. (Yeh et al., 1995).
SeW is believed to be involved in skeletal and cardiac muscle metabolism
(Rayman, 2002; Yeh et al., 1995).
In primates, SeW was found in several tissues with highest amounts in
skeletal muscle and heart and lowest levels in liver. Similarly, mRNA levels
were highest in monkey skeletal muscle and heart. However, as in the
monkey, selenium concentrations were highest in human kidney and lowest
in skeletal muscle and heart (Gu et al., 2000). Therefore SeW protein levels
correlated with SeW mRNA levels but not with tissue selenium
concentrations.
In contrast to primates and sheep, rodent cardiac does not affected by Se
deficiency. Furthermore, there are species-specific differences in SeW
structure and amino acid composition. For-example, SeW in rats and mouse
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cells with overexpressed SeW did not show any increase in resistance to
H 2O 2.
An important metabolic function of this protein is its involvement in
antioxidant defence, but clearly there is a need to further characterise this
selenoprotein
The human SeW gene maps to chromosome 19q13.3, spans approximately 6.3
kb and comprises six exons and SeW is expressed in all of the 22 tissues assayed,
and shows highest expression in skeletal muscle and heart (Bellingham et al.,
2003).
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SeP is expressed in various tissues including liver, heart, brain, kidney, testis and
muscle in rats and also in placenta and uterus in the mouse (Hill and Burk, 2001)
and associated with cell membranes (Brown and Arthur, 2001). It is interesting
that SeP can be synthesised in the brain (Yang et al., 2000). It has been suggested
that tissues like liver can take up small-molecule forms of selenium whereas
presence of the element in selenoprotein P facilitates uptake by tissues like brain
(Burk et al., 2003). Four isoforms of the protein have been identified in rat plasma,
including the full length protein containing 10 SeCys, and three shortened isoforms
(Chen and Berry, 2003). SeP genes in mammals include 10-12 UGA codons and
two SECIS elements in the 3I untranslated region (Chen and Berry, 2003). It is
interesting to note that in Se deficiency SeP mRNA and protein expression are
preferentially retained in comparison to other selenoproteins (Chen and Berry,
2002) showing high position of SeP in selenoprotein hierarchy.
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deduced amino acids). Moreover, mRNA from both this SeP-like protein
and SeP were expressed in all areas of the brain but most prominently in the
cerebellar cortex, hippocampus and olfactory bulb. The authors suggested
that this selenoprotein is a major Se carrier in the brain and has a role in the
morphological response of nerve or ganglial cells (Saijoh et al., 1995).
Selenophosphate synthetase-2 (SPS) is an enzyme involved in selenocysteine
biosynthesis providing an autoregulatory mechanism for selenoprotein
expression (Guimaraes et al., 1996). Selenophosphate is synthesised from
selenide and ATP by the selD gene product, selenophosphate synthetase,
and is required for selenocysteine synthesis and its subsequent incorporation
into selenoproteins (Low et al., 1995). There are two forms of SPS in
mammals with SPS-2 being a selenoprotein. For example, SPS was detected
using immunoblotting in extracts of rat brain, liver, kidney, and lung (Kim
and Stadtman, 1995). A magnesium ion and a monovalent cation, either
K+, NH 4+ or Rb +, are required for catalytic activity. Polyphosphates and
other common nucleotide triphosphates do not replace ATP as substrate
(Verez et al., 1994). The reaction catalysed by SPS proceeds as follows:
ATP+selenide+H2O selenophosphate+Pi+AMP
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100
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and its function is related to the regulation of cellular redox balance. Recently
the induction of the SelS gene in HepG2 cells has been characterized and
its function examined as an antioxidant (Gao et al., 2004). Indeed SeS gene
expression was shown to be up-regulated in the liver of Psammomys obesus
after fasting. In fact SelS is regulated by glucose deprivation and endoplasmic
reticulum (ER) stress in HepG2 cells. For example, glucose deprivation and
the ER stress inducers tunicamycin and thapsigargin increased SelS gene
expression and protein content several-fold. The overexpression of SelS
increased Min6 cell resistance to oxidative stress-induced toxicity (Gao et
al., 2004). Therefore, this study widen existing knowledge about regulatory
functions of selenoproteins and showed for the first time that selenoprotein
can be induced by ER stress and glucose starvation.
Selenoprotein U was characterised by Castellano et al. (2004). They used a
comparative genomics approach that relies on the genome-wide prediction
of genes with in-frame TGA codons, and the subsequent comparison of
predictions from different genomes. They applied this method to various
genomes and identified a novel selenoprotein family, named SelU, which
present in fish, chicken and sea urchin. In particular, selenium incorporation
into chicken SelU was demonstrated and the SelU expression pattern in
zebrafish embryos was characterized (Castellano et al. (2004). In great
contrast, mammals, worms and land plants contained cysteine homologues.
Data of Castellano et al. (2004) indicate a scattered evolutionary distribution
of selenoproteins in eukaryotes, and suggest that other taxa-specific
selenoproteins probably exist.
Selenoprotein T and selenoprotein X have been recently identified as
members of the family of selenoproteins, however their functions are as yet
unclear (Lescure et al., 1999; Kryukov et al., 1999).
As shown above the a selenoprotein family is getting bigger every year and it
seems likely that selenoproteins are a key element in regulating antioxidant system
of the body. For example, important roles of selenoproteins in endothelial cell
function have been recently reviewed (Brigelius-Flohe et al., 2003) and the authors
showed that selenoproteins are involved in the regulation of :
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Antioxidant
defence
Methionine sulfoxide
reductase B
Thioredoxin reductases
(3 forms)
Iodothyronine
5 -deiodinases
(3 forms)
Sperm capsule
selenoprotein,
PH -GSH -Px
Redox regulation
of gene
expression
Thyroid
metabolism
Sperm structure
integrity
Diseases:
Muscular dystrophy
Impaired fertility
Increased incidence of
mastitis,retained placenta and
cystic ovaries
Decreased: growth,
immunocompetence,
meat quality, feed efficiency
Elevated milk somatic cell
count
Poor thermoregulation and
Increased cold sensitivity
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conditions in farm and laboratory animals where all major organs are affected
(Table 2.20). In many cases a combination of these two compounds is needed to
achieve the best effect in their treatment or prevention.
Table 2.19 Nutritional diseases of rats related to selenium or/and vitamin E deficiency (Adapted from
Levander et al., 1995)
+
+
+
+
-
+
+
+
+
+
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Syndrome
Tissue or organ
affected
Species
Encephalomalacia
Cerebellum
Exudative diathesis
Microtic anaemia
Liver necrosis
Pancreatic fibrosis
Erythrocyte haemolisis
Muscular degeneration
Vascular
Blood, bone marrow
Liver
Pancreas
Erythrocytes
Skeletal muscle
Microangiopathy
Heart muscle
Kidney degeneration
Embryonic degeneration
Poor hatchability
Steatitis
Testicular degeneration
Kidney tubules
Vascular system
Egg embryo
Adipose tissue
Testes
Retained placenta
Impaired fertility
Ill-thrift
Placenta
Spermatozoa
Thyroid, pituitary
The condition can occur at any age but is most prevalent in young growing chickens
and turkeys (Whitehead and Portsmouth, 1989). In the chicks, obtained from
laying hens depleted of vitamin E and selenium exudative diathesis was observed
at hatching, indicating that the deficiency lesions had developed during the
embryonic period, whereas these signs were not observed in chicks obtained
from commercial laying hens adequate in vitamin E and Se on the depletion diets
until they were 2 weeks old (Hassan et al., 1990).
ED will only occur if the diet is deficient in selenium and Se is 200 times more
effective than vitamin E in preventing this disease. Therefore this symptom is
primarily considered as a selenium deficiency (Machlin and Gordon, 1962). ED
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Vitamin E
Selenium
It seems likely, that there are some specific stress conditions where protective
effect of selenium cannot be replaced by vitamin E. For example, GSH-Px provided
protection of the mice body against paraquat-induced lethality, while a-tocopherol
(20 mg/kg diet) was not effective (Cheng et al., 1998; 1998a). Using the GSHPx1 knockout mice it has been shown that even high vitamin E doses (750 or
7500 mg/kg diet) while inhibiting lipid peroxidation in the liver were not able to
replace the protection of GSH-Px against paraquat-induced lethality in mice
(Cheng et al., 1999). In the same study a linear increase in PHGSH-Px activity in
the liver as a result of increase in dietary vitamin E supplementation. Furthermore
it seems likely that normal GSH-Px expression is necessary to protect mice against
lethality and hepatic protein oxidation due to diquat (a herbicide generating
superoxide radical) toxicity (Fu et al, 1999). It is interesting that vitamin E can
affact GSH-Px activity in Se-deficient animals (Table 2.21). Futhermore, Se and
vitamin E deficiency affact the expression of a range of genes (Table 2.22). Indeed,
vitamin E deficiency alone did not induce any significant changes in expression
profile among the genes evaluated (Fisher et al., 2001). Se deficiency caused a
down-regulation of GSH-Px and induction of genes, encoding for detoxifying
enzymes in liver. However, combined vitamin E and Se deficiency affected much
more genes (Fisher et al., 2001). This means that a simplistic presentation of the
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Table 2.21 Effect of Se and vitamin E on antioxidant defences in rats (Adapted from Fisher et al., 2002)
Parameter
Selenium, g/kg
-Tocopherol, g/g
GSH-Px1, mU/mg protein
TrxR, mU/mg protein
GST, mU/mg protein
-Se, -vit.E
-Se, +vit.E
+Se, -Vit.E
+Se, +vit.E
25.8
1.03
5.45
1.32
209
31.8
29.5
10.9
1.16
226
892.3
1.15
154.2
9.46
141
881.5
32.9
161.0
9.25
162
Table 2.22 Effect of Se and vitamin E deficiencies on gene expression in rat liver (Adapted from Fisher
et al., 2002)
GenBank
-Se
Gene
Accession no. -Vit.E, fold
Function
X12367
J05181
18.8
3.4
PO4800
2.5
Stress response
GSH-Px1
Antioxidant protection
Glutamate-cysteine ligase GSH synthesis
catalytic subunit
Cytochrome P-450 3A1
Xenobiotic metabolism
J03969
D14014
2.9
3.1
Cell cycle
Nucleophosmin
G1/S-specific cyclin D1
Y13336
2.0
AF081503
2.6
U72350
3.2
Y22424
2.2
L49379
2.3
Apoptosis
Deefender against cell
death 12 protein
Inhibitor of apoptosis
protein I
Bcl2-L1
Inflammation
11--Hydroxysteroid
dehydrogenase 2
CMOT (canalicular multispecific organic anion
transporter)
role of these two compounds as just antixidant components is not valid anymore.
Even though selenium and vitamin E are working together, there are various
functions for these two distinctive antioxidants where they cannot be replaced by
each other. For a nutritionist who is looking for the best productive and reproductive
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Later the techniques used in those early experiments to detect ROS were questioned
(Seko and Imura, 1997). However, a great deal of evidence analysed by Spallholz
(1994) clearly showed that selenite Se is a pro-oxidant catalyst. Spallholz concluded
that Se compounds are toxic owing to their pro-oxidant catalytic activity to produce
superoxide (O2.-), hydrogen peroxide, and very likely other cascading oxyradicals.
The proposed scheme of superoxide radical generation was as follows (Seko et
al., 1989; Spallholz, 1997; Shen et al., 2000):
4GSH
SeO32-
GSSG
GSH GSSG
GSSeSG
GSH
GSSeH
GSSG
O2
H2Se
O2*Se0
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Selenite
Selenium dioxide
Selenocystine
Diselenodipropionate
Diphenyldiselenide
Selenomethionine
Selenate
Elemental selenium
Selenobetaine
K-selenocyanate
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of selenite increased intracellular ROS level and c-Jun N-terminal kinase1 (JNK1)
phosphorylation. It is interesting that antioxidants such as GSH, N-acetyl cysteine,
curcumin, epigallocatechin gallat and epicatechin inhibited selenite-induced
intracellular ROS elevation and JNK1 phosphorylation (Kim et al., 2004).
The experimental results of Stewart et al. (1999) suggested that selenite and
selenocystamine generated 8-hydroxydeoxyguanosine DNA adducts, induced
apoptosis and were found to be cytotoxic in mouse keratinocytes. On the other
hand SeMet was not cytotoxic, did not generate 8-hydroxydeoxyguanosine adducts
and did not induce cellular apoptosis at any of the Se concentrations studied.
Therefore, in keratinocytes, apoptosis may be initiated by superoxide (O2*-) and
oxidative free radicals that are generated by selenite and selenocystamine, but
not by SeMet. Co-incubation of ascorbic acid or copper sulfate with selenite
appeared to protect primary human keratinocytes against selenite-induced
cytotoxicity. However, synergistic effects were observed between selenite and
trolox resulting in enhanced cytotoxicity (Shen et al., 2001).
Menter et al. (2000) showed that sodium selenite is a potent inducer of apoptosis
in normal and cancerous prostate cells. At the same time SeMet selectively induces
apoptosis in cancer but not primary cells of the human prostate. Similarly, Sundaram
et al. (2000) showed that selenite had a significant inhibitory effect on growth of
tumor cells but had little effect upon dermal fibroblasts that had been passaged
numerous times. Selenium also induced mitochondrial damage and high rates of
apoptosis in two brain tumor cell lines and in minimally passaged fibroblasts.
Therefore these results clearly showed the damaging effect of selenite on cells
and indicated that some types of cells after repeated passages can develop resistance
to Se damage.
Sodium selenite exerted clear cytotoxic effects on a human hepatoma cell line.
Shen et al. (1999) showed that Se-induced cell death occurs predominantly in the
form of apoptosis. The involvement of glutathione in selenite-induced oxidative
stress was further demonstrated by the concurrent decline of intracellular reduced
glutathione and an increase in oxidized glutathione content of Se-treated cells.
Moreover, the finding that selenite-induced oxidative stress and apoptosis was
significantly attenuated by superoxide dismutase, catalase and deferoxamine
provides additional evidence to suggest that Se-induced oxidative stress mediates
the induction of apoptosis. Recently, Shen et al. (2001) provided convincing
evidence that the intracellular O2- formed through the reaction of selenite with
GSH is a potent proapoptotic agent and mainly acts on mitochondria to trigger
the apoptotic signalling pathway.
The prooxidant effect is probably tissue-dependent, reflecting an antioxidant
composition and concentrations in each tissue studied. For example, the effect of
sodium selenite (0.05, 0.1, and 0.2 mg/kg body weight, i.p.) on the thiobarbituric
acid reactive substance (TBARS), and sulfhydryl group in the striatum and thalamus
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Introduction
Selenomethionine (SeMet) was first researched as a possible cause of toxicity of
seleniferous wheat in the 1950s and was later proven to be synthesised from
inorganic selenium sources by various plants, including yeast, marine algae,
Candida albicans, as well as by Escherichia coli and rumen bacteria (for review
see Schrauzer, 2000; 2003). Detailed analysis of the literature suggested that
organic selenium in the form of various selenoamino acids is a natural form of
selenium in animal and human diets and that the digestive system adapted to this
nutrient form during evolution, thereby explaining why there are principal
differences in assimilation and metabolism between organic and inorganic forms
of selenium (Surai, 2002). Therefore there is an inconsistency in common practise
of selenium supplementation of animal diets. On one hand, naturally occurring
organic selenium is represented by a mixture of selenoamino acids with SeMet
comprising more than 50% of total selenium in many feed ingredients, including
grains and forages, etc. On the other hand, until recently the supplemental form
of selenium for farm animals and poultry has been inorganic, either selenite or
selenate. Recent approval by the US Food and Drug Administration of organic
selenium in the form of selenized yeast (Sel-Plex, Alltech Inc.) for poultry, pigs
and cows is going to resolve the discrepancy between natural and supplemental
selenium sources.
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form of selenite, selenate and selenide as well as in the metallic (Se0) form. In
contrast, selenium in feed ingredients (forages, grains, oilseed meals, etc.) is an
integral part of the amino acids selenomethionine and selenocysteine and exists
in the Se-2 oxidation state. As a result, in nature animals receive Se mainly in the
form of selenomethionine (SeMet; Combs and Combs, 1986). Indeed SeMet is
considered a natural nutritional form of selenium for animals and human (Table
3.1).
15
16
P
39.974
33
As
74.922
17
Cl
32.06
35.453
34
35
Se
Br
79.904
78.96
51
52
Sb
121.75
53
Te
127.60
I
126.91
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Year
Se-Met-related events
1930
1949
1950-1960
1970
1984
1980-2000
Numerous experiments proved that SeMet and Se-Yeast are suitable forms of
Supplemented Se
1990-2000
1998-2001
2001-2002
2000-2003
Se-Yeast is used to produce Se-enriched eggs, pork, chicken and milk which
found their way on supermarket shelves in various countries
2001-2002
2002
2003
2004
mainly in sedimentary rocks and shales formed during the cretaceous period,
while lower concentrations of Se are characteristic for igneous (volcanic) rock,
sandstone, granite and limestone (Van Metre and Callan, 2001). Investigations
conducted in China indicated that soils developed under tropic and subtropic
conditions (laterite, yellow soil and red soil) are characterised by comparatively
high Se levels (>0.3 ppm; Tan et al., 2002). In contrast, the soils developed under
the temperate (warm) steppe and desert conditions (chernozem, chestnut soil,
calcic brown soil, desert soil and solonchak) have moderate Se concentrations
(0.14-0.30 ppm). Finally, such soils as brown earth, drab soil, dark brown soil,
loessial soils, purple soil, red drab soil, developed under the temperate (warm)
humid/sub-humid conditions are quite poor in Se (Tan et al., 2002). In particular,
low Se soils occur mainly in the northeast to the southwest of China.
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In the case of acidic soils or poor soil aeration, Se can form insoluble
complexes with iron hydroxide and become poorly available. For example,
at pH 6, only 47% of labelled Se was transferred from soil to ryegrass leaves.
Increasing pH to 7 increased Se assimilation to 70% (Haygarth et al., 1995).
Indeed, Se in alkaline soils occurs in selenate form, where it is soluble and
easily available to plants.
Since sulfate competes with selenate for uptake by the sulfate transporter,
high soil sulfate decreases Se uptake by plants (Terry et al., 2002). This
explains low Se availability from soils following application of certain types
of fertilizers.
Selenium can also be leached from the topsoil in areas of high rainfall.
Therefore areas with higher rainfall have lower forage selenium content.
Solubility is the critical determinant of Se bioavailability to plants and the
amount of water-soluble Se in soils varies substantially and does not correlate
with total soil Se (Combs and Combs, 1986).
Selenite is strongly adsorbed by soils while selenate is only weakly absorbed
and leaches easily.
Selenide and elemental Se are usually found in reducing environments and
are unavailable to plants and animals.
Selenite is present in mildly oxidizing, neutral pH environments and typically
humid regions, while selenate is the predominant form under ordinary
alkaline and oxidized conditions (Goh and Lim, 2004). The authors also
showed that the adsorption of selenite and selenate by soils appeared to be
influenced by the variable pH-dependent charges on the soil particle surfaces.
In particular, phosphate had more profound effects than sulfate on Se
adsorption in the soil.
Application of gypsum (calcium sulfate) to soils decreased Se availability
for plants (Selenium in Nutrition, 1983)
Leaching during the soil development process and irrigation water decreased
Se level in plants (Selenium in Nutrition, 1983)
Forage Se is reported to be low on sandy soils and lower on mineral upland
soils than on organic moorland soils in the British Isles (MacPherson, 2000).
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Biological materials
Rats injected with selenite
Rats injected with SeMet
1 day
5-35 days
Wheat grain
Corn
Rice
Soybeans
Phytoplankton
Astragalus
Se enriched garlic
Se enriched onions
Se enriched Broccoli Floretes
Se enriched Broccoli Sprouts
SeMet
SeCys
6-10
64-70
63
14-25
56-83
61-64
68-81
>80
3.2
37
1-6
1-4
5
12
22
46-57
4-12
15-16
6-10
1-13
7-38
11
-
Plasma components
Se, ng/ml
GSH-Px, %
SeP, %
Albumin
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Controls
Se-enriched yeasta
127
15
67
18
214
17
52
31
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Se-compound
Se-compound
Selenate
Selenite
SeCys
SeMet
Selenohomocysteine
Se-methylselenocysteine
-glutamyl-selenocystathionine
Selenomethionine selenoxide
-glutamyl-Se-methylselenocysteine
Se-adenosylselenohomocysteine
4-selenouridine
Selenosugars
Selenocysteineselenic acid
Se-proponylselenocysteine selenoxide
Se-methylselenomethionine
Selenocystathionine
Dimethyl diselenide
Selenosinigrin
Selenopeptide
Selenowax
Selenobiotin
Selenolanthionine
3-butenyl isoselenocyanate
-glutamyl-Se-methionine
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Compound
Selenate
Selenite
selenocystine
Selenocystathione
Se-Methylselenocysteine
-Glutamyl-Se-methylselenocysteine
SeMet
-Glutamyl-selenomethionine
Se-Adenosylselenohomocysteine
Selenolanthionine
Total Se, %
2
nd
0.5
0.5
3
73
13
4
Nd
Nd
96
Nd
1
0.5
1
0.5
0.5
85
Nd
3
1.5
93
Nd not detected
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Name
Boletus edulis
Rozites caperata
Agaricus campester
Clitocybe clavipes
Amanitopsis crocea
Clitocybe nebularis
Agaricus bispoirus
Suilus bovinus
Lactarius vellereus
Lycoperdon pyriforme
Lepista nuda
Leccinum aurantiacum
Lactarius torminosus
Tricholoma portentosum
Boletus erythropus
Suilus luteus
Verpa Bohemica
Russula fietens
Tricholomopsis rutilans
Lactarius scrobiculatus
Lactarius flexuosus
Se, mg/kg
Range of concentrations
21.06
10.9
6.5
5.1
3.9
3.4
3.0
3.1
2.9
2.7
2.2
2.1
1.8
1.5
1.5
1.4
1.3
1.3
1.2
1.2
1.1
14.1 27.9
1.25 14.5
1.1 11.5
3.8 6.0
2.1 5.7
2.0 4.2
1.9 4.1
1.9 4.0
1.1 3.6
1.2 3.1
1.3 3.0
0.9 3.4
1.7 2.0
0.7 2.4
0.8 2.1
0.1 2.4
1.0 1.5
0.8 - 1.7
0.9 1.4
0.9 1.3
0.6 1.2
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Selenite (SeO3-2)
Selenate (SeO4-2)
+GSH
+ATP
Selenoglutathione
trisulfide
Adenosyl-phosphoselenate
Hydrogen selenide
(H2Se)
Non-acumulator
plants, marine algae,
bacteria and yeast
Plant proteins
Plant
selenoproteins
+Serine
Accumulator plants
Selenocysteine
+Homoserine
Selenomethylcysteine
Selenocystathionine
Selenohomocysteine
Selenomethionine
Figure 3.2 Selenomethionine biosynthesis in plants, marine algae and brewers yeast (Adapted from
Shchrauzer, 2000; 2003).
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chemical form of Se
other dietary components
selenium status
physiological status
species
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164
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Food
Selenomethionine
Selenocysteine
Supplements
Selenoproteins
Selenophosphate
Selenate
Selenite
GS -Se-SG
Breath
Trimethylselenonium:(CH3)3Se+
Urine
Figure 3.3 Metabolism of selenomethionine, selenite and selenate (Adapted from Schrauzer, 2000;2003;
Combs, 2001; Meuillet et al., 2004).
In fact, Burk et al. (2001) demonstrated that Se from SeMet, but not that from
selenate or selenocysteine, can be incorporated into albumin, presumably as SeMet
in the methionine pool. In another study, albumin was purified from plasma of a
human before and after 28 days of supplementation with 400 g Se/day as SeMet.
It was shown that the albumin contained 1 Se atom, presumably as SeMet, per
8000 methionine residues before supplementation and 1 per 2800 after
supplementation (Hondal et al., 1999). These findings support the view that SeMet
is a non-specific form of Se that is metabolized as a constituent of the methionine
pool where it is randomly distributed and is unaffected by specific Se metabolic
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First, studies in our laboratory (Surai, 2000) indicated that chicks hatched
from eggs enriched with Se by means of using Se yeast (Sel-Plex) had
higher liver GSH-Px activity not only at hatching, but more importantly,
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even at 5 days posthatch. More recent observations with quail and chickens
indicates that when organic Se in the form of Sel-Plex was included in the
maternal diet, Se concentration in the liver of the progeny was alleviated up
to 3 weeks posthatch (Surai et al., unpublished). This could be explained
by usage of SeMet accumulated in tissues as a result of Se transfer from the
egg during embryogenesis.
Secondly, the bioavailability of the Se pool in maintaining liver GSH-Px
activity during a period of Se deprivartion, following excess selenite or
SeMet loading was assessed in rats (Ip and Hayes, 1989). In this study halflife of decay of the enzyme was calculated to be 4.2 and 9.1 days,
respectively, in rats that had already been exposed to 3 ppm Se as either
selenite or SeMet.
Thirdly, in a human study Persson-Maschos et al. (1998) showed that in
individuals who had been supplemented with organic Se, the decline in the
level of selenoprotein P following a period of supplementation was slower
than in individuals who had been supplemented with selenite.
Fourthly, when wheat and selanate were used as Se sources in a
supplementation study in Finnish men it was shown that once the
supplements were withdrawn, platelet GSH-Px activity declined less in the
group given wheat Se (Levander et al., 1983).
Finally, after several weeks supplementation with high-Se bread, plasma
Se of New Zealand subjects increased from 50-70 ng/ml to 120-175 ng/ml
(Robinson et al., 1985). Plasma Se remained elevated when supplementation
ceased.
In addition, in SeMet or Se-yeast supplemented mice, liver GSH-Px activities
declined more slowly during Se depletion than in mice given selenite
(Spallholz and Rafferty, 1987 cited by Schrauzer, 2003).
Furthermore, in children the relative bioavailability of Se-yeast versus
selenite measured as GSH-Px activity was similar in plasma, red blood cells,
and platelets, however, Se-yeast provided a longer lasting body pool of Se
(Alfthan et al., 2000).
These data are in agreement with the suggestion that SeMet is the major
selenocompound found initially in animals given this selenoamino acid, but is
converted with time afterwards to selenocysteine when incorporated in functional
selenoproteins (Whanger, 2002). For example, the chemical forms of Se were
determined in erythrocyte and liver proteins after injection of 75Se as either sodium
selenite or SeMet in male weanling rats. Void volume proteins contained principally
selenocysteine (75Se-Cys) in [75Se]selenite-injected animals. This material contained
both 75Se-Met and 75Se-Cys 1 d postinjection in 75SeMet-injected animals, but
primarily 75Se-Cys at 20 d afterwards (Beilstein and Whange, 1986). This means
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that Se-Cys was synthesised from SeMet and with time all SeMet was converted
to Se-Cys. In acid hydrolysates of whole liver 75Se was recovered principally as
75
Se-Cys from animals injected with [75Se]selenite. However, for animals injected
with 75Se-Met, liver 75Se was present initially as 75SeMet, but after five days the
majority of liver 75Se was as Se-Cys. The long-term fate in rats of an oral dose of
[75Se]selenocystine was compared with that of an oral dose of [75Se]SeMet. It was
shown that intestinal absorption of [75Se]selenocystine was 81% of the administered
dose and that of [75Se]SeMet was 86% (Thomson et al., 1975). The initial utilization
of [75Se]selenocystine was different from that of [75Se]SeMet. However, after the
first week 75Se from both sources appeared to be metabolized similarly, suggesting
that dietary Se of both forms is ultimately incorporated into the same metabolic
pool (Thomson et al., 1975).
Weanling male rats were fed a basal Se-deficient diet or this diet plus 2 ppm Se
as either selenite, SeCys or SeMet for nine weeks (Deagen et al., 1987). Except
for the kidney, the tissue Se concentrations were similar in rats fed selenite or
SeCys, but the Se content in testis, muscle, pancreas, heart, spleen, whole blood,
erythrocytes and plasma was significantly higher in rats fed SeMet than in those
fed either selenite or SeCys. The greatest increase due to SeMet compared with
the selenite and SeCys treatments was about 10-fold in the muscle compared with
1.3- to 3.6-fold for the other tissues (Deagen et al., 1987). In general SeMet has
a slower whole body turnover in comparison to sodium selenite and there is greater
efficiency in the re-utilization of Se from SeMet (Swanson et al., 1991). Indeed,
the average whole body half-lives of SeMet and selenite in humans were shown
to be 252 and 102 days, respectively, confirming re-utilization of SeMet in the
body (Patterson et al., 1989). It should be noted that only a small proportion of
the methionine pool can be replaced by SeMet, since only part of methionine
could be replaced by SeMet in the diet. Furthermore, protein turnover prevents
accumulation of SeMet to toxic levels in the organism (Schrauzer, 2003).
In fact, rapid turnover of various selenoproteins and dependence of this process
on Se status were described. For example, the half-life of GSH-Px is approximately
3 days (Sunde et al., 1989), and 2-iodothyronine deiodinase has a half-life of
only 30 - 45 minutes (Kim et al., 2003; Botero et al., 2002; Curcio et al., 2001),
while that of selenoprotein P in plasma is 3-4 hrs (Burk and Hill, 1994). In growth
medium there was an increase in TR mRNA levels of 2-5-fold at 1 microM Se and
an increase in the stability of TR mRNA with a half-life for degradation of 21 hrs
compared to 10 hrs in the absence of Se (Gallegos et al., 1997). Similarly, the
selenoprotein W mRNA half-life in myoblasts is about 57 hrs for cells grown in a
low Se medium while Se treatment increased half-life by 2-fold (Gu et al., 2002).
Therefore, it is clear that Se reserve development could be an important regulatory
mechanism for maintaining effective antioxidant defence during periods of
increased demand. Therefore, from a nutritional viewpoint, SeMet is superior to
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Age
Gender
Social class
Parental smoking
Ethnic group
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Despite higher reported Se intakes, males and females had similar mean plasma
Se values. RBC Se and RBC GSH-Px activity were significantly higher in females
in comparison to males (Short et al., 1997).
There is a great discrepancy in results of comparative evaluation of Se
bioavailability from various sources. The problem is that it is difficult to choose
a proper end point for such evaluation. A range of techniques has been used for
this purpose:
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Activity of GSH-Px (plasma, RBC, whole blood, liver and other tissues).
Since GSH-Px is only one of more than 20 selenoproteins described to date
in animal and human tissues, it is not clear at present if the requirement for
Se needed to maintain GSH-Px activity is higher than for the expression of
other important selenoproteins. The important limitations of this approach
are also related to variations in methodology of GSH-Px activity assessment
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174
Feedstuff
Biological availability, %
210
89
86
86
80
71
65
60
25
22
18
16
15
9
Sodium selenite was used as standard and prevention of exudative diathesis in chicks was
used as an index of Se bioavailability.
Newly-hatched White Leghorn chicks with low-Se status were fed a low-Se basal
diet for two weeks post-hatch followed by either continued depletion or a repletion
period of four weeks with graded levels of Se (0.03, 0.06, 0.09 and 0.12 mg/kg)
provided via sodium selenite, wheat or fish meal. The bioavailability of Se in
wheat and fish meal in comparison to selenite for increasing the activity of GSHPx was 78% and 58%, respectively; and for increasing Se whole blood Se
concentration was 123% and 107%, respectively (Hassan et al, 1993). Based on
the activity of plasma GSH-Px, the biological availability of Se in soybean meal,
lucerne, fish meal and SeMet was 33, 85, 82 and 92%, respectively (Ikumo and
Yoshida, 1981). On the other hand, Se availability in fish meal, based on the
prevention of incidence of ED, was 74%. In contrast to previous data, Se in fish
meal was poorly able to prevent deficiency in chickens (Martello and Latshaw,
1982) and the results of Whitacre and Latshaw (1982) clearly showed that the
commercial preparation of fish meal significantly decreased Se utilization.
Availability of Se in feeds was estimated in relation to restoring blood serum
GSH-Px activity in Se-depleted chickens. The availability of the Se (relative to Se
in selenite) in capelin fish meal was 48.0 (38.5-60.0), mackerel fish meal, 34.1
(32.3-35.8), soybean meal, 17.5, maize gluten meal, 25.7, and SeMet, 78.3%
(Gabrielsen and Opstvedt, 1980). These data on Se availability from fish sources
are in line with others studied, but data on soybean meal are somewhat low. It is
possible that thermal treatment of processed soya could affect Se availability.
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176
Table 3.8 Bioavailability of Se (%) in comparison to sodium selenite (100%) (Adapted from
Hakkarainen, 1993).
Source of Se
Induction of
GSH-Px
Plasma
Wheat
Barley
Oats
Fishmeal
Meatmeal
Selenomethionine
Bioassay
Se concentration
79
83
90
71
77
80
33
37
62
66
61
73
21
20
26
77
80
83
46
56
21
-
100
83
41
80
30
77
109
107
151
102
90
107
85
86
40
47
-
Whole
blood
Liver
Heart
123
104
99
107
69
-
140
151
82
90
67
98
113
119
26
-31
-
108
87
60
100
42
114
selenite 81%, SeMet 80%, beef 80%, chicken 77%, veal 77%, and lamb 58%
(Wen et al., 1997). It seems likely that all cuts of beef are characterised by high
bioavailability of dietary Se when compared with selenite or L-SeMet. For example,
after eight weeks of dietary Se repletion, relative activity of liver GSH-Px from
the different dietary treatments compared with that of control animals (100%)
was (%): selenite 91, SeMet 122, beef liver 108, striploin 105, round 106, shoulder
106, brisket 103 (Shi and Spallholz, 1994). The authors also showed that Se
recovery for liver GSH-Px was generally highest from SeMet > beef muscle =
beef liver > selenite. Similar trends were seen for muscle tissue deposition of Se
which was highest from SeMet > beef muscle > selenite = beef liver. Furthermore,
the faecal excretion of Se was lowest from the SeMet dietary group and highest
from the selenite dietary group. The nutritional biopotency of Se in Brazil nuts
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Table 3.9 Selenium concentration in seafood collected from Supermarkets in Taiwan, g/g wet weight
(Adapted from Chien et al., 2003).
Species
Mean
Range
Fish
Salmo salar Linnaeus
Scomberomorus commersoni
Sebastiscus albofasciatus
Eleutheronma tetradactylum Show
Liza macrolepis
Lateolabrax Japonicus Cuvier
Oreochromis sp.
Mola mola Linneaus
2.01
1.75
1.55
1.55
1.55
1.54
1.34
0.81
1.56 - 2.39
1.66 1.90
1.57 1.67
1.44 1.72
1.20 1.77
1.43 1.70
1.25 1.44
0.71 0.93
Crustaceans
Panulirus longipes
Ovalipes punctatus
Metapenaus ensis
Portunus sanguinolentus
1.30
1.28
1.27
1.09
0.87 1.76
1.15 1.35
1.00 1.38
0.91 1.33
Bivalve molluscs
Crassostrea gigas
Raphia amabilis
Rudiapes variegates
Meretrix lusoria
Amusium pleuronectes
1.18
1.33
0.87
0.73
0.63
0.77 1.59
1.00 1.49
0.67 1.20
0.53 1.00
0.56 0.70
The Se intake of a Japanese fisherman may be as high as 500 g/day and in Greenlanders it
was 652.7g/day (Chien et al., 2003).
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Fish
Angler fish
Cod
Grayfish
Hake
Halibut
Salmon
Swordfish
Tuna
Whiting
Goby
Gurnard
Mackerel
Mullet
Ox-eye bream
Perch
Porgy
Redfish
Scorpion fish
Sole
Anchovy
Red banfish
Sardine
Selenium
Mercury
Se/Hg ratio
0.173
0.188
0.139
0.466
0.134
0.195
0.283
0.734
0.290
0.279
0.372
0.356
0.426
0.337
0.073
0.286
0.247
0.417
0.262
0.225
0.299
0.678
0.089
0.077
0.282
0.086
0.069
0.086
0.579
0.249
0.187
0.145
0.138
0.126
0.103
0.104
0.139
0.204
0.096
0.134
0.057
0.075
0.058
0.156
1.94
2.44
0.49
5.42
1.94
2.27
0.49
2.95
1.55
1.92
2.70
2.83
4.14
3.24
0.53
1.40
2.57
3.11
4.60
3.00
5.16
4.35
These results indicate that Se in Se yeast is more bioavailable than selenite Se,
and therefore is the preferred form for supplementation. The bioavailability of Se
in the form of yeast is higher than that of other Se compounds used for preterm
infants (Bogye et al., 1998). A daily supplement of 0.75 mg Se from the yeast
product maintained whole blood and milk Se concentrations at the same levels as
3.0 mg Se as sodium selenite. Further, 3.0 mg Se from the yeast product increased
whole blood Se by about 40% and that of milk by about 100%. The activity of
GSH-Px in erythrocytes of the group given selenite was not significantly different
from that in either of the groups given the yeast product. The concentrations of Se
in the tissues of two cows from each group were marginal to adequate, and there
was a trend for the concentrations to be higher in the tissues of the cows
supplemented with the yeast product (Ortman and Pehrson, 1997).
Supplementation of the feed either with 0.2 ppm organic Se or sodium selenite
for eight weeks increased the blood Se level within this period from a background
level of about 5.6 g/L to 167 (Se-yeast) and to 91 g/L (selenite; Malbe et al.,
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1995). The respective change in whole blood GSH-Px was from 0.22 to 3.0 (Se
yeast) and to 2.3 (selenite) microKat/g Hb. The bioavailability of yeast Se was
superior to selenite: the relative bioavailability (selenite = 1) of yeast Se was 1.4 if
blood GSH-Px, 1.9 if blood Se, and 2.7 if milk Se was used as the response
criterion (Malbe et al., 1995). Feeding organic Se from SeMet or selenized yeast
results in much higher tissue and milk selenium concentrations than are obtained
with selenite (Spears, 2003). Cows fed selenoyeast had greater whole blood Se
concentrations than cows fed sodium selenite. At birth, calves from cows fed
selenoyeast had greater whole blood Se concentrations and GSH-Px activities
than calves from cows fed sodium selenite (Gunter et al., 2003).
Utilization (absorption, retention and appearance in milk and blood) of two
different chemical forms of Se (selenite and SeMet) in lactating, non-lactating
and never pregnant women using stable isotope tracers was studied. It was shown
that significantly more Se from SeMet than from selenite was absorbed and
appeared in the plasma in all groups. Milk contained more Se from apparently
absorbed SeMet than from selenite. All groups retained significantly more Se
from SeMet than from selenite (Moser-Veillon et al., 1992). It has been shown
that SeMet-Se was more effective in raising plasma and RBC Se than was selenite
in men (Luo et al., 1985).
A purified, casein-based diet without added Se was fed to nine groups of rats
throughout pregnancy to produce a marginal Se deficiency. During lactation,
groups (n = 8) were fed experimental diets containing either 0.1, 0.25 or 0.5 ppm
Se as selenite, SeMet, or Se yeast. On day 18 of lactation, tissue Se and GSH-Px
activities of dams and pups were determined. Based on slope-ratio analyses, the
bioavailability of SeMet and Se yeast was greater than that of selenite in both
lactating dams and their nursing pups (Smith and Picciano, 1987). Weanling rats
were fed a basal diet or this diet plus 0.2, 1.0, 2.0 or 4.0 mg/kg Se as either
selenite or SeMet. Except at the 0.2 mg/kg Se level, Se accumulated in all tissues
at higher levels when SeMet was fed than when selenite was given, and the
magnitude of difference became more pronounced with increasing levels of dietary
Se (Whanger and Butler, 1988). This was particularly true for muscle and brain. It
appears that after saturation of activity of GSH-Px and other selenoproteins, Se in
inorganic form cannot be stored in the body and is released with urine and faeces.
In great contrast, SeMet was unspecifically incorporated in body proteins, especially
muscle, providing higher Se concentration in tissues. Although the tissue Se
concentrations differed markedly, there were no differences in the GSH-Px activity
in tissues of rats fed SeMet or selenite. This means that GSH-Px expression reached
maximum level at this high Se supplementation condition. The percentage of Se
associated with GSH-Px was lower in all tissues from rats fed SeMet than in those
from rats fed selenite, reflecting more Se accumulating in the form of SeMet
(Whanger and Butler, 1988).
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(Ip, 1988). Altered Se metabolism at suboptimal dietary Met may occur because
more SeMet is incorporated into protein and thus less Se is available for GSH-Px
synthesis. Supplementation of cell culture with L-Met significantly reduced 75SeMet
incorporation, but significantly increased the proportion of cellular 75Se recovered
as SeCys (Beilstein and Whanger, 1987). More Se is incorporated into sheep wool
and plasma protein from feed supplemented with SeMet when dietary sulphur is
limiting than when it is not (White, 1980).
Did you know that selenium assimilation depends on dietary
mineral and vitamin balance?
Copper (Cu) (100 mg/kg) reduced the incorporation of selenium (when used at
level of 2 ppm) into protein fractions of egg white (Davis et al., 1996). Long-term
ingestion of moderate levels of Cu (10 mg /kg) influenced the metabolism of Se,
possibly through effects of chronic Cu toxicity on liver function. For example, at
19 weeks, ram lambs were allocated randomly to individual metabolism cages
and received a single oral dose of 75Se-SeMet. Sheep given Cu supplements had
reduced levels of 75Se activity in muscle compared with control animals (White et
al., 1989). This decrease in muscle 75Se in Cu-supplemented lambs was associated
with a nonsignificant increase in 75Se content of other tissues and a nonsignificant
increase in faecal excretion of 75Se. Marginal deficiency of zinc (Zn) (8.6 mg/kg
diet) in rats can decrease Se availability and a small excess of Zn increases Se
availability for hepatic GSH-Px activity. In fact, Se concentrations in plasma,
erythrocytes, muscle, heart, and liver were significantly elevated by Zn (Yin et
al., 1991). Furthermore, Zn had an antagonistic effect on Se absorption by Znadequate rats (House and Welch, 1989). Chronic magnesium (Mg) deficiency in
rats decreases Se absorption and retention and erythrocyte concentrations of this
mineral, and increases Se in plasma, kidney and heart (Jimenez et al., 1997).
There are also interactions between Se and heavy metals, which will be considered
separately.
Vitamin B6 is involved in the metabolism of selenium. For example, the addition
of pyridoxal phosphate to the media resulted in elevated GSH-PX activity in all
human lymphoblast cells regardless of the chemical form of Se used (Beilstein
and Whanger, 1992). The levels of Se and GSH-Px in erythrocytes and muscle
were significantly higher in vitamin B6-supplemented groups than in vitamin B6deficient groups (Yin et al., 1991). It seems likely that this vitamin is involved in
the transport and deliverance of Se in plasma to the other tissues and the
incorporation of Se from SeMet to GSH-Px in liver. Therefore the conversion of
SeMet to a form available for GSH-Px synthesis is reduced by vitamin B6 deficiency.
For example, tissue retention of 75Se provided as SeMet was increased in vitamin
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Substrate
Control area
Corn, g/g
Rice, g/g
Water, g/ml
Urine, g/ml
Hair, g/g
High Se area
0.096
0.029
0.05
0.38
1.37
46.52
6.53
0.352
3.27
43.88
Table 3.12 Average Se content (ppm, wet bases) of selected food reported in 1970th (Adapted from
Selenium in Nutrition, 1983).
Food
USA
0.22
1.62
0.43
0.53
0.38
0.012
0.006
0.082
0.098
0.38
0.01
0.006
Canada
0.14
2.77
0.43
1.25
1.08
0.015
0.003
0.08
0.039
0.52
0.027
0.007
Germany
UK
Ukraine
0.27
0.44
1.54
0.14
1.01
0.03
0.0.3
0.06
1.39
0.13
0.37
<0.01
<0.01
0.12
0.11
0.11
<0.01
<0.01
0.29
0.10
0.30
0.022
0.28
0.125
0.004
Table 3.13 Selenium contents (g/100g as consumed) in foods (Adapted from Combs, 2001).
USA,
1999
Red meats
Poultry
Fish
Milk
products
Eggs
Cereal
products
Vegetables
Fruits
8- 50
5 - 14
1 - 26
5 - 15
13 -148 10 - 61
1 - 26
1-8
China2
13- 28
1- 4
1- 3
5- 25
5 - 15
5 - 10
2-6
5 - 10
24 - 53 3 - 31
3 - 20 10 - 60
1 - 10 0.3 - 2.5 0.2 - 1.0 1 - 3
6- 20
6 - 66
5- 20
2 - 53
5 - 20
3 - 88
24-98
4-9
2-6
0.5- 2
0.1- 14
0.5- 6
1- 9
0.5- 1
4- 0
0.2- 4
0.1- 2
0.1-0.4
0.2 - 2
0.1- 0.3
China3 Venezuela
17- 83
10 - 70
32 - 93
11-43
5 - 15
5 - 15
1.7 - 11 106- 690 12- 51
0.2- 9 34 - 4570 0.2- 298
0.5- 4
0.5 - 6
/before selenized fertilizers were introduced; b/after selenized fertilizers were introduced;
/Low Se-area; 2/ Moderate Se-area; 3/High Se-area
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Table 3.14 Selenium content in some common foods, g/100 g (Adapted from McNaughton and Mars,
2002).
Food item
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UK1991
UK1995
USA1999
New
Zealand2000
Australia2002
Cereals
Bread
28-35
Corn flakes
2.0
Muesli
Oats (cooked)
trace
Rice (white, cooked)
4.0
Pasta (white, cooked)
Trace-4.0
Wheat breakfast biscuits
4.0
4.3-9.2
4.7
4.2
3.1
13.0
4.8
2.3
28.2-36.6
5.1
17.3
8.1
7.5
21.3-21.7
4.7
3.2-5.9
1.8-2.0
2.2-2.5
2.0
0
0.4-5.5
2.4-12.5
9.3-12.5
6.3
12.8
11.0
2.5
3.9
17.8
Dairy
Milk
Cheese (cheddar)
Yoghurt
Ice-cream
Butter/margarine
1.0
12.0
1.0-2.0
trace
1.0-1.5
7.4
1.4-1.5
1.5-1.7
-
2.0-2.1
13.9
2.8-4.9
2.6
0-1.0
0.1-1.4
2.3
0.7-1.0
0.6
0.6-1.6
2.3-2.6
7.0-7.9
2.0
4.5
1.1-1.4
Eggs
Whole
Yolk
White
9.0-12.0
20.0
6.0
22.5-30.8
45.2
17.6
15.7-16.1
39.2
4.4
19.0-41.4
26.0
9.0
Fats
Butter/margarine
Oils
Fish
Fruit, fresh
trace
trace
20.-50
Trace-0.3
0.4
0-1.0
0
12.6-50.2
0.2-1.1
0.6-1.6
0
19.5-51.2
0-3.0
1.1-1.4
0.5-0.53
12.0-63.2
0.1-1.4
Meat products
Beef
Chicken
Lamb
Pork
Processed meat
Liver
3.0
6.0-7.0
1.0
14.0
20.0-22.0
7.0
7.6
3.8
14.0
42.0
13.4-19.0
19.0-27.6
26.4-27.4
14.4-45.0
57.0-116.0
2.2-8.3
13.7-14.5
3.7-5.6
1.9-15.0
0-18.0
8.7-42.0
7.2-12.1
11.6-28.0
13.0-22.0
9.4-20.5
4.7-23.3
30.0-37.9
Vegetables
Peeled, raw/cooked
canned
Mushrooms, raw
Trace-3.0
trace
9.0
0.2-3.0
-
0.2-1.9
0.5-0.7
8.8
0-1.1
0-0.25
7.7
0.05-3.3
0.7-3.1
25.5
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Region
Pskov
Gomel
Rjazan
Brjansk
Irkutsk
Altaiskij
Bashkortostan
Archangelsk
Vladimir
Karelia
Norilsk
Tula
Gorkiy
Perm
Chelabinsk
Sachalin
Se in wheat, ppb
40 2
68.0 25.1
77.7 26.8
84.9 26.1
88.0 26.6
91.3 40.0
99.6 21.3
100.0 52.9
108.0 61.5
110.0 81.3
119.0 39.0
126.0 42.1
146.0 40.4
161.0 68.0
161.0 65.2
257.0 114.0
72
79.3
83.0
84.0
75.0
87.0
90.0
93.0
101.0
90.0
102.0
89.0
108.0
103
101
137.5
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188
Table 3.16 Selenium content in American diet (Adapted from Holben and Smith, 1999).
Food
Grains and cereals
Bread, whole wheat
Bread, white
Spaghetti/macaroni,
cooked
Egg noodles, cooked
Rice, white, long
grain, cooked
Corn or wheat flakes
Crispy rice cereal
Oatmeal, cooked
Puffed wheat cereal
Serving
size, g
Selenium Food
content, g
28
25
140
10.2
7.1
29.8
160
158
37.4
11.9
28
28
234
12
1.4
4.3
19.0
14.8
28.1
19.4
57.0
23.9
2.9
13.0
49.9
Serving
size, g
Selenium
content, g
80.4
15.4
10.7
6.7
2.5
839.2
1.4
3.3
2.1
2.4
245
5.1
Cheddar cheese
28
Mozzarella cheese,
part skim
28
Yogurt, plain, low fat 245
3.9
4.6
8.1
Vegetables
Garlic, raw
Mushrooms, raw
0.4
4.3
2.8
35
33.2
Selenium content in various feed ingredients is also highly variable (Table 3.20)
and average data on Se content in feedstuffs presented in various tables are not
suitable for diet balancing and Se supplementation is a routing practice in
commercial animal and poultry production. It has been shown that through usage
of Se in selenized fertilizers or by foliar application it is possible to substantially
increase Se content in various food and feed ingredients (Table 3.21). Data from
Finland are an example of successfully increasing Se status of the population by
using Se with fertilizers. However, environmental concern about washing out Se
from soil and contaminating water reservoirs is a limiting factor for the wide
application of such a technology. It seems likely that production of Se-enriched
animal-derived products is a safe and commercially valuable way of resolving Se
deficiency on a global scale.
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Food
Eggs
Beef
Pork
Lamb
Poultry
Turkey (white meat)
Fish
Liver
Kidney
Milk whole
Milk skimmed
Dairy produce
Cheese cheddar
Cheese parmesan
Cheese stilton
Bread white
Bread wholemeal
Cereals cornflakes
Cereals muesli
Cereals bran based
Rice brown
Rice white
Flour white
Flour brown
Flour wholemeal
Fruit
Vegetables
Mushrooms
Cashew nuts
Brazil nuts
11.0-19.4
7.6
14.0
3.8
18.5
10.0
36.0
42.0
145.0
1.5
1.0
3.2
7.4
12.0
16.0
6.0
9.0
4.7
4.2
3.6
10.0
13.0
2.3
4.4
10.0
1.0
2.0
9.0
27.0
254.0
Table 3.18 Estimated human dietary intake of selenium from dietary sources (Adapted from Selenium in
Nutrition, 1983).
Food
Total, g
Meat, fish, %
Cereals, %
Dairy products, %
Others, %
56.2
67.1
7.07
15.0
10.2
132.0
52.0
33.7
10.2
4.1
Canada
150.7
30.5
49.4
15.5
4.6
Japan Venezuela
88.3
63.0
27.1
2.6
7.3
325.8
46.8
27.1
21.6
4.5
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50
41
24
9
26
30
50
10
30
190
Table 3.19 Estimated intake of Se from different food groups in the UK in 1997 (Adapted from BNF,
2001).
Group
Item
Bread/
cereals
Meat
Bread
Miscellaneous cereals
Carcass meat
Offal
Meat products
Poultry
Fish
Oils & fats
Milk
Dairy produce
Eggs
Green vegetables
Other vegetables
Canned vegetables
Fresh fruit
Fruit products
Nuts
Beverages
Sugars and preserves
Potatoes
Estimated total intake
Fish
Fats
Dairy
products/
eggs
Vegetables
Fruit
Other
Total
Selenium
Consumption
content
g/100 g fresh weight g/day
4.4
3.9
11.5
49.2
13.0
18.5
36.0
0.3
1.4
3.2
19.4
0.8
2.2
1.4
0.1
0.07
25.1
0.04
0.9
0.3
191.7
108
101
22
1
47
19
14.0
27
281
60
14
34
76
33
69
44
2
937
63
123
2075
Estimated contribution
to total Se intake
g/day
%
4.8
3.9
2.5
0.5
6.1
3.5
5.0
0.1
3.9
1.9
2.7
0.3
1.7
0.5
0.07
0.03
0.5
0.4
0.6
0.4
39
Table 3.20 Selenium concentrations in various feed ingredients, ppm (Adapted from Selenium in
Nutrition, 1983).
Ingredient
Alfalfa meal
Barley
Brewers grains
Corn
Fish meal
Linseed meal
Meat meal
Oats
Poultry by-product
Soybean meal
Wheat
Wheat bran
Wheat middling
Whole soybeans
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USA
Canada
0.01-2.00
0.05-0.5
0.15-1.00
0.01-1.00
1.0-5.0
0.5-1.2
0.08-0.5
0.01-1.00
0.5-.10
0.06-1.00
0.1-3.00
0.1-3.0
0.15-1.0
0.07-0.90
0.02-0.27
0.02-0.99
0.29-1.10
0.01-0.33
1.3-3.4
0.7-1.5
0.2-0.81
0.01-1.10
0.04-0.78
0.02-1.5
0.24-1.3
0.41-0.89
-
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12.2
10.0
6.3
1.3
15.5
8.9
12.7
0.3
10
4.8
6.9
0.8
4.3
1.3
0.2
0.1
1.3
1.0
1.5
1.0
100
Sample
Soil application
Soybean
Soybean protein
Soybean okra
Foliar application
Control
Selenite
Se fertilizer
Selenite
Se fertilizer
0.055
0.110
0.040
0.198
0.511
0.115
0.196
0.553
0.131
1.126
1.890
0.868
1.211
1.947
1.111
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192
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Selenite
Selenate
Seleno methionine
+ Ascorbate
Metallic Se
Not
absorbed and
not
assimilated
+ Ascorbate
Se-Met
Absorbed and
assimilated
Table 3.22 Selenium content (mg/kg dry matter) of Finish retail-store foodstuff (Adapted from Varo et
al., 1994).
Foodstuffs
1984 a
1990b
1992 c
0.05
0.07
<0.01
0.17
0.35
0.06
0.09
0.69
0.23
0.06
0.11
0.64
1.09
0.23
0.42
1.27
0.14
0.04
0.07
0.54
0.84
0.17
0.29
1.16
/Before Se fertilization;
b
/The amount of sodium selenate in fertilizers used for grain production (16 mg/kg) and in
fertilizers used for fodder and hey (6 mg/kg);
c/ The amount of sodium selenate in all fertilizers (6 mg/kg) since 1991
Application of Se supplemented fertilizers was started in 1985, the amount of Se in
fertilizers was reduced in 1991
Table 3.23 Selenium balance in pigs as affected by premix (Adapted from Groce et al., 1973)
Item
Stored Se premix
Exp. 1
Exp. 2
Se intake, g/day
Se excretion, % of intake
Faecal
Urinary
Se retention, g/day
Se retention, % of intake
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53.2
132.2
52.0
129.5
51.7
29.8
9.8
18.6
25.9
47.7
34.9
26.5
41.0
12.7
23.7
46.2
22.5
41.7
46.5
35.9
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194
It is well known that chemical and physical properties of Se and sulphur are very
similar, reflecting similar outer-valence-shell electronic configurations and atomic
sizes (Combs and Combs, 1984). Therefore plants cannot distinguish between
these two elements when synthesising amino acids. As a result they can synthesize
SeMet when Se is available. This biological feature was the basis for development
of the commercial technology of organic Se production from yeast (Sel-Plex,
Alltech Inc., USA). Selenium composition in this product matches closely that
found in most grains with more than 50% of total Se being in the form of SeMet.
Analysis of the protein fraction of Se yeast has shown that Se is present in all
the major soluble proteins. SeMet was identified as the major Se-containing
compound in the protein fraction as well as in the whole cell (Korhola et al.,
1986). Therefore, yeast cells can take up Se in the form of selenite or selenate
from media and synthesise selenoamino acids. In particular certain strains of yeast
are capable of accumulating as much as 3000 ppm Se in organic form when in
the growth medium sulphur is replaced by selenium compounds and proper growth
conditions are provided (Gassner et al., 1999; Demirci et al., 1999). The influence
of various Se concentrations from organic (SeMet) and inorganic (sodium selenite)
Se compounds on growth pattern and cell viability and the alterations in the
antioxidant enzyme system of yeast were evaluated (Bansal and Kaur, 2002). A
continuous decrease in cell and colony-forming units counts was observed with
increasing concentrations of Se from either source. Increasing Se status of yeast
cells was found with increasing concentrations of Se with both forms, with much
greater uptake for organic Se at maximum Se concentrations. However, high
concentrations of sodium selenite in the culture medium have a strong inhibitory
effect on the growth of this yeast (Suhajda et al., 2000). Sodium selenite had
stronger inhibition on the yeast growth than sodium selenate and the ratio of
selenium to protein was higher with sodium selenate than with sodium selenite.
Did you know that a composition of selenocompounds in the
selenized yeast is similar to that of grains with more than 50% of
selenium in the form of SeMet?
As mentioned above, SeMet is the major selenocompound in Se-enriched yeast.
For example, SeMet in yeast and nuts comprised respectively 65% and 75% of
total Se (Wrobel et al., 2003). Similarly, a proteolytic enzyme extract of Se yeast
was found to contain Se as SeMet (74.8%), selenocystine (9.9%), selenite (5.1%)
and as at least three unknown Se compounds (10.2%, Yoshida et al., 2002). SeMet
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A, %
B, %
C, %
Water soluble Se
Water insoluble polysaccharide-bound Se
Water insoluble protein-bound Se
Residual protein-bound Se
Residual hydrolysable Se
11.5
15.5
19.0
38.0
16.0
28.0
27.0
38.0
6.0
1.0
21.0
72.0
4.0
1.0
1.0
Did you know that not all Se-yeasts are the same and data
obtained with one product can not be automatically applied
for another product?
When selecting a Se supplement, another important consideration is composition
of organic Se compounds in the supplement. While SeMet represents the dominant
Se form in Se-enriched yeast, each yeast has a unique combination of organic Se
compounds which must be considered when beneficial effects of organic Se are
expected. This means that SeMet alone sometimes less effective that Se-enriched
yeast. For example, in mice high-Se yeast caused the largest increase of GSH-Px
activity followed by sodium selenite and SeMet (Bergman and Slanina, 1986).
Furthermore, SeMet in purified form is unstable and easily oxidised. For example,
recently it has been shown that in the freeze-dried samples of oyster total Se and
the Se species evaluated are stable for at least 12 months, under all the conditions
tested. However, after purification of Se species, including SeMet, in the enzymatic
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196
extracts they are only stable for 10 days if stored at 4C in Pyrex containers
(Moreno et al., 2002). In contrast, SeMet is quite stable in the yeast. Indeed,
analysis of high-Se yeast stored at room temperature for more than 10 years showed
SeMet as the major Se product (Block et al., 2004). Furthermore, the shelf life of
Se yeast at 25C predicted from the Arrhenius plot exceeded 1126 days (Szulc et
al., 2003).
Did you know that pure SeMet is unstable and easily oxidised?
In contrast, in the form of selenized yeast SeMet is quite stable.
The most common dietary supplement form of Se for human is Se-enriched yeast.
The development and commercial application of Sel-Plex with guaranteed
composition and evidence from research and commercial trials (which will be
presented in this book) opens a new era in animal nutrition providing opportunities
not only for improvement of animal health and productivity but also for production
of Se-enriched meat, milk, eggs and other foods considered to be important steps
in improvement of human diets. Indeed, a comparison between Sel-Plex and
selenite, based on published data, presented in this book in various chapters,
clearly showed advantages of the natural form of Se in comparison to selenite
(Table 3.25). Indeed sodium selenite has a range of various properties, which are
not shared by other forms of selenium (Table 3.26). Therefore, it seems appropriate
that selenite be considered as a drug, which should be used accordingly. For
example, when Se deficiency is diagnosed based on clinical signs, selenite would
be the preparation of the choice. Using it via feed, water or injection will solve the
short-term or acute Se deficiency, which has been demonstrated under various
experimental conditions with chickens, pigs and cattle. However, when the goal
is meeting physiological requirements of animals in order to maintain high
productive and reproductive performance, optimum food animal product quality
and immunocompetence, a Se supplement that allows tissue reserves is needed.
Did you know that based on the mode of action selenite should be considered
and used as a drug and Sel-Plex as a feed additive?
Did you know that based on the mode of action selenite
should be considered and used as a drug and Sel-Plex
as a feed additive?
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Absorption
Accumulation
Toxicity
Bioavailability
Higher bioavailability in
comparison to selenite to
animals and human
Antioxidant activity
Effect on DNA
Transfer to eggs,
milk and meat
Protective effect in
stress conditions
Environmental issues
Stability during
storage and feed
processing
Stable
Stable
Classification based
on the mode of action
Feed additive
Drug
197
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198
Table 3.26 Reactions of selenite which are not shared by other forms of selenium (Adapted from
Whanger, 2002).
Reactions
Reactions
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Introduction
Human and animal defence against various diseases depends on the efficacy of
the immune system responsible for elimination of foreign substances (e.g. parasites,
bacteria, moulds, yeast, fungi, viruses and various macromolecules) or the creation
of specific inhospitable conditions within the host for a wide range of pathogens.
This protective capacity is based on the effective immune system which is
considered to be an important determinant of animal health and well being. In
that sense, a remarkable ability of components of the immune system to distinguish
between self and non-self is a great achievement of animal evolution.
Commercial animal production is based on balanced feed, providing
requirements in major nutrients and optimised environmental conditions. However
it is very difficult to avoid various nutritional or environmental stresses which are
responsible for immunosuppression and increased susceptibility to various diseases
and as a result decreased productive and reproductive performance of animals.
For example, mycotoxins are among major immunosupressive agents in animal
diet. In such situations immunomodulating properties of certain macro- and
micronutrients are of great importance for the animal industry. In fact, almost all
nutrients in the diet play a crucial role in maintaining an optimal immune
response, and both insufficient and excessive intakes can have negative
consequences on the immune status and susceptibility to a variety of pathogens.
Information has been actively accumulated for the last 10 years indicating that
selenium is among major immunostimulating agents and its requirement for such
action is usually higher than that for animal growth and development. In fact,
selenium has been shown to have immunomodulatory effects in a variety of species
when administered in quantities in excess of established dietary requirements.
Therefore, selenium as an essential component of selenocysteine-containing
proteins is involved in most aspects of cell biochemistry and function and immune
cell activity is also Se-dependent. For example, Se deficiencies are found in chronic
renal failure, malnutrition malabsorption, long term parenteral nutrition, AIDS
and probably Se deficiency interferes with chronic infections which often go with
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Immunity
Natural (Innate)
Physical
bariers
Phagocytes
Mast
cells
NK cells
Macrophages
ROS, RNS,
Small peptides
Kill pathogen
Humoral
Dendritic
cells
B-lymphocytes
T-lymphocytes
Immunoglobulins
(antibodies)
Direct contact
with target cells
Neutrophils
Eicosanoids
Cytokines
Complement,
Acute phase protein
Inflammatory
response
Cell-mediated
Figure 4.1 General scheme of the immune system (adapted from Surai 2002).
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Cells
Significance
Monocytes, macrophages
Neutrophils
Eosinophils
Destruction of parasites
Basophils
Mast cells
B cells (B lymphocytes)
Cytotoxic T cells (T
lymphocytes)
Suppresser T cells (T
lymphocytes)
Macromolecules
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Immunoglobulins
Interleukins
Leucotrienes
Lysozymes
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Table 4.2 Macromolecules produced in immune cells (adapted from Kolb, 1996; Lydyard et al., 2000).
Name
Site of production
Principal effects
IL-1
IL-2
IL-3
T-helper cells
IL-4
IL-5
IL-6
IL-9
IL-10
IL-11
IL-12
Mesenchymal cells
B cells, MPH
INF-
T cells, NK cells
TNF-
MPH, T cells
IL-7
IL-8*
PlateletMPH
activating
factor (PAF)
Fibroblast
MPH
growth factor
Complement MPH
-I-Protease MPH
inhibitor
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Name
Site of production
-2-Macro- MPH
globin
Leukotrienes MPH
Principal effects
Inhibits proteases and breakdown of healthy
tissues
Promote leukocyte accumulation at inflammatory
foci
Increases the production of granulocytes
(neutrophils) in the bone marrow
Granulocyte MPH
colonystimulating
factor
Granulocyte- MPH
macrophage
colonystimulating
factor
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intensive flow of superoxide radical (Gille and Sigler, 1995). Superoxide radical
can disproportionate to H2O2 which penetrates into the bacterium with a production
of hydroxyl radical, which is ultimately a deadly weapon able to damage any
biological molecules. In general, the production of ROS and reactive nitrogen
species is a characteristic for both mammalian and avian macrophages (Qureshi
et al., 1998). It is interesting that, people whose phagocytes possess no functional
NADPH oxidase, are shown to suffer from chronic infections of the skin, lung,
liver and bones leading to a premature death (Halliwell and Gutteridge, 1999).
Microbe
Activation
O2
Nucleus
NADPH
oxidase
NADPH oxidase
O2*-
NADPH
oxidase
SOD
H 2O2
Fe2+
OH*
Mieloperoxidase
Granules
O2-
HOCl
OH* H O
2 2
O2* Microbe
O2* HOCl
Fe2+
OH*
Phagosome
Figure 4.2 Respiratory burst in neutrophils (adapted from Kettle and Winterbourn, 1997; Nordberg and
Arner, 2001).
In general ROS, RNS and eicosanoids (e.g. leukotrienes and prostaglandins) have
recently received substantial attention as major metabolites produced by
macrophages (Dietert and Golemboski, 1998). Because of this powerful weapon,
macrophages bind, internalize, and degrade foreign antigens (e.g. bacteria) quite
quickly. It takes only 15 minutes for chicken macrophages to kill more than 80%
of the internalized Salmonella (Qureshi et al., 1998). Therefore natural immunity
works rapidly, gives rise to the acute inflammatory response. They contain various
substances (including enzymes producing free radicals and small peptides with
an antibiotic activity) involving in microbial killing. They have also receptors for
chemoattractive factors released from microbes. In addition to ROS, RNS and
eicosanoids mentioned above, macrophages also synthesize and secrete a great
number of such communicational molecules as cytokines, including the proinflammatory cytokines called interleukin 1 (IL-1), interleukin 6 (IL-6), and tumour
necrosis factor- (TNF). They also produce cytokine inhibitors, endocrine
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Th1
Th2
Th3
Th0
Humoral
T Supressor/Cytotoxic
(CD8)
Type 1
Plasma cells
(B-lymphocytes)
Type 2
Antibody production
IL-2
INF-
Delayed-Type hypersensitivity
(cell-mediated inflammatory response)
Figure 4.3 General scheme of adaptive immune system (adapted from Field et al., 2001).
Table 4.3 Main immunoglobulin classes (adapted from Lydyard et al., 2000).
Immunoglobulin
Characteristics
IgG1; IgG2;
IgG3; IgG4
IgA
IgM
IgD
IgE
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Table 4.4 Key features of innate and adaptive immunity (adapted from Hansson et al., 2002; Calder,
2001).
Innate
Adaptive
Appearance in
evolution
Primitive organisms
Vertebrates
Induction time
Recognizes
Common pathogen-associated
microbial pstterns (PAMPs)
Cellular
components
Generation of
specificity
Effector
mechanisms
Complement (Alternative
Antibodies; cytotoxic T cells
pathway); cytokines; chemo(CTL); classical complement
kines; cell-mediated cytotoxicity activation; antibody-dependent
cell-mediated cytotoxicity;
cytokines; chemokines
Soluble mediators
Macrophage-derived cytokines
Lymphocyte-derived cytokines
Characteristic
transcription
factors
NF-B (+JNK/AP1)
Physiological
barriers
Skin
Mucosal membranes
Lysosyme
Stomach acid
Commensal bacteria
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Insult
Antigen presentation
Cytokines
Macrophages
Endothelial cells
Cytokines
T-lymphocytes
B-Lymphocytes
Macrophages
Neutrophils
Cytokines
Inflamatory mediators
(TNF, IL-1, Il -6, Il -8, PGE2
ROS, RNS)
Tissue damage
Brain: reduced
appetite, fever
Antibodies
Skeletal muscle
Adipose tissue
Nutrient release
Liver
Altered metabolism
Acute phase proteins
Complement
Pathogen
destruction
Homeostasis
Immunological memory
Figure 4.4 The natural and adaptive immunity interactions (adapted from Calder, 2001).
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Effect and role of animal immune system in modern animal production systems is
difficult to overestimate. Banning feed grade antibiotics in Europe will make
immune system competence the major factor determining efficiency of poultry
production. Molecular immunology is developing very quickly and mechanisms
of immunocompetence recently have received substantial attention (McCorkle,
1998; Saif and Swayne, 1998) and nutritional modulation of resistance to infectious
diseases (Klasing, 1998) is a frontline for future research.
Did you know that the total number of lymphocytes represent a
little more than 1% of the body weight of an animal?
Among different nutrients playing an important role in modulating immune
response (Klassing, 1998) are natural antioxidants. Selenium (Turner and Finch,
1991; Larsen, 1993; MacPherson, 1994; McKenzie et al., 1998) is shown to be
very promising. However, antioxidant roles in animal and human immune system
modulation received only limited attention. Mechanisms of nutritional modulation
of resistance to infectious diseases were divided by Klasing (1998) into seven
categories. However, Selenium was not mentioned there.
It is well known that several indicators of immune responsiveness are depressed
when animals are selenium and/or vitamin E deficient. In particular, decreases in
both cellular and humoral immune function in man, laboratory and farm animals
and chickens are also observed (Combs and Combs, 1986). Since these nutrients
serve as antioxidants, membrane integrity may be affected by a deficiency.
However, cellular integrity is very important for receiving, and responding to the
messages needed to co-ordinate an immune response (Latshaw, 1991). Therefore
the antioxidant status of the host is a critical consideration in the optimal functioning
of the immune system. Furthermore, modulating immune responses of birds by
nutritional means in many cases targets specific immunity (Korver and Klasing,
2001).
Phagocyte functions
Phagocytosis is the process by which leukocytes and other cells ingest particulate
ligands whose size exceeds about 1 m. By ingesting microbial pathogens,
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Species
Effect of Se
Cattle
Goat
Chicken
Rat
Rat
Antibody production
Research data accumulated for the last 30 years clearly indicate that antioxidant
deficiency is associated with impaired immune system in human and laboratory
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(Larsen et al., 1997). Following chilling, antibody titre response was substantially
reduced and the titre reduction was prevented with dietary additions of Se between
0.1 and 1.2 mg/kg (Larsen et al., 1997). Furthermore, significantly higher antibody
titres at 10 d post-immunisation for Newcastle Disease Virus were attributed to
0.06 mg/kg and 150 IU/kg Se and vitamin E, respectively (Swain et al., 2000).
The antibody immune response in White Leghorn chicks was also significantly
improved for the Se enrichment diet in the Salmonella or Salmonella and aflatoxin
inoculated groups (Hegazy and Adachi, 2000). Selenium can also be used with
drinking water. For example, Deng et al. (1999) studied effects of sodium selenite
solution on the humoral immunity and erythrocyte immunity function against
Newcastle disease in chicks. Their results showed that the HI titre of the control
group was much lower than that of the 4 selenium-supplemented groups. At the
same time, the erythrocyte immune function of the control group was lower than
that of the tested groups. Similarly, when chicken were vaccinated with attenuated
strain C30-86 of Newcastle disease virus via drinking water at 3 and 21 days of
age supplementation with selenium through water increased HI antibody titre by
stimulating antibody production (Yang et al., 2000).
Immunomodulating properties of antioxidants were shown with a range of
animal species, including various farm and laboratory animals. For example,
increased selenium supplementation was beneficial to swine immunity. In particular,
the most consistent trend was seen in the humoral immune response to lysozyme,
where diets with Se 0.9 mg/kg and various concentrations of vitamin E invariably
produced higher titres than diets which contained Se 0.3 mg/kg (Blodgett, et al.,
1988). However, cell-mediated immunity as measured by the in vivo skin response
to phytohaemagglutinin was similar among diets. Injections of Se (5 mg) to sows
on d 100 of gestation resulted in higher IgM levels in colostrum and in serum
from pigs at birth (Hayek et al., 1989). However, concentrations of colostral and
serum IgA or IgG were not affected by treatment. Piglets receiving the higher
doses of vitamin E (200 ppm) and selenium (0.3 ppm) had more antibodies present
against Aujeszky vaccine in the third week post-weaning than piglets receiving
the lower doses (30 and 0 ppm) (Daza et al., 2000). Also in pigs, haemagglutination
assays indicated that vitamin E (220 IU/kg diet) and Se (0.5 mg/kg diet)
independently increased the immune response, particularly during the latter weeks
of the experiment (Peplowski et al., 1980). The combination of both nutrients
provided in the diet or by injection, resulted in a further increase in
haemagglutination titres, suggesting an additive response.It seems likely, that
immunomodulating properties of Se are observed at doses usually much higher
than commercially used. For example, the effect of dietary Se on the immune
response was evaluated in 96 crossbred weanling swine. Six groups of 16 pigs
were fed diets with Se supplemented as sodium selenite at 0, 0.3, 0.6, 0.9, 1.2,
and 1.5 mg/kg. The basal diet contained 0.068 mg of Se/kg. Whole blood
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increase of Chlamydia antibody response but not when it was given in combination
with vitamin E.
Six groups of three six-month-old lambs were fed a basal diet containing 0.13
mg Se/kg supplemented with either 0.1, 0.5 or 1.0 mg Se/kg in the form of sodium
selenite or as selenomethionine. Experimental animals generally showed enhanced
antibody response to tetanus toxoid, parainfluenza-3 virus and Corynebacterium
pseudotuberculosis, and their total serum IgG concentrations were higher than in
unsupplemented control animals (Larsen et al., 1988a). In two field studies
significantly higher titres to tetanus toxoid were detected in ewes injected with
100 mg selenium as barium selenate. Lambs from selenium supplemented ewes
had significantly higher titres to tetanus toxoid than lambs from ewes in the control
group (Larsen et al., 1988a).
Fifteen Shetland ponies were used in a 7-wk trial to study the effect of
supplemental Se on humoral antibody production (Knight and Tyznik, 1990).
Before the experiment, the ponies were depleted of Se and assigned randomly to
either a low Se (0.02 ppm) or higher Se (0.22 ppm) diet. In comparison with those
ponies receiving the low Se concentrate, ponies receiving the Se-supplemented
diet had higher GSH-Px activities and blood Se concentrations during the later
weeks of the experiment. Se-supplemented ponies were also characterised by an
enhanced primary immune response as evidenced by increased hemagglutination
titers and higher IgG concentrations.
Eleven men were fed foods naturally high or low in selenium for 120 d. Selenium
intake was stabilised at 47 g /d for 21 d, then changed to either 13 or 297 g/d
for 99 d, leading to significantly different blood selenium and GSH-Px activity.
Antibody titers against diphtheria vaccine were 2.5-fold greater after reinoculation
in the high selenium group (Hawkes et al., 2001).
It is interesting to note that immune system can be adapted to low Sesupplementation and in some conditions antibody production can be less
compromised in Se-deficient animals in comparison to control once. For example,
the selenium-deficient mouse-trypanosome system was used to study the effects
of selenium deficiency in Swiss Webster mice infected with Trypanosoma musculi
(Ongele et al., 2002). The results indicated that there was a severe depression in
primary and secondary antibody responses to sheep red blood cells in all inoculated
mice. However, these responses were significantly less depressed in seleniumdeficient mice. In particular, in Se-deficient mice, a low parasitemia was observed
and infection was cleared by day 16 post-inoculation (PI), whereas control mice
sustained the parasitemia until day 24 PI.
Selenium is toxic at high doses so it is necessary to be careful choosing the
correct dose of this trace element for an experiment. For example, mice were
supplemented parenterally with Se 1.5-1.9 mg/kg body weight, vitamin E 500600 IU/kg body weight or Se plus vitamin E (25-100 g and 7.5-30 IU/mouse,
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Species
Effect of Se
Lamb
Sheep
Cattle
Calves
Horse
Pig
Chicken
Mouse
Lymphocyte functions
It has been suggested that Se and vitamin E deficiencies affected T lymphocytes
to a greater extent than B lymphocytes (Larsen, 1993). This was explained to be
a result of higher level of PUFAs in T lymphocytes and higher membrane fluidity.
In fact, vitamin E and Se deficiencies may affect both the maturation of specific
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1988). Three groups of lambs from a flock in which white muscle disease had
been diagnosed were used in another experiment (Ramos et al., 1998). Group 1
was inoculated with 6 mg of sodium selenite and 200 mg of vitamin E, group 2
was inoculated with 200 mg of vitamin E, and group 3 was the untreated control
group. Lambs in the group 1 treated with selenium and vitamin E showed a greater
response to the delayed hypersensitivity test compared to the other groups. It is
concluded that supplementation of lambs with selenium and vitamin E has a
positive effect especially on cell-mediated immunity. Similarly, the Con Astimulated lymphocyte proliferation was significantly lower in Se-deficient cows
(Cao et al., 1992). The effect of dietary selenium on leukocyte migration inhibitory
factor (LMIF) production was examined in vitro using lymphocytes from goats
fed a diet deficient in selenium. The ability of peripheral blood lymphocytes to
produce LMIF induced by Con A was significantly inhibited in Se-deficient cells
(Aziz and Klesius, 1985). Se deficiency in goats decreased the production of
leukotriene B4 (LTB4) by PMN and LTB4-mediated neutrophil chemotaxis (Aziz
and Klesius, 1986). Therefore dietary Se deficiency in goats is associated with
depressed PMN generation of factors that stimulate neutrophil chemotaxis and
neutrophil chemiluminescence (Aziz and Klesius, 1986a).
Dietary (2 ppm for 8 wk) or in in vitro (1 x 10-7M) supplementation with Se
results in a significant enhancement of the proliferative responses of spleen
lymphocytes from mice in response to stimulation with mitogen or antigen. In
contrast, Se deficiency (0.02 ppm for 8 wk) had the opposite effect. The alterations
in the ability of the cells to proliferate were related to the ability of Se to alter the
kinetics of expression of high-affinity IL2 receptors on the surface of activated
lymphocytes (Kiremidjian-Schumacheret et al., 1992). The results also suggested
that Se most likely affects processes in the cytoplasmic and/or nuclear
compartments of activated lymphocytes.
It seems likely that selenium can restore the age-related defects in cell
proliferation through an increase in the number of high-affinity IL-2 receptors.
For example, supplementation with selenium (2.00 ppm for 8 weeks) of aged
male mice resulted in a significant increase in the ability of spleen lymphocytes to
undergo blastogenesis (Roy et al., 1995). Furthermore, Se supplementation
restored the age-related deficiency of the cells to respond to stimulation by nuclear
DNA synthesis and cell proliferation. In the same experiment populations of in
vivo, alloantigen-activated lymphocytes from Se-supplemented aged animals
contained significantly higher numbers of cytotoxic lymphocytes than those from
Se-normal aged animals, which resulted in an enhanced capacity to destroy tumor
cells. The significant increase in the number of cytotoxic effector cells within
these activated T-lymphocyte populations was explained as the result of an
enhanced clonal proliferation of cytotoxic precursors cells, followed by the
differentiation of greater numbers of cytotoxic effector cells (Roy et al., 1995).
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depression in forced antibody response was found in both high seleniumsupplemented groups. A substantially diminished mitogenic response to Con A
and pokeweed mitogen was also observed in the 12-ppm selenium group. Similarly,
Se administered to female Sprague-Dawley rats for 10 weeks at 0.5 and 2.0 ppm
resulted in significant enhancement of splenic NK activity while the NK response
in the 5.0 ppm Se-treated rats was equivalent to the non-Se-treated controls.
Conversely, the DTH response was significantly suppressed at all three dosages,
while antibody synthesis and prostaglandin E2 activity were significantly reduced
compared to the controls at the highest dosage of Se (Koller et al., 1986).
As mentioned above, Se is proven to have an important function in maintaining
proliferative capacity of T- and B-cells (Turner and Finch, 1991). It has been
suggested that Se may modulate the expression of IL-2 receptors on the cell surface,
which could lead to the altered ability of lymphocytes from Se-deficient animals
to respond to mitogen and antigens (Larsen, 1993). Again this phenomenon is a
characteristic for various farmed and laboratory animals (Larsen, 1993; Turner
and Finch, 1991; MacPherson, 1994; Table 4.7).
Table 4.7 Effect of Se on Lymphocytes (adapted from Larsen, 1993; Turner and Finch, 1991; Roy et al.,
1990; Petrie et al., 1989).
Species
Effect of Se
Lamb
Sheep
Cattle
Goat
Pig
Chicken
Dog
Rat
Mouse
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Neutrophil migration and O22- activity (cow) Lymphokine activated killer cell activity
High-affinity IL-2 receptor (mouse)
Disease resistance
The final goal of the improvement of the immune system is to increase resistance
to various diseases. Indeed, this option was extensively studied with chickens.
For example, a combination of vitamin E with Se resulted in reduced mortality
and increased body weight gain in chickens infected with Eimeria tenella (Colnago
et al., 1984). The same authors showed that dietary supplementation with selenium
or vitamin E reduced mortality and increased body weight gain of nonimmunized
chickens infected with E. tenella in three of four experiments. When chicks were
inoculated with virulent Mareks disease (MD) virus at 10 days of age, selenium
(0.6 mg/kg) decreased the morbidity and mortality from MD. In particular, selenium
increased the ability of cells to remove ROS and lipid peroxides, and decreased
the degree of tissue damage caused by ROS (Huang and Chen, 1996). In another
experiment, from one day of age chicks were fed on a basal diet containing
selenium at 0.086 mg/kg (group I) or the basal diet supplemented with selenium
at 0.3 mg/kg (group II) or 0.6 mg/kg (group III). The chickens were infected with
infectious bursal disease virus at 39 days of age. Ten days later the mortality rates
in groups I, II and III were 33.3, 12.4 and 10.6%, respectively, and the infection
induced inhibition of T lymphocyte transformation was less in the selenium
supplemented birds (Bu et al., 1996). When Se was added to the feed of White
Leghorn chickens prior to challenge with either E. coli or sheep erythrocyte
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antigen, the incidence of death or lesions was reduced from 86 to 21% at the
optimal dose of Se (0.4 mg/kg feed; Larsen et al., 1997). Lower Se values were
measured in infected with Ascaridia galli chickens compared with controls, and
this was related to a lower degree of Se absorption and the regeneration of the
intestinal mucosa in infected birds (Damyanova et al., 1995).
Immunostimulating and disease-preventing effects of natural antioxidants are
not restricted to avian species, but obvious with other farm animals. For example,
vitamin E alone or in combination with Se was shown to have a protective effect
against swine dysentery infection, when challenged with Trioeponema
hyodysenteriae (Teige et al., 1982).
Se deficiency in cows is related to a range of various diseases including metritis,
mastitis, cystic ovaries, retained placenta, increased somatic cell count (SCC) and
some others. Decreased incidence of metritis in selenium-treated dairy cows
provides a good example of an association between selenium deficiency and
decreased disease resistance (Suttle and Jones, 1989). Incidence of metritis was
60% for cows injected with selenium and 84% for those not receiving selenium
(Harrison et al., 1984). Days to minimum uterine size were significantly less in
cows with metritis and selenium treated when compared with cows with metritis
and not selenium treated (32.9 vs. 35.8; Harrison et al., 1986).
Numerous studies have linked low Se and/or vitamin E status to increased
susceptibility of dairy cows to intramammary infections (Spears, 2000). An
increased Se status is protective against various infections. For example, in an
experiment, dairy cows were fed rations containing 0.05 or 0.35 mg Se/kg dry
matter and were inoculated intracisternally with Escherichia coli at 14-16 weeks
of lactation. Milk bacteria numbers were significantly higher between 16 and 24
h postinoculation in the Se-deficient group and three of the four cows in this
group required euthanasia, whereas all four cows in the Se-supplemented group
recovered without therapeutic intervention (Maddox et al., 1991). Seleniumsupplementation (0.2 ppm) showed a positive effect on udder health. The
percentage of quarters harbouring mastitis pathogens dropped from 22.9 to 13.0
in the Se-yeast group and from 18.4 to 7.4 in the selenite group during the
supplementation period. It is interesting that in cows supplemented with Se duration
of clinical symptoms was reduced by 46% and a combination of Se and vitamin E
was even more effective decreasing duration of mastitis by 62% (Smith et al.,
1984; Figure 4.5). These findings were confirmed in following studies from the
same department of the Ohio State University. A combine Se-vitamin E
supplementation of cows prior to calving and during lactation was associated
with a reduction of the following: in the prevalence of infected quarters at calving
(by 42.4%) and in quarter lactation days infected (by 59%), in clinical mastitis
over the entire lactation (by 32.1% and during the first four days of lactation (by
57.2%) and SCC (Smith and Conrad, 1987). Similarly, diets of first lactating
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60
50
40
30
20
10
0
Se
Vitamin E
Se+vitamin E
Figure 4.5 Clinical cases of mastitis (adapted from Smith et al., 1984).
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The frequency of quarter atrophy and agalactia, and reduction in wholeudder milk yield in the first 4 days after challenge exposure, were greater in
the SeD cows.
Log10 peak bacterial concentrations in milk were higher in SeD than in SeS
cows.
Mean log bacterial concentration was significantly higher from 12 to 20
hours after challenge exposure in SeD than in SeS cows.
Duration of infection was significantly greater in SeD than in SeS cows .
Milk somatic cell counts increased significantly more slowly in SeD than in
SeS cows from 8 to 16 hours after challenge exposure.
Ratios of milk somatic cells to bacteria in milk were significantly lower in
SeD than in SeS cows at 12 and 16 hours after challenge exposure.
Peak SCC were reached earlier in SeD than in SeS quarters.
Supplementing the feed of selenium-deficient diary cows with selenium (Se)yeast or selenite at a level of 0.2 ppm induced self-cure of subclinical mastitis; the
prevalence of quarters harbouring subclinical mastitis decreased to about one
half during the 8 week supplementation period. Possible mechanisms of such
protective effects of Se on mastitis include (Ali-Vehmas et al., 1997):
In 1999 Harrison and Hancock from the Washington State University conducted
a detailed meta-analyses of publications related to the effect of selenium and/or
vitamin E on mastitis in cows. They showed that from 19 studies conducted all
over the world including Mexico, Italy, UK, Israel, Canada, Portugal and Korea
and published in peer-review journals in 13 studies (68%) positive effect of
selenium/vitamin E supplementation was reported. On average, in control animals
mastitis was registered in 32% and in Se-supplemented cows mastitis was evident
only in 10% (Harrison and Hancock, 1999). The total costs associated with clinical
mastitis is about $107 per case (Hoblet et al., 1991).
The first report on relationship between retained placenta and selenium/vitamin
E status of cows was published by Trinder et al. (1969). Since then, this subject
has received substantial attention. It is interesting that Se-vitamin E combination
not only significantly reduced the incidence of retained placenta but also reduced
the days required for calving the first service (Kim et al., 1997). It is unclear why
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Species
Immunological response
References
Cattle
Intracellular killing
Extracellular hydrogen peroxide
Metritis, Cystic ovaries
Se+vit.E: AB (IgG ) to Pasterella haemolitica
Antibody titres
Serum and colostrum IgG
Neutrophil kill
Lymphocyte proliferation
Mastitis
IgM
Antibody titres after vaccination
Incidence and duration of mastitis by 46%
Colostrum IgG
Virus titer after vaccination
Limphocyte proliferation
Lymphocyte proliferation
Antibody titres after vaccination
Se+vit.E: prevalence of infected quarters at
calving by 42%; Quarter lactation days infected
by 59%; clinical mastitis by 32%; somatic cell
numbers
AB to egg lysozyme
IgM , IgG by stress , AB to Pasterella
haemolitica ; AB to IBRV
IgM , IgG, AB to IBRV
Lymphocyte proliferation
In vitro lymphocyte killing ability
Antibody titers , IgG
Lymphocyte proliferation
Cell-mediated immunity
AB to Leptospira
IgM , primary AB to PIV-3
IgG , AB to tetanus tox.
Lymphocyte response to PWM, PHA, ConA
Lymphocyte response to PHA and PWM
Se+VitE: response to PHA, ConA, PWM
Primary AB to Brucella abortus, AB to RBC
Chlamydia antibody response
Neutrophil activity
Low Se: lymphokin (MIF)
Cattle
Cattle
Beef cattle
Beef cows
Dairy cow
Dairy cows
Dairy cows
Dairy cows
Dairy cows
Dairy cows
Dairy cows
Dairy cows
Heifers
Heifers
Heifers
Calves
Calves
Calves
Calves
Lambs
Lambs
Lambs
Lambs
Lambs
Lambs
Lambs
Lambs
Lambs
Sheep
Sheep
Sheep
Ewes
Goat
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Table 4.10 Effect of Se supplementation on humans (adapted from Hawkes et al., 2001; Larsen, 1993;
McKenzie et al., 1998).
Killing by macrophages
Lymphocyte counts
IgG ; IgA
NF-kB activation
Table 4.11 Effect of Se deficiency on human (adapted from Hawkes et al., 2001; Larsen, 1993;
McKenzie et al, 1998).
Spontaneous abortion
Skeletal myopathy
Psoriasis-severity
infected at one day of age with virulent Mareks disease virus (vMDV). They
showed that in infected birds the Se-GSH-Px activity was significantly decreased
and lipid peroxidation was enhanced. Therefore preventing these changes in
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Species
Effect of Se
Cattle
Chicken
Pig
Mouse
Rat
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252
Decreased immunity
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Antioxidant status
Poor Good
Increased NFB
activity
Enhanced
inflammation
Cytokines
Oxidants
Sulphur
amino acids
GSH
Normal NFB
activity
+
Lymphocyte
activity
Inflammation
PGE2
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256
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Disbalancein eicosanoid
production by phagocytes
and inflammation
Phagocyte functions
compromised
Compromised AO system
and oxidative stress
Decreased activity
of NK cells
Damages to healthy
tissues by free
radicals
produced by
phagocytes
Compromised antibody
production by
B-lymphocytes
Figure 4.8 Oxidative stress and the immune system (adapted from Surai 2002).
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Conclusions
Selenium affects all components of the immune system, including the development
and expression of nonspecific, humoral, and cell-mediated responses. In general,
a deficiency in Se appears to result in immunosuppression, whereas
supplementation with low doses of Se appears to result in augmentation and/or
restoration of immunologic functions. On the one hand, a deficiency of Se has
been shown (Kiremidjian-Schumacher and Stotzky, 1987) to inhibit
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First two weeks posthatch represent most important period of immune system
development and maternal diet is shown (Klasing, 1998; Surai and Sparks,
2001) to have a profound effect on this process. In particular, the first week
of chick life is a period of rapid expansion of leukocyte population, seeding
of lymphoid organs and other events ultimately leading to the production
of unique clones of lymphocytes that will mediate immunity in postnatal
development (Klasing, 1998). In this respect effects of various combinations
of natural antioxidants and n-3 PUFA await investigation.
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5
SELENIUM AND SEMEN QUALITY
We never know the worth of water till the well is dry
Introduction
Animal and human spermatozoa are unique in structure and chemical composition
and are characterized by high proportions of polyunsaturated fatty acids (PUFAs)
in the phospholipid fraction of their membranes (Surai, 2002). This feature of
these highly specialized cells is a reflection of the specific needs of their membranes
for high levels of fluidity and flexibility, which are necessary for sperm motility
and fusion with the egg. This functional advantage conferred by PUFAs is, however,
associated with disadvantages in terms of the susceptibility of sperm to free radical
attack and lipid peroxidation. Therefore antioxidant protection is a vital element
in maintaining sperm membrane integrity, motility and fertilizing ability. It has
been suggested (Surai, 2002) that natural antioxidants (vitamin E, ascorbic acid
and glutathione) together with antioxidant enzymes (superoxide dismutase and
glutathione peroxidase) build an integrated antioxidant system in avian semen
capable of protecting it against free radicals and toxic products of their metabolism.
The delicate balance between free radical production and antioxidant defense is
considered to be an important determinant of semen quality and in particular its
fertilising ability.
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18:2n-6
20:2n-6
20:3n-6
20:3n-9
20:4n-6
22:3n-9
22:4n-6
22:5n-3
22:6n-3
Total PUFA
Peroxidability Index
Chicken
Turkey
Guinea fowl
Duck
2.6a
1.5a
1.6
<0.5
11.7 a
3.8a
27.9 a
0.5
2.1a
52.1
215.0
4.6b
4.0ab
0.9
2.7a
9.4b
9.2b
12.6b
0.7
2.7a
46.8
184.0
3.8abc
3.3ab
<0.5
1.0b
15.8c
4.3a
19.4c
<0.5
1.7a
49.3
195.1
3.8abc
1.6a
1.0
<0.5
18.9d
0.6c
19.9c
0.6
8.0b
54.4
231.3
Goose
5.7bd
3.9b
0.5
0.9b
13.3 ac
1.4c
17.5cd
<0.5
2.8a
46.0
185.6
*/Values within a row that do not share a common letter are significantly different (P<0.05).
Phospholipids are the major lipid fraction in avian spermatozoa comprising 66.470.7% of total sperm lipid in the case of the chicken (Cerolini et al., 1997; Kelso
et al., 1997a; Surai et al., 2000) or 59.5% for the turkey (Cerolini et al., 1997). It
seems that the phospholipid proportion in the chicken spermatozoa increases with
aging reaching 72.1% of total lipid at 60 weeks of age (Kelso et al., 1996), but
decreasing by 72 weeks of age down to 51% (Kelso et al., 1997). In some cases
the phospholipid proportion is reported to be 84.5% of the total lipids in the cockerel
spermatozoa (Blesbois et al., 1997), but the authors did not detect any
triacylglycerol in the spermatozoa, which could comprise up to 4.2% (Cerolini et
al., 1997) or 7.3% (Kelso et al., 1997) depending on the age of cockerels.
Our data indicated that docosatetraenoic (DTA; 22:4n-6) and arachidonic acid
(AA; 20:4n-6) are the major PUFAs in the phospholipid fraction of the chicken
spermatozoa (Surai, 2002). Their proportions varied depending on the cockerels
age. DTA is the characteristic PUFA of avian spermatozoa in contrast to mammalian
spermatozoa where docosahexaenoic acid (DHA; 22:6n-3) is the major PUFA
(Poulos et al., 1973). In fact, DHA is generally the most important spermatozoan
PUFA in mammals, including man (Nissen and Kreysel, 1983), bull (Kelso et al.,
1997), monkey, (Lin et al., 1993), ram and boar (Poulos et al., 1973). However,
in dog and rabbit spermatozoa, docosapentaenoic acid (DPA; 22:5n-3) is the main
PUFA (Poulos et al., 1973). The importance of C22 polyunsaturates in relation to
male fertility has been shown in humans where the amount of DHA in spermatozoa
is positively correlated with sperm motility (Nissen and Kreysel, 1983; Zalata et
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al., 1998; Conquer et al., 1999) and with the normal morphology of sperm cells
(Lenzi et al., 2000). Therefore the best morphological pattern corresponded to
the highest DHA concentration in the human semen (Lenzi et al., 2000a). In
general, in mammalian spermatozoa long chain PUFAs containing 20-22 carbon
atoms comprise more than 50% of total fatty acids in the phospholipid fraction
and the level of DHA in semen depends on stage of sperm maturation (Ollero et
al., 2000). In mammals, long chain PUFAs are actively produced during the
maturation of spermatozoa after testicular release (Lenzi et al., 2000a). In contrast
to mammals, avian spermatozoa are less unsaturated and are characterised by the
presence of DTA and AA which comprise more than 30% of total PUFA (Surai et
al., 1998b).
The biological reason for these species-specific differences in the PUFA profiles
of spermatozoa is not clear at present. However, there is a growing body of
evidence indicating that the fatty acid composition of sperm membranes, especially
levels of PUFA, determine their biophysical characteristics such as fluidity and
flexibility as appropriate for their specific functions, including sperm motility and
fertilising capacity (Ladha, 1998). For example, increased PUFA concentrations
in human spermatozoa were associated with increased sperm membrane fluidity
(Comhaire et al., 2000). It is also necessary to appreciate that phospholipids
containing DHA are not evenly distributed throughout the membranes of
mammalian spermatozoa, being located mainly in the tail (Connor et al., 1998).
Whether this is the case for avian spermatozoa, with DTA being confined to the
tail, awaits investigation. However, the very high proportion of long chain PUFA
in the avian spermatozoa predisposes them to lipid peroxidation and it seems
reasonable to suggest that antioxidant protection plays a crucial role in the
maintenance of spermatozoan membrane integrity and their fertilising ability.
Clearly, avian and mammalian spermatozoa are rich in PUFAs (Figure 5.1) and
are vulnerable to lipid peroxidation.
Did you know that the fatty acid composition of sperm
membranes, especially levels of PUFA, determine their
biophysical characteristics as appropriate for their specific
functions, including sperm motility and fertilising capacity
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Ram
Chicken
50
40
30
20
10
0
20:4n-6
22:4n-6
22:5n-6
22:6n-3
PUFAs
Figure 5.1 PUFAs in spermatozoa phospholipids, % (adapted from Surai, 2002)
20:4n-6 arachidonic acid; 22:4n-6- docosatetraenoic acid; 22:5n-6-docosapentaenoic acid; 22:6n-3
docosahexaenoic acid.
major attention to this subject came in 1970 after publication of several milestone
papers by Jones and Mann based on the results of experiments conducted in the
Agricultural Research Councils Unit of Reproductive Physiology and
Biochemistry, University of Cambridge (Jones and Mann, 1973; 1976; 1977;
1977a; Jones et al., 1978; 1979). These publications clearly showed that lipid
peroxidation:
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et al., 2000), bull (Dawra et al., 1983; Beconi et al., 1991; Slaweta et al., 1988;
OFlaherty et al., 1997; Beorlegui et al., 1997), buffalo (Singh et al., 1989), rabbit
(Castellini et al., 2000) and horse (Baumber et al., 2000; Ball and Vo, 2002).
Furthermore, lipid peroxidation in human semen has been studied further in detail
and several comprehensive reviews have discussed their findings (Aitken 1994;
1995; 1999; Lenzi, 2000, 2000a). The conclusion is that lipid peroxidation in
mammalian semen is considered to be one of the most important factors causing
infertility in man as well as causing decreased sperm quality during the storage of
semen from farm animals.
Research on lipid peroxidation in avian semen started much later in comparison
to mammalian species, This is probably because such research has largely been
driven by a practical need to understand molecular mechanisms of semen
deterioration during storage for further artificial insemination. In poultry production,
however, artificial insemination was introduced several decades later than for
mammalian species, and its practical usage is related mainly to turkey production.
Therefore, a publication by Fujihara and Howarth showing that, during incubation
at 41C, chicken spermatozoa produced a thiobarbituric acid-reactive product
and that susceptibility to peroxidation was enhanced by the addition of ascorbate
was a starting point in research related to lipid peroxidation in avian semen (Fujihara
and Howarth, 1978). The authors concluded that chicken spermatozoa, following
ejaculation and exposure to air, could undergo peroxidation with a consecutive
decrease in their viability. The conclusion was very important for future
improvement of techniques for avian semen storage in vitro. Further evidence for
lipid peroxidation in avian semen was presented by Wishart (1984), who found
that the formation of high concentrations of malondialdehyde (MDA) during a 5hour aerobic incubation of chicken semen was associated with a partial or complete
loss of fertilizing ability. Most importantly, the fertilizing ability of samples that
produced low or negligible concentrations of MDA remained unimpaired (Wishart,
1984). Semen samples incubated under anaerobic conditions produced only a
negligible amount of MDA. There was a considerable (70-fold) variability between
individual males in relation to MDA production (Wishart, 1984). This could be
explained as a result of compositional and functional differences in sperm
membranes among individual male chickens (Fujihara and Koga, 1992).
Did you know that lipid peroxidation in semen is considered
to be one of the most important factors causing infertility in
man as well as causing decreased sperm quality during the
storage of semen from farm animals
Independently of the work conducted in the UK by Wishart (1984), investigation
of lipid peroxidation in turkey semen was started approximately at the same time
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in Ukraine (Surai, 1983; 1984). In particular an induced (by Fe2+) lipid peroxidation
in turkey semen was used to assess semen susceptibility to lipid peroxidation,
since the initial level of peroxides in fresh or even stored semen was shown to be
comparatively low. At that time an idea of the possible protective effects of natural
antioxidants in the male diet on the lipid peroxidation in the semen was developed
(Surai, 1984). In particular, it was shown that dietary supplementation of male
turkeys with vitamin E was associated with a significant decrease in semen
susceptibility to lipid peroxidation. This work was further developed by Cecil
and Bakst (1993) who showed that during aerobic storage of turkey spermatozoa,
lipid peroxidation was time and temperature-dependent. The authors also suggested
that turkey spermatozoa are more sensitive to lipid peroxidation than semen from
other species. It seems likely that lipid peroxidation in semen is also age-dependent.
For example, Donoghue and Donoghue (1997) reported that MDA concentrations
were 10-fold higher in semen from older turkey males (56 wk of age) than for
younger ones (30 wk of age). Turkey male ageing (between 31 and 52 weeks)
was accompanied by a 30% increase in lipid peroxidation at 47 and 52 weeks of
age. Furthermore in vitro storage did not cause lipid peroxidation in sperm obtained
during the first half of the reproductive period but MDA levels significantly
increased in sperm obtained during the second half of this period (Douard et al.,
2003). In general lipid peroxidation is a significant factor affecting the fertility of
stored turkey sperm and males varied in production of MDA during in vitro storage,
with most pairs exhibiting a threefold increase (Long and Kramer, 2003). This is
in agreement with the data of Kelso et al. (1996) indicating a fall in the antioxidant
defence (activity of GSH-Px) of chicken spermatozoa between 25 and 60 weeks
of age. Accumulation of TBARS in duck semen as a result of lipid peroxidation
has also been recently described (Surai et al., 2000). It seems likely that MDA
accumulation is associated with mid-piece abnormality in human spermatozoa
(Aitken et al., 1993) and with a decrease in the fertilising capacity of chicken
(Wishart, 1984) and turkey (Cecil and Bakst, 1993) spermatozoa. It is interesting
that lipid peroxidation was particularly strong in the midpiece and tail of frozen/
thawed bovine spermatozoa and significantly less intense in the head (Brouwers
and Gadella, 2003).
The molecular mechanisms of lipid peroxidation in avian semen have received
little attention. Recently it has been shown that during sperm storage, lipid
peroxidation is associated with a significant decrease in PUFA concentration in
spermatozoa. In particular, the main PUFA in the chicken semen (22:4n-6) was
most susceptible to peroxidation. Its proportion in the phospholipid fraction was
significantly decreased as a result of incubation of chicken sperm for 12 hours at
20C (Surai et al., 1998b). The inclusion of a promoter of lipid peroxidation
(Fe2+) in the incubation medium further increased the rate of lipid peroxidation,
significantly decreasing the proportions of not only 22:4n-6, but also of 20:4n-6,
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chromatin destabilisation
marked alterations in the DNA-protein complex
changes in the activities of various enzymes, including cytochrome oxidase,
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1996). This conclusion was based on the results of a range of different experiments
with mammals showing that:
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organisation. Mouse and rat Sptrx-2 proteins display a high homology to their
human ortholog and the coding genes are located at syntenic positions (MirandaVizuete et al., 2003). The authors also showed that Sptrx-2 is required during the
final stages of sperm tail maturation in the testis and/or epididymis, where extensive
disulfide bonding of the fibrous sheath proteins occurs.
It seems likely that it is just the beginning of deeper understanding of roles of
new proteins in sperm function. Recently a novel member of the thioredoxin
family, thioredoxin-like protein 2 (Txl-2) has been cloned and characterised (Sadek
et al., 2003). In the study light and electron microscopy analyses showed that the
protein was associated with microtubular structures such as lung airway epithelium
cilia and the manchette and axoneme of spermatids. In fact full-length Txl-2 weakly
associates with microtubules and the author suggested that this thioredoxin might
be a novel regulator of microtubule physiology. Therefore it is just a matter of
time before TR in the sperm is characterised and its essential role in sperm
antioxidant protection and structural integrity maintenance is shown.
There are species-specific differences in the Se level in semen. For example,
the level of Se in seminal plasma was lowest in the human and the stallion, higher
in ram and boar, with the highest levels in the bull (Saaranen et al., 1989). In
bulls, 75Se appeared in the epididymal caput within 5 days and passed through
the epididymis in 20 days (Vanha-Perttula and Remes, 1990). Furthermore, in
semen 75Se was first mainly found in seminal plasma, where a plateau level was
reached at 5 d followed by a gradual decline after 12 d. The total semen level,
however, increased after 14 d and this increase was due to a rapid appearance of
the label in spermatozoa. In the sperm 75Se level reached a plateau at 20 d and
remained high until 40 days, after which a gradual decline ensued (Vanha-Perttula
and Remes, 1990). Selenocysteine is shown to be the main form of Se in rat
sperm and selenocysteine and selenomethionine were found in ovine sperm (Alabi
et al., 2000).
Since hydrogen peroxide and lipid peroxides are toxic for the spermatozoa
(Alvarez et al., 1987; De Lamirande and Gagnon, 1992), GSH-Px plays an
important role in protecting cell membrane lipid from peroxidation, thus
maintaining the integrity of the cell (Flohe and Zimmermann, 1970). In fact,
GSH-Px in the sperm is considered to be the main enzyme which removes peroxides
and thereby protects cells against damage caused by free radicals and the products
of lipid peroxidation in vivo (Griveau et al., 1995).
There is species and tissue-specificity in GSH-Px expression. For example,
GSH-Px activity has been found to be expressed in the semen of several mammalian
species including ram, dog, human, goat, bull (Li, 1975; Kantola et al., 1988;
Kelso et al., 1997b). However, there are species-specific differences in expression
of this enzyme in semen. In bulls, for example, GSH-Px is exclusively associated
with the seminal plasma and not found in spermatozoa (Brown et al., 1977; Smith
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et al., 1979). In contrast, GSH-Px activity in seminal plasma was low in man and
ram and not detectable in boar and stallion (Saaranen et al., 1989). Bull and ram
seminal plasma GSH-Px activities per mg protein were comparable, but when
expressed per ml seminal plasma, activity of the bull was more than 7 times the
highest activity of ram seminal plasma (Pond et al., 1983). It has been shown that
approximately two thirds of GSH-Px activity in bull semen was non-Se-GSH-Px
(Slaweta et al., 1988). In the same experiment it was found that the MDA level
was negatively correlated with Se-GSH-Px activity and it has been suggested that
Se-GSH-Px plays a role in protecting the acrosome membranes against disruption.
GSH-Px has been found to be expressed in chicken seminal plasma and
spermatozoa (Figure 5.2; Surai et al., 1998, 1998b). There are species-specific
differences in activity and distribution of GSH-Px in avian semen. For example,
in seminal plasma total GSH-Px activity was the highest in turkey and lowest in
duck and goose (Figure 5.3; Surai et al., 1998). In spermatozoa, on the other
hand, the highest GSH-Px activities were found for goose and duck and much
lower GSH-Px activity was recorded for guinea fowl, turkey or chicken (Figure
5.4). Recently, it has been shown that despite a high proportion of PUFAs and a
low level of vitamin E, duck spermatozoa have the same susceptibility to lipid
peroxidation as chicken spermatozoa (Surai et al., 2000). It has been suggested
that an increased activity of Se-GSH-Px in duck semen compensates for the
relatively low concentrations of other antioxidants.
180
Se-GSH-Px
160
Non-Se-GSH-Px
140
120
100
80
60
40
20
0
Senimal plasma
Spermatozoa
Figure 5.2 Distribution of GSH-Px in chicken spermatozoa (adapted from Surai et al., 1998).
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250
Se-GSH-Px
Non-Se-GSH-Px
200
Units
150
100
50
0
Chicken
Turkey
Guinea fowl
Duck
Goose
Figure 5.3 GSH-Px activity in avian seminal plasma (adapted from Surai et al., 1998a).
160
Se-GSH-Px
140
Non-Se-GSH-Px
120
Units
100
80
60
40
20
0
Chicken
Turkey
Guinea fow l
Duck
Goose
Figure 5.4 GSH-Px activity in avian spermatozoa (adapted from Surai et al., 1998a.
If selenium is limiting in the diet (which is the case in many countries in the
world), then dietary supplementation of this trace element should have a beneficial
effect on the antioxidant defense in various tissues including sperm. This was
confirmed in our studies. Inclusion of Se in the diet of male chickens significantly
increased Se-GSH-Px activity in the liver, testes, spermatozoa and seminal plasma
(Figures 5.5 and 5.6; Surai et al., 1998c). As a result, a significant decrease in the
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sperms and tissue susceptibility to lipid peroxidation was observed (Figures 5.7
and 5.8). This protective effect was more expressed in stored semen as compared
to fresh. In this respect, it is extremely important that an inducible form of the
enzyme (Se-GSH-Px) represents more than 75% of the total enzymatic activity in
chicken spermatozoa and more than 60% in the testes and liver of cockerels.
25
Liver
Testes
GSH-Px, Units
20
15
10
0
20E
20E+Se
200E
200E+Se
Dietary supplementation
Figure 5.5 Effect of selenium and vitamin E on GSH-Px activity in chicken liver and testes (adapted from
Surai et al., 1998c).
700
Seminal plasma
600
Spermatozoa
GSH-Px, Units
500
400
300
200
100
0
20E
20E+Se
200E
200E+Se
Dietary supplementation
Figure 5.6 Effect of selenium and vitamin E on GSH-Px activity in chicken semen (adapted from Surai et
al., 1998c).
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60
Fresh semen
Stored semen
MDA (nmol/g)
50
40
30
20
10
0
1
Groups
Figure 5.7 Effect of Se and vitamin E on lipid peroxidation in chicken spermatozoa (Adapted from Surai
et al., 1998c). Vitamin E and Se were supplemented in the feed (mg/kg) as follows: group 1, no
supplementation; group 2, vitamin E 20 and Se 0; group 3, vitamin E 200 and Se 0; group 4, vitamin E 20
and Se 0.3; group 5, vitamin E 200 and Se 0.3.
25
L
MDA (nmol/g)
20
15
10
0
1
Groups
Figure 5.8 Effect of Se and vitamin E on lipid peroxidation in chicken liver (L) and testes (T) (adapted
from Surai et al., 1998c). Dietary groupd are the same as in Figure 7.
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epididymis. They used 10 mature boars which were fed from weaning to 18 month
of age diets fortified with two levels of supplemental Se (0 or 0.5 ppm) or vitamin
E (0 or 220 IU/kg diet). The low-Se diet caused changes in spermatozoa: the
mitochondria in the tail midpiece were more oval with wider gaps between
organelles and the plasma membrane connection to the tail midpiece was not
tightly bound as when boars were fed Se. Furthermore, sperm ATP concentration
was decreased and percentage of immature spermatozoa with cytoplasmic droplets
increased when boars were fed the low-Se diet (Marin-Guzman et al, 2000). It
seems likely that, Se has a role in establishing the number of boar spermatozoal
reserves and Sertoli cells (Table 5.2). For example, when boars diet was
supplemented with Se 0 or 0.5 ppm for 18 months, testicular sperm reserves were
higher in boars fed on the high Se diet (Marin-Guzman et al., 2000a). In addition,
the boars fed dietary Se had also a greater number of Sertoli cells and round
spermatids at 6.2 month of age and by 18 month of age they also had more
secondary spermatocytes. It is well known that both Se and vitamin E are involved
in a regulation of animal reproduction. However, it seems likely that low Se in the
diet had a greater detrimental effect on semen quality that diets inadequate in
vitamin E. In particular, boars fed the nonfortified Se diets had sperm with lower
motility and a higher percentage of sperm cells with bent and shoehook tails
(Marin-Guzman et al., 1997). Therefore Se-supplementation improved sperm
motility and prevented its decline over the 16 week collection period and the
percentage of normal sperm was approximately 3-fold higher when the Se-fortified
diet was fed to boars (Mahan et al., 2002). At the same time the semen from boars
fed the nonfortified Se diet had a lower fertilisation rate of oocytes with fewer
accessory sperm penetrating the zona pellucida.
Did you know that low Se in the diet had a greater detrimental
effect on semen quality than diets inadequate in vitamin E
Positive effect of Se on semen quality was also shown with rams. For example,
thirty-three 8-month-old ram lambs were kept at grass and fed a supplement of
barley and peas, with ad libitum access to grass silage when grazing became
restricted. On day 0, the rams were allocated to two groups by restricted
randomisation of live weight. One group had a zinc, cobalt and selenium soluble
glass bolus administered with the other group not receiving a bolus to act as a
control (Kendall et al., 2000). Semen was collected once a week between days 44
and 86. The bolused lambs had a significantly increased erythrocyte GSH-Px on
all samplings after bolusing and had significant increases in motility, proportion
of live sperm and proportion of intact membranes.
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Item
Dietary selenium
0.0
0.5
0.54
2.3
0.30
1.19
1.15
13.9
0.80
1.24
Semen
Volume, ml
Sperm concentration, no. x 109
Total sperm, no. x109
GSH-Px, U/ml
Se, mg/kg
160
290
43.5
33
0.031
163
253
43.5
71
0.134
Seminal plasma
Se, mg/kg
GSH-Px, U/ml
0.024
12.3
0.060
37.7
Sperm
Se, mg/kg
GSH-Px, U/g
0.42
579
0.94
977
39.4
65.9
64.0
92.4
50.7
73.0
89.8
163.8
Semen quality
ATP concentration, nmoles ATP/106 spermatozoa
Sperm motility, %
Normal sperm, %
Fertilization rate, % of eggs
Accessory sperm, no./oocyte
1.15
60.4
24.2
73
14
1.55
87.9
61.9
99
60
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300
Inorganic Se
Organic Se + vitamin E
25
20
15
10
5
0
Summer
Winter
Figure 5.9 Selenium source and sperm number in boars (adapted from Jacyno et al, 2002).
250
Inorganic Se
Organic Se + vitamin E
200
150
100
50
0
Summer
Winter
Figure 5.10 Selenium source and boar sperm concentration (adapted from Jacyno et al, 2002).
50
Inorganic Se
Organic Se
40
30
20
10
0
Summer
Winter
Figure 5.11 Selenium source and defective sperm in boars (adapted from Jacyno et al, 2002).
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Sperm morphology
Basal diet
Selenite
Sel-Plex
Normal sperm
Bent midpiece
Swollen midpiece
Ruptured midpiece
Swollen head
Corkscrew head
Coiled
Fragment/other
57.9
18.7
1.6
0.9
1.3
15.4
3.2
1.0
89.4
6.2
0.4
0.1
0.2
1.8
0.8
1.1
98.7
0.7
0.1
0.0
0.2
0.2
0.0
0.1
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Figure 5.12 Image analysis of normal and Se-deficient chicken spermatozoa (adapted from Surai, 2002).
Table 5.4 Effect of selenium on semen quality (Hubbard Ultra-Yield Roosters (adapted from Edens,
2002)).
Variable
254
91.6
1.3
3.8
1.3
2.0
254
85.4
1.9
6.2
3.2
3.3
300
97.7
0.6
0.8
0.6
0.4
Table 5.5 Effects of sodium selenite or selenomethionine of productive parameters of Hubbard breeder
hens (adapted from Edens, 2002).
Farm 1
Variable
Egg production, %
Fertility, %
Daily settable eggs
Hatchability, %
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Farm 2
Selenite
Sel-Plex
Selenite
Sel-Plex
82.87
96.12
8799
83.10
85.46
96.68
9111
84.40
84.22
96.23
8379
82.40
84.74
97.26
8647
82.64
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insemination as well as during sperm transport and storage (avian species) in the
female reproductive tract. However, the form of dietary selenium is a key for
success in optimising Se supplementation. In fact organic selenium can help
maintaining reproductive performance in farm animals and poultry. The general
relationship between selenium and male fertility is shown in Figure 5.13.
Se in the feed
Spermatozoa:
Figure 5.13 Selenium and male fertility (adapted from Surai, 2002).
Conclusions
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Mammalian and avian spermatozoa are rich in PUFAs that make them
vulnerable to lipid peroxidation which is considered as an important
mechanism of impaired sperm quality and reduced fertilizing ability.
Antioxidant systems of semen play an important role protecting spermatozoa
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membranes against the damaging effects of free radicals and toxic products
of their metabolism.
GSH-Px and other selenoproteins are considered to be the major antioxidant
protection in mammalian and avian semen
Organic selenium supplementation is shown to be effective means in
improving semen quality
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6
SELENIUM AND MYCOTOXINS
Prevention is better than cure
Introduction
Silent killers, invisible thieves, unavoidable contaminants, natural toxicants all these names have been given to a group of fungal metabolites called mycotoxins
and their negative effects on animal production are incalculable. In general
mycotoxins are considered to be unavoidable contaminants in foods and feeds
and are a major problem all over the world (Wood, 1992). Interest in these naturally
occurring chemical compounds is intense due to their detrimental effect on human
health (carcinogenicity) and animal productive and reproductive traits. More than
300 mycotoxins have been shown to induce signs of toxicity in mammalian and
avian species (Fink-Gremmels, 1999; Leeson et al., 1995) and this number is
increasing. It has been estimated that 25% of the worlds crop production is
contaminated with mycotoxins (Fink-Gremmels, 1999). The most significant
mycotoxins in naturally-contaminated foods and feeds are aflatoxins, ochratoxins,
zearalenone, T-2 toxin, vomitoxin and fumonisins (Devegowda et al., 1998). In
many cases these mycotoxins can be found in combination in contaminated feed.
Mycotoxins appear at different stages of grain production. For example,
Fusarium species are known to invade grains during the growth of the plant and
they produce so-called field mycotoxins. On the other hand, Aspergillus and
Penicillium species generally develop during grain storage and so may be called
storage mycotoxins. This simple classification tends to over-simplify the situation.
However, two facts are clear: mycotoxin contamination depends on moisture
content of grain which should be less than 15% and drought stress can also increase
fungal contamination of grain. In practice, a range of mycotoxins can be found in
contaminated feeds, the type and level depending on climatic and storage
conditions. Temperate climates with high moisture conditions, e.g. Canada, USA
and Europe, encourage the growth of Fusarium and Penicillium species, producing
DON, zearalenone, ochratoxin A and T-2 toxin that are of concern for human
health. On the other hand, warm and humid climatic conditions, e.g. Latin America,
Asian countries and some parts of Australia, are ideal for the growth of Aspergillus
and the production of aflatoxin, considered to be a carcinogen. The winter season
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Mycotoxins of concern
Three major economically important genera of mycotoxin-producing fungi are
Aspergillus, Penicillium and Fusarium. It is necessary to take into account that
there are many different species of each of the mentioned genera. For example,
Aspergillus species comprise a group of more than 150 members. About 45 of
them, 75 Penicillium species and 25 Fusarium species are known to produce
mycotoxins. Furthermore, mycotoxins can be toxic at very low concentrations.
When 19 mycotoxins were injected to chicken embryos, the maximum toxic effect
was exerted by T-2 toxin, which produced 100% embryonic mortality at doses as
low as 0.01mg.
The most significant mycotoxins in feeds are:
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- The main toxic effect is associated with inhibition of protein synthesis via
binding to the ribosome.
- DON in moderate to low doses can cause a number of detrimental effects
associated with reduced performance and immune function
- The main effect at low dietary doses is a reduction in food consumption
(anorexia), while higher doses induce vomiting (emesis).
- It alters brain neurochemicals
- Animals fed low to moderate doses are able to recover from initial weight
losses, while higher doses induce more long-term changes in feeding
behaviour.
- Swine are more sensitive to DON than mice, poultry, and ruminants, with
males being more sensitive than females.
It seems likely that DON widely contaminates feed ingredients. For
example, wheat and barley from the 1993 crop year in the USA (630
samples collected in 25 states) were analysed for DON. The mycotoxin
contamination in the 483 wheat samples averaged 2.0 g/g and ranged
from < 0.5 to 18 g/g . DON contamination in the 147 barley samples
averaged 4.2 g/g and ranged from < 0.5 to 26 g/g. About 40% for the
wheat samples and 57% of the barley samples contained DON levels
that were greater than the U.S. Food and Drug Administration 1982
advisory level of 2 g/g for DON in wheat designated for milling (human
consumption; Trucksess et al., 1995). In the period September 1998January 2000, the average DON concentration in wheat was 0.446 g/g
(n= 219) in The Netherlands (Pieters et al., 2002).
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World-wide, the most economic losses are due to the aflatoxins, followed by
ochratoxin A. A summary of the main effects of mycotoxins on poultry are shown
in Table 6.1.
In general, lipid peroxidation and stimulation of apoptosis seemed to be
important mechanisms of mycotoxin action. The wide range of mycotoxins that
can contaminate poultry feed and their different chemical compositions make
protection against mycotoxin-related toxicity a difficult task. There are several
problems that complicate mycotoxin prevention issues:
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Table 6.1 Effects of common mycotoxins (adapted from Yaroshenko et al., 2003).
Mycotoxin
Effects on poultry
Aflatoxin B1
Ochratoxin A
Fumonisin B1
Zearalenone
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Decreased performance
Reduced egg production and hatchability
Decreased serum proteins
Increased liver and kidney weight
Liver and kidney lesions
Decreased semen volume and testes weights
Disruption of germinal epithelium
Decreased hatchability
Enlarged, fatty livers and enlarged spleens
Reduced weight gain and feed consumption
Impaired feed efficiency
Reduced egg production
Reduced egg quality
Induced immunosuppression
Kidney lesions
Rickets like deformities in chickens with leg weakness
Hepatocellular hyperplasia
Increased kidney and proventriculus weights
Liver lesions
Immunotoxicity
Reductions in feed consumption and weight gain
Severe oral lesions
Abnormal behaviour
Altered feathering
Decreased resistance to pathogens
Decreased egg production
Impaired shell quality
Decreased egg production
There are no safe doses of mycotoxins. A dose that does not affect animal
at short exposure could be toxic at longer consumption. Doses that may be
safe under laboratory conditions can have detrimental effects on growth
and reproduction under conditions of commercial poultry production.
International trade of feed ingredients, e.g. maize and soybeans, especially
long shipments from Latin America to European and Asian countries, is
another important risk factor.
Most of mycotoxins are stable compounds that do not degrade during
storage, milling or high-temperature feed manufacturing processes.
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Rats
Rats
Phospholipid vesicles
OA in vitro
OA in feed
OA in feed
OA in vitro
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OA in vitro
OA in feed
OA in feed
OA in vitro
Rats
Rat kidney microsomes
Rat liver
TBARS
A model oxidation system OH* single-strand
cleavage DNA
Astrocytes and neurones
TBARS , LDH release
Mouse and rat kidney
DNA adduct formation
OA in diet
OA in vitro
TBARS
OA in vitro
Ethane exhalation
MDA in serum,
liver, kidney
TBARS ,
oxygen uptake
Free radical generation
(EPR)
TBARS
Chicken liver
TBARS
References
SOD, catalase
aspartame
-
Vitamin E
Protective effect of
antioxidants
Lipid peroxidation
measurement
OA in feed
Tissue
Mycotoxin
326
Mycotoxin
Tissue
Lipid
Protective
peroxidation effect of
measurement antioxidants
References
T-2 in feed
Rat liver
TBARS
T-2 in feed
T-2 in feed
T-2 in feed
T-2 in feed
T-2 in feed
T-2 in feed
T-2 in feed
T-2 in feed
Se, ascorbic
acid, vitamin E
CoQ10 and
vitamin E
Lycopene
FB1 also stimulated lipid peroxidation in rat liver, rat liver nuclei fraction, primary
rat hepatocytes, Vero cells in culture and PC bilayers. In those systems TBARS
accumulation and DNA strand breaks were increased (Table 6.5). DON increased
TBARS formation in rat and mice liver and decreased GSH in rat brain and spleen.
There are also data available indicating pro-oxidant properties of zearalenone
(Karagezyan et al., 1995; Ghedira-Chekir et al., 1999) and citrinin (Ribeiro et al.,
1997). Aurofusarin is shown to decrease antioxidant defences and stimulate lipid
peroxidation in quail egg and tissues of newly hatched quail (Dvorska et al.,
2002; 2003; Dvorska and Surai, 2004).
It is clear from the data that mycotoxins strongly promote lipid peroxidation in
various in vitro and in vivo systems. This effect was obvious no matter which
measurement was used to assess the process of lipid peroxidation.
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Rat liver
Rat testis
Cultured rat hepatocytes
Rat liver
AFB1 in feed
AFB1 in feed
AFB1
AFB1 in diet
AFB1 in vitro
AFB1
intraperitoneally
Aflatoxin B1 in feed Rat liver
AFB1
Rat liver and kidney
intraperitoneally
AFB1
Rat liver
intraperitoneally
AFB1 in vitro
Primary rat hepatocytes
Rat liver
Rat liver
AFB1 in feed
AFB1 in feed
Tissue
Mycotoxin
Se, vitamin E
Picroliv
-
GSH-Px
TBARS
GSH, SOD, Catalase,
GSH-Px
TBARS , GSH ,
ROS generation
Vitamin E, ternatin
TBARS , ROS
formation
TBARS
MDA , NO
MDA
TBARS , LDH release
TBARS
Se , vitamin E
TBARS ,
conjugated dienes
MDA , NO
References
Protective effect of
antioxidants
Lipid peroxidation
measurement
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Phosphatidylcholine
bilayers
Vero cells
Glioma cells, mouse
fibroblasts
Macrophage cell line
Rat liver
FB1
FB1
FB1
FB1
FB1
FB1
DON + 3-AcDON
Mice liver
in feed
Aurofusarin in feed Quail egg yolk
FB1
DON in feed
Tissue
Mycotoxin
Vitamin E
-
TBARS
TBARS
TBARS
TBARS
TBARS
TBARS
Rate of peroxidation ,
free radical formation ,
accelaration of chain
formation
TBARS
TBARS ,
DNA fragmentation
MDA
TBARS
Catalase, mannitol
Protective effect of
antioxidants
Lipid peroxidation
measurement
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References
328
Selenium in Nutrition and Health
329
apoptosis (Sastre et al., 1996) since it is thought that cells activate an intrinsic
death program contributing to their own demise. Several processes, such as
initiation of death signals at the plasma membrane, expression of pro-apoptotic
oncoproteins, activation of death proteases, endonucleases etc., ultimately coalesce
to a common irreversible execution phase leading to cell demise. A balance
between cell death and cell survival factors plays a major role in the decision
making process as to whether a cell should die or must live (Ray et al., 2000).
Apoptosis is distinguishable from necrosis. When cell death is induced by
osmotic, physical or chemical damage, early disruption of external and internal
membranes takes place with subsequent liberation of denatured proteins into the
cellular space and induction of an inflammatory response in the vicinity of the
dying cell (Sastre et al., 1996). In contrast, apoptosis is characterised by cell
shrinkage, nuclear pyknosis, chromatin condensation, DNA cleavage into
fragments of regular sizes and activation of the cystein-proteases called caspases
(Dare et al., 2001). Reactive oxygen species are thought to play a major role in
apoptosis (Hockenbery et al., 1993; Ratan et al., 1994), being involved in the
initiation as well as execution of apoptosis (Herdener et al., 2000). GSH depletion
increases the percentage of apoptotic cells in a given population; and increased
GSH concentration is shown to decrease the percentage of apoptosis in fibroblasts
(Sastre et al., 1996). In fact, GSH depletion sensitised cells for intracellular
induction of apoptosis (Zucker and Bauer, 1997). Therefore, a decrease in GSH
concentration, or an increase in GSSG concentration or perhaps a change in the
ratio of GSH/GSSG constitutes a trigger for apoptosis (Beaver and Waring, 1995).
For example, ROS from mitochondria can cause apoptosis after GSH depletion
(Zucker et al., 1997). Therefore, apoptosis is induced by oxidative damage either
directly from oxygen free radicals or hydrogen peroxide or from their generation
in cells by injurious agents. For example, H2O2 is considered a common mediator
for the apoptosis induced by various anticancer drugs (Simizu et al., 1998). In
line with those findings there are data showing protective effects of catalase and
SOD from different inducers of apoptosis (Sandstrom and Buttke, 1993; Herdener
et al., 2000). Indeed, intracellular induction of apoptosis depends on ROS
production and can be efficiently blocked by antioxidants (Langer et al., 1996;
Schaefer et al., 1995).
In many cases mycotoxins decreased cellular level of GSH, which can trigger
apoptosis. In general, T-2 toxin is a most potent apoptotic agent. However, there
are also reports indicating apoptosis caused by FB1, OA and AFB1 (for review
see Surai, 2002; Surai and Dvorska, 2005). For example, based on the DNA
fragmentation profile in gel electrophoresis and the morphological changes in
electron microscopy, the induction of apoptotic nuclear changes by various
mycotoxins was investigated in HL-60 human promyelotic leukemia cells (Ueno
et al., 1995). The results showed that T-2 toxin, nivalenol, deoxynivalenol, OA,
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citrinin, AFB1 and some other mycotoxins were positive for the induction of
DNA fragmentation. However, fumonisin B1 was not active in DNA fragmentation.
The toxicity of the mycotoxins nivalenol (NIV), DON and FB1 were studied in
the K562 human erythroleukemia cell line (Minervini et al., 2004). Morphological
evidence of apoptosis was related to the toxicity of the mycotoxins and more
toxic NIV and DON resulted in more late stage apoptotic events than FB1. The
results suggested that DNA damage and apoptosis rather than plasma membrane
damage and necrosis may be responsible for the observed cytotoxicity. Fumonisin
B1, T-2 toxin, Fusarenon-X and Deoxynivalenol could induce apoptosis of liver
cell, kidney cell, gastrointestinal epithelial cell, immunological cells as well as
several cell lines of human and animals. Furthermore the rank order of the potency
of trichothecene mycotoxins to induce internucleosomal DNA fragmentation was
found to be T-2, satratoxin G, roridin A >> diacetoxyscirpenol > baccharin B-5 >>
nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, fusarenon-X, baccharin B-4
vehicle control (Nagase et al., 2001). The apoptosis-stimulating effect of FB1 has
been clearly demonstrated and stimulation of lipid peroxidation by FB1 and
decreased antioxidant concentrations including GSH in tissues could lead to
changes in redox status of the cell and trigger a cascade of apoptotic changes.
In general, apoptosis is considered a common mechanism of toxicity of various
mycotoxins. Since antioxidant-prooxidant balance in the cell (redox status) is
responsible for regulation of apoptosis it seems likely that selenoproteins such as
GSH-Px, TR and MSR could be potentially involved in prevention of mycotoxinrelated apioptosis. Therefore, Se status of the animals could be an important factor
in their resistance to mycotoxicoses.
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Species
Dose
Reference
Broiler
chicks
Broiler
chicks
1 ppm,
7-49 days
2.5 mg/kg
diet for 21
days
2.5 mg/kg
diet for 21
days
Broiler
chicks
Broiler
breeder
hens
0.2; 1; 5 or
10 mg/kg
Broiler
chicks
Broiler
chicks
5 ppm
Broiler
chickens
2.5 g/g of
feed for 42
days
2.5 g/g diet
from hatching
to 4 weeks of
age
Chickens
Chickens
400 ppb of
AFB1+AFB2
for 5 weeks
0.3 ppm
from 0 to 6
weeks
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Cell-mediated immunity
Giambrone et al., 1978
(graft-versus-host reaction) ;
DTH skin reactions to
tuberculin ; Humoral immunity
(AB to rabbit red blood) ;
serum IgG and IgA; IgM
Cell mediated immune
Kadian et al., 1988
response (delayed type hypersensitivity reaction) suppressed
at all the three periods tested
(30, 45 and 60 days of age).
Cell-mediated immunity
Giambrone et al., 1985
measured by a delayed hypersensitive skin test ; humoral
immunity; development of
the acquired immunity to ND
or fowl cholera vaccination
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Species
Dose
Reference
4-month-old
wild
turkeys
Chick
embryo
100-400g/kg
feed for 2 wk
Lymphoblast transformation
6-day chick
embryos
6-day chick
embryos
Weanling
piglets
Weaned pigs
Pigs
Female
lambs
White-tailed
deer
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1.09-17.4 g/g
Mitotic index B cells ;
Potchinsky and Bloom,
embryo at day 18 Mitotic index T cells;
1993
of incubation
sister chromatid exchanges in
B lymphocytes (up to 8-fold);
sister chromatid exchanges in
T lymphocytes (up to 2 fold)
0.1, 0.5, and
Macrophage recruited in the
Neldon-Ortiz and
1 g/per embryo peritoneal cavity after i.p.
Qureshi, 1992
Sephadex elicitation, substrate
adherence potential of peritoneal
exudate cells at 1 mg AFB1;
macrophage phagocytic
potential at 0.5-1.0 mg AFB1
0.1 g
Incidence of sister chromatid
Dietert et al., 1985
exchanges in blood cells
(5-fold); cell-mediated immunity
(graft vs host and cutaneous
basophil hypersensitivity
reactions)
140-280 ppb,
AF decreased proinflammatory Marin et al., 2002
4 weeks
(IL-1beta, TNF-alpha) and
increased anti-inflammatory
(IL-10) cytokine mRNA expression
140, or 280 ppb, Skin thickness (PHA skin test) van Heugten et al., 1994
for 3 weeks
linearly with increasing dietary
AB1
300 or 500 mg/ The humoral immune response Panangala et al., 1986
kg of feed
to Erysipelothrix rhusiopathiae,
measured by enzyme-linked
immunosorbent assay; the
proliferative responses of
lymphocytes to mitogens;
complement titers; serum
immunoglobulin G and M values
2 ppm for 37 d
Response to intradermal
Fernandez et al., 2000
injection of PHA
0.8 ppm for 8
Lymphocyte proliferation and Quist et al., 1997
weeks
delayed type hypersensitivity
reactions
333
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334
Dose
Weanling
rats
Reference
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Species
Dose
Reference
Broiler
chicks
2 ppm
Chicks
5 ppm
Broiler
chicks
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335
Species
Dose
Broiler
chicks
Up to 4 ppm for
20 days from
hatch
0.5-8g/g from
day-old to 3
weeks of age
Reference
Immunoglobulin-containing
Dwivedi and Burns, 1984
cells in lymphoid organs;
total Ig levels at 2-4 ppm
Broiler
Total circulating lymphocytes Chang et al., 1979
chickens
of blood ; monocytes at
2.0 g/g; circulating
heterophils
Salmonella- 3.0 mg/kg
PHA- and ConA-stimulated
Elissalde et al., 1994
challenged
blastogenesis. Salmonella
broiler
typhimurium alone had no
chicks
affect on the variables measured
Growing
2.5 mg/kg of
Cutaneous basophil hyperHarvey et al., 1992
gilts
feed for 35 days sensitivity response to PHA,
delayed hypersensitivity to
tuberculin, stimulation index
for lymphoblastogenesis,
interleukin-2 production when
lymphocytes were stimulated
with concanavalin A, number and
phagocytic activity of
macrophages
Rats
Gavage with 0,
Thickening of the basement
Dortant et al., 2001
0.07, 0.34 or
membrane and reduction in
1.68 mg OTA/kg splenic T-cell fraction. IgG at
body weight for 0.34-1.68 mg/kg OTA
4 weeks
Rats
Dams exposure The proliferative response of
Thuvander et al., 1996b
to a single dose splenocytes to the T-cell
of OA (0, 10, 50 mitogen Con A in pups from
or 250 g/kg
dams given 10 or 50 g OA/kg
BW) on day 11
body weight; proliferation of
of lactation
thymocytes in response to Con
A in pups from dams exposed
to 50 g OA/kg body weight
Mice
Dams, OA, 200
In offspring on days 14 and 28 Thuvander et al., 1996
g/kg before and postpartum percentages of
during gestation splenic CD4+ and CD8+ cells
Mice
Dams exposure Proliferation of splenic and
Thuvander et al., 1996a
to OA (500g/kg thymic lymphocytes in
BW) on day 16
response to mitogens in the
of gestation
pups at 15 days of age;
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Species
Dose
Mice (contd)
Mice
Mice
Mice
Reference
Species
Dose
White
Leghorn
chicks
Reference
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Species
Dose
Reference
Mice
Mice
Mice
Macrophage 1-10M
cell line
Murine
1-100 M
macrophage
cell line
Murine
1, 10, and
cell
100 M
macrophage
Rat splenic 1-100 g/ml
macrophages
and lymphocytes
LPS-induced
NO production
Dombrink-Kurtzman et
al.,. 2000
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Species
Dose
Reference
Young
poults
7-week-old
pigs
Up to 1 ppm
for 32 days
0.5, 1.0, 2.0 or
3.0 mg/kg diet
for 3 weeks
AB to antigens
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Species
Dose
Reference
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Species
Dose
Mice
4.0 mg/ kg of
exposed to BW
Listeria
infection
Mice
20 ppm for
1- 4 weeks
Human
1 -5 ng/ml
lymphocytes
Reference
Species
Dose
Growing
pigs
Young pigs
Mice
Mice
Mice
Mice
Weanling
mice
Weanling
mice
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Species
Dose
Murine
0.1 ng/mlperitoneal 1 g/ml
macrophages
Human
216 ng/ml
lymphocytes
Murine
25-100 ng/ml
macrophage
cell line
Reference
Phagocytosis, microbicidal
Ayral et al., 1992
activity; superoxide anion
production at 1 ng/ml; phagosome-lysosome fusion at
100 ng/nl
PHA-induced lymphocyte
Miller and Atkinson,
proliferation at 0.005- 0.5 ng 1986
DON/ml; at 50-100 ng/ml
lymphocyte proliferation
50% inhibition in cell
Meky et al., 2001
proliferation
Ji et al., 1998
Production of H2O2 after LPS
stimulation, but at 250 ng/m;
NO production at 25-250 ng/ml
unknown and continue to be a frontier for future research (Bondy and Pestka,
2000). It seems likely that trichothecenes are potent immunosuppressive agents
that directly affect immune cells and modify immune responses because of tissue
damage elsewhere. Immunomodulating properties of DON have been studied
mainly with rodents (Table 6.10). The evidence is quickly accumulating showing
that DON can be immunosuppressive or immunostimulatory, depending upon
the dose and duration of exposure. While immunosuppression is probably related
to the inhibition of translation, immunostimulation can be a result of interference
with various cellular regulatory mechanisms (Rotter et al., 1996).
Mycotoxin-induced immunosuppression is related to both natural and adaptive
immunity (Table 6.11):
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Table 6.11 Summary of effects of selected mycotoxins on the immune system (adapted from
Yaroshenko et al., 2003)
Mycotoxin
Aflatoxin B1
Ochratoxin A
Fumonisins
T-2 toxin
DON
In fact, high levels of polyunsaturated fatty acids in the immune cells and presence
of sensitive receptors on their surface make them an important target for free
radical attack (Surai, 2002). As mentioned above, oxidative stress caused by
mycotoxin consumption could be responsible for breaking effective
communications between immune cells which ultimately would misregulate
immune system and cause immunosuppression. Although the molecular basis for
many of the specific immunosuppressive effects of mycotoxins are presently
unclear, inhibition of DNA, RNA and protein synthesis via a variety of different
mechanisms appears to be directly or indirectly responsible for the immunosuppressive action of many mycotoxins (Corrier, 1991). Furthermore, detrimental
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against toxic effects of mycotoxins were observed. For example, Burguera et al.
(1983) indicated that selenium (Se) has a protective effect against AFB1 toxicity
in turkey poults. To investigate the biochemical mechanism of the protective effect
of dietary Se against aflatoxin toxicity, hepatic metabolism of AFB1 in turkey
poults was examined at various dietary Se concentrations (0.2, 2.0 or 4.0 ppm).
The experimental results provided clear evidence of Se-induced enhancement of
aflatoxin detoxification (Gregory and Edds, 1984). It was suggested that the
protective action of Se was not mediated by an increase in glutathione availability
for aflatoxin conjugation or by effects on the activities of these enzymes as
measured in vitro. Therefore dietary supplements such as Se are considered effective
in the reduction of aflatoxicosis in poultry (Dalvi, 1986). Recently the protective
antioxidant effect of organic Se in combination with vitamin E has been shown in
chicks exposed to cold stress and aflatoxin-contaminated feed (Stanley, 1998).
Similar protective effects of Se against aflatoxicosis have been shown with
mammalian species. Pigs fed a diet containing 2.5 mg Se/kg of feed were protected
from toxic effects of AFB1 (Davila et al., 1983). The effects were measured by
alteration of clinical responses and hematologic (prothrombin times),
electrophoretic and clinical chemistry values. Effects of a single intramuscular
injection of Se-vitamin E (5 mg of Se + 68 IU of -tocopherol/60 kg of BW) as a
pretreatment 14 days before an oral dose of AFB1 (1.0 mg/kg) were studied in 24
dairy calves. Although aflatoxin exposure caused a significant decrease in body
weight and feed intake, Se was demonstrated to interact significantly with AFB1
for feed intake, causing an improvement of this parameter (Brucato et al., 1986).
Milks et al. (1985) showed that Se is able to protect against the
hepatocarcinogenic effects of AFB1 in the rat. Moreover inhibitory activity of Se
on mutagenesis induced by AFB1 in the presence of a rat liver microsomal
activation system has been shown in Salmonella typhimurium tests (Francis et al.,
1988). The results of another experiment showed that Se could effectively protect
cells from AFB1 cytotoxicity in cultured cells but had no effect on aflatoxin B1DNA adduct formation or mutagenesis (Shi et al., 1995). In a different study, the
same authors reported that Se could effectively inhibit AFB1-induced DNA damage
(Shi et al., 1994).
It has been revealed that Se can inhibit the formation of hyperplastic foci and
enzyme-altered foci as well as hepatocarcinogenesis induced by AFB1, but Se
can neither prevent the enlargement nor accelerate the regression of the foci already
developed after administration of carcinogens (Wang, 1990). Therefore Lei et al.
(1990) concluded that Se had an inhibitory effect on the initiation and promotion
stages of AFB1-induced preneoplastic foci and nodules. Selenium also prevented
progression of these nodules to hepatocellular carcinoma even after cessation of
AFB1 administration. Additional experiments were conducted to verify the effect
of Se on the mutagenic activity of AFB1. After 14 days of Se administration to
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simultaneously with zeolite was significantly higher compared with birds fed the
diet containing T-2 toxin alone. Therefore, zeolites alone were not effective in
prevention of toxic effects of T-2 toxin. These data are in agreement with
observations of Kubena et al. (1990; 1998) indicating absence of protective effects
of aluminosilicate sorbents against T-2 toxicosis. Superactivated charcoal
(Edrington et al., 1997) and organic sorbents (Bailey et al., 1998) were also
ineffective against T-2 toxicosis. Therefore, zeolite probably was not able to bind
a substantial amount of T-2 toxin in the digestive tract; and as a result did not
interfere with pro-oxidant properties of this mycotoxin.
25
alpha-tocopherol
gamma-tocopherol
carotenoids
ascorbate
20
15
10
0
Control
T-2
T-2+Zeolite
T-2+Mycosorb
Figure 6.1 Effect of T-2 toxin on antioxidant concentrations in quail liver, g/g tissue (Adapted from
Dvorska and Surai, 2001).
In marked contrast, inclusion of yeast glucomannans (Mycosorb) in T-2 toxincontaining diets fed quail significantly slowed the depletion of natural antioxidants
and vitamin A in the liver (Dvorska and Surai, 2001). This protective effect can
be attributed to the high adsorbent capability that esterified glucomannans have
for T-2 (Dawson, 2001). It could well be that mycotoxin binding by Mycosorb
also prevents T-2 toxin participation in development of oxidative stress in the
intestine. As a result, damage to the enterocytes is prevented thereby maintaining
effective antioxidant absorption, assimilation and delivery to the target tissues.
Did you know that a combination of several mycotoxins in low
doses could have higher detrimental effects on animal health
than one mycotoxin in higher doses?
It seems likely that consumption of mycotoxins could compromise Se metabolism
in animals. Therefore, a combined usage of adsorbents and antioxidants would
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T-2 toxin
T-2+Mycosorb
T-2+Mycosorb
+ Sel-Plex
Figure 6.2 Effect T-2 toxin, Mycosorb and Sel-Plex on Se level in chicken liver, ng/g
160
140
120
100
80
60
40
20
0
Control
T-2 toxin
T-2+Mycosorb
T-2+Mycosorb
+ Sel-Plex
Figure 6.3 Effect T-2 toxin, Mycosorb and Sel-Plex on lipid peroxidation in chicken liver, (MDA, g/g)
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Conclusions
Recent results show that in many cases membrane-active properties of various
mycotoxins determine their toxicity. Indeed, incorporation of mycotoxins into
membrane structures causes various detrimental changes. These changes are associated
with alteration of fatty acid composition of the membrane structures and with
peroxidation of long chain PUFAs inside membranes. This ultimately damages
membrane receptors, causing alterations in second messenger systems; then to
inactivation of a range of membrane-binding enzymes responsible for regulation of
important pathways. Finally, this causes alterations in membrane permeability, flexibility
and other important characteristics determining membrane function. Detrimental effects
of mycotoxins on DNA, RNA and protein synthesis together with pro-apoptotic action
further compromise important metabolic pathways. Consequently, changes in
physiological functions including growth, development, reproduction etc. occur. An
importance of lipid peroxidation in all these processes is confirmed by protective
effects of natural antioxidants against mycotoxin toxicity. As a part of a range of
selenoproteins selenium is involved in prevention of lipid peroxidation, membrane
alteration and apoptosis and could decrease mycotoxin toxicity. However, protective
effects of antioxidants including selenium are of limited value and a combination of
mycotoxin binders with organic selenium could be the next step in preventing
damaging effects of mycotoxins in animal and poultry production.
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Miller, K. and Atkinson, H.A. (1986). The in vitro effects of trichothecenes on the
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7
SELENIUM IN POULTRY NUTRITION
Introduction
After long heated debates about eggs, cholesterol and human health it seems
likely that the storm has at last calmed and today we can say, An egg a day is
OK and start thinking about egg improvement. The last 20-40 years have been
associated with rapid progress in the poultry industry through out the world. Indeed
between 1961 and 2002, world chicken egg output increased more than threefold,
reaching 53.8 million tones and the output is predicted to grow further in the next
25-30 years. Similar achievements have been seen in poultry meat production
reaching 72.5 million tons in 2003. Indeed genetic progress, together with
substantial achievements in poultry nutrition, is responsible for those successes.
However increased productive parameters are often associated with a decrease of
reproductive characteristics and more importantly with increased susceptibility to
various stresses. Therefore an optimal balance of various antioxidants in the diet
is considered to be an important way forward in modern poultry nutrition. Among
many compounds with antioxidant activities found in poultry feed, selenium is
considered to be a chief executive of the antioxidant system regulating whole
antioxidant network in the body. Indeed, Se deficiency is related to decreased
productive and reproductive performances of poultry. Commercial poultry
production is associated with various stresses and Se as a part of various
selenoproteins can help maintaining antioxidant defences preventing damages to
various tissues. In accordance with NRC (1994) Se requirement is quite low,
however, those data are not related to commercial conditions and it seems likely
Se requirement for optimal poultry health is much higher. In fact,
immunomodulating properties of Se are shown with doses which are substantially
higher than those needed for chicken growth and development. For the last few
years a great body of information was accumulated indicating preferences of
organic Se in comparison to selenite or selenate. In fact, in poultry production
organic Se is proven to be an effective feed supplement to meet chicken requirement
in this element in various commercial conditions. Important features of Se
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metabolism and its practical applications in poultry will be presented in this chapter
with a special emphasis to optimal Se forms for the dietary supplementation.
Selenium deficiency
Se deficiency in the chicken, especially in combination with low vitamin E supply,
is responsible for the development of a range of diseases. Indeed, vitamin E and
selenium deficiency is related to many different conditions in farm and laboratory
animals where all major organs are affected (Table 2.20, Chapter 2).
In general selenium and selenium+vitamin E deficiency are associated with
development of a variety of pathological conditions, which target major body
systems including muscles, heart-vascular, reproductive and nervous systems.
Various animal species are shown to have different susceptibility to selenium
deficiency and different tissues show various degrees of severity upon a selenium
deficient diet. In general, some signs of selenium and Se+vitamin E deficiency
(nutritional muscle dystrophy) are similar in various species; others
(encephalomalacia) are species-specific.
Did you know that Se deficiency in poultry is associated with
decreased productive and reproductive performances and
development of various diseases targeting major body systems
including muscles, heart-vascular and nervous systems?
In many cases a combination of these two compounds is needed to achieve the
best effect in their treatment or prevention. In poultry Se- or Se + vitamin E
deficiencies are related to the development of:
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Reduced fertility. Fertility and hatchability were low on the basal (low Se)
diet and were corrected partly by vitamin E and completely by Se (Latshaw
and Osman, 1974). Details of effects of Se on male reproduction are
presented in Chapter 5.
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hyaline capillary thrombi affecting the cerebellar cortex and adjacent white
matter (Klein et al., 1994). NE in turkey poults leads to neurological signs
such as tremor, incoordination, and recumbency shortly after being moved
to new quarters. Associated lesions included ischemic necrosis of the
cerebellum and spinal cord (Jortner et al., 1985; Frank and Bergeland, 1988).
The disease usually occurs during the second or third week after hatching,
which may be related to the cerebellar PUFA accumulation that occurs at
that time (Budowski et al., 1987). In an experiment conducted by Fuhrmann
and Sallmann (1996) NE started at day 9. In other experiments of the same
authors (Sallmann et al., 1991; Fuhrmann et al., 1994) with similar type of
linoleic acid-rich diet and commercial chicks, the disease began after day
16 reflecting differences in the initial levels of vitamin E in the tissues of the
newly hatched chick. The lower the level of vitamin E in the incubation
eggs and chick tissues the earlier the disease appears. The effect of the
breeder diet on the severity of encephalopathy was observed in 4-week-old
chicks (Bartov and Bornstein, 1980).
As mentioned above, experimentally NE in chicks can be induced by diets
low in vitamin E and containing large amounts of linoleic acid (see Bartov
and Budowski, 1979). There are other nutritional and environmental factors
predisposing chickens to encephalomalacia, including deficiency in animal
protein and decreased temperature in hatcheries (Chen and Shi, 1985).
However, outbreaks of this disease were registered in commercial conditions
with diets containing sufficient levels of vitamin E and synthetic antioxidants
(Hislop and Whitte, 1967a,b; Marthedal, 1973).
It is well recognised that NE developed on the diet rich in linoleic acid, but
linolenic acid had a protective effect against this disease (Budowski and
Crawford, 1985). Therefore it has been postulated that n-6 fatty acids
especially arachidonic acid degradation products including pentane
(Fuhrmann and Sallmann, 1995) and hexanal (Hu et al., 1989) have a prooxidative toxic effect which could lead to encephalomalacia development
(Budowski et al., 1979; Fuhrmann and Sallmann, 1996). Indeed, an
overproduction of arachidonic acid-derived eicosanoids is considered as a
factor in the etiology of the cerebellar lesion and possibly a structural change
due to a loss of docosahexaenoic acid and gain of arachidonic acid
(Budowski et al., 1987).
Molecular mechanisms of NE are still not clear. In the vitamin E-deficient
cerebellum the cytosolic phospholipase A2 activity was increased (Fuhrmann
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dietary Se used, since organic Se is more efficiently deposited in the egg yolk
(Cantor, 1997; Paton et al., 2000; 2002).
Therefore, during egg incubation fat-soluble antioxidants are transferred to
the developing embryonic tissues mainly during the last week of incubation (Surai
et al., 1996; Surai, 1999). Only vitamin A transfer to the embryonic liver started
earlier than that of vitamin E or carotenoids. As for Se transfer, it is still not clear
from the present results at what stage of embryo development this trace element
is transferred to the embryonic tissues. It seems likely that Se from the albumin is
transferred to the embryo during first 2 weeks of the embryonic development,
while Se from egg yolk is delivered to the embryo during last week of incubation.
The form of selenium in the egg yolk and embryonic tissues needs further
investigation as well. It could well be that SeMet could represent a substantial
proportion of the total Se in the embryo when Se is supplied to laying hens in the
organic form. Water soluble antioxidant ascorbic acid started to be synthesised in
the yolk sac membrane (YSM) at early stages of incubation and its concentration
in this tissues progressively declined during embryonic development (Surai et
al., 1996; Surai et al., 1995). Reduced glutathione and antioxidant enzymes
glutathione peroxidase, superoxide dismutase and catalase are also expressed in
the embryonic tissues at various stages of their development (Surai, 1999a).
Did you know that selenoprotein synthesis in embryonic tissues
could be considered as major antioxidant defence strategy?
Our results indicate that there are tissue-specific features in antioxidant defence
strategy during embryonic development of the chicken. They can be summarised
as follows.
LIVER
At the time of hatching, liver phospholipids contain high levels of highly
polyunsaturated fatty acids, particularly arachidonic (20:4n-6) and
docosahexaenoic (22:6n-3) acids (Surai et al., 1999). It is clear that high levels of
PUFA require a considerable degree of antioxidant defence against peroxidation.
The three antioxidant enzyme activities in the liver changed in a different manner
during development. SOD activity increased between days 10 and 11 of
development, then significantly decreased in activity up to day 15 and remained
at the same level during the rest of the developmental period (Surai, 1999a).
Similarly there was no difference in SOD activity in the embryonic liver between
days 12 and 18 (Wilson et al., 1992). GSH-Px activity in the embryo liver increased
through the time of development: from 84.7 mU/mg protein on day 10 to 254.9
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mU/mg protein on day 22 (Surai, 2002) which is in agreement with Gaal et al.
(1995) and Wilson et al. (1992).
Comparison of GSH-Px activity in various tissues showed that the highest
activity was found in the embryo liver at all stages of development (Table 7.1). At
the early stages of embryo development (10-11 days) it was 1.2-1.3 times higher
compared to the yolk sac membrane and 2.5-2.6 times higher than that in the
brain. At day 17 of development it was higher by 4.0; 2.8; 2.0; 1.7; 1.3 and 1.2
times compared to the brain, skeletal muscle, heart, YSM, kidney and lung
respectively. In general, changes in GSH-Px activity patterns during the
development were very similar to those found for vitamin E, carotenoids (Surai et
al., 1996) and vitamin A (Gaal et al., 1995). Probably GSH-Px plays a primary
role in antioxidant defence of the liver by effectively removing hydrogen peroxide
and lipid hydroperoxides from cells whereas catalase may be less important,
decreasing throughout the development. Nevertheless, the concentration of GSH,
a primary cell water-soluble antioxidant, gradually decreases throughout the
development.
Table 7.1 GSH-Px activity in the chick embryo tissues, mU/mg protein (Adapted from Surai, 1999a)
10
Liver
84.7
Brain
34.4
YSM
70.1
Kidney
Lung
Heart
Leg Muscles
11
13
106.9
40.2
83.6
162.7
41.4
126.2
206.6
47.3
153.0
131.4
200.7
109.3
86.0
211.3
53.3
124.4
158.5
179.4
105.6
74.9
226.8
55.2
123.2
173.2
148.4
106.8
68.9
21
22
229.3
48.9
105.2
207.8
206.6
126.1
64.1
254.9
42.0
100.4
205.8
210.7
114.1
60.2
CAT had two peaks of activity at day 10 of embryonic development and in day
old chicks (Surai, 2002). After hatching the liver displayed CAT activity similar to
that in the kidney but much higher compared to other tissues. In rat postnatal
development CAT activity significantly decreased with ageing (Matsuo, 1993).
Did you know that a cooperative action of selenoproteins with
vitamin E provides an effective antioxidant defence in the
developing chicken liver?
An accumulation of natural antioxidants vitamins A, E and carotenoids,
comparatively high vitamin C concentration and an increased GSH-Px specific
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BRAIN
It may be envisaged that the brain of the chick embryo may be at particular risk
from peroxidative damage due to presence of very high levels of C20 and C22
polyunsaturated fatty acids in neuronal phospholipids (Surai et al., 1999). The
antioxidant system of the developing brain is poorly understood. It includes
natural antioxidants such as vitamins E and C, ubiquinols, glutathione, antioxidant
enzymes - superoxide dismutase, glutathione peroxidase and catalase and other
elements such as dopamine, noradrenaline, taurine, carnosine (Gaal et al., 1995,
Surai et al., 1996; Matsuo, 1993). The chick embryonic brain is characterised by
comparatively low level of vitamin E which is almost 100 fold lower compared to
the liver and remains practically constant during the development (Surai et al.,
1996) and this apparent deficiency in the brains antioxidant defence capacity is
further compounded by the relatively low levels of vitamin A, selenium, glutathione
peroxidase, and carotenoids (Speake et al., 1996). Our previous results (Surai et
al., 1993) also indicate that comparatively low levels of vitamin E persist in the
brain of adult chicken. Thus it is a characteristic of poultry brain to contain low
levels of vitamin E. At the same time, the brain generates especially high levels of
free radicals (Reiter, 1995) which are necessary for physiological responses
(Westermarck et al., 1993).
Did you know that during embryonic development and in
early postnatal development chicken brain is characterised by
low antioxidant defences and high levels of polyunsaturated
fatty acids?
Our data (Surai, 1999a) indicate that SOD is the main enzyme in antioxidant
defence of the brain, especially during the last days of incubation when SOD
activity in the brain was significantly higher compared to other tissues. However,
GSH-Px and CAT activities in the brain were low and it is unlikely that they are
the primary defence in this tissue.
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The antioxidant profile of the chicken brain is shown in Table 7.2. The
cerebellum had some distinctive features in antioxidant concentration compared
to the other brain regions, in particular the cerebrum. The cerebellum was
characterised by significantly increased alpha-tocopherol concentration and
catalase activity, but decreased ascorbic acid level and Mn-SOD activity compared
to that in the cerebrum. In general, the brain was characterised by low level of
vitamin E, low GSH-Px and catalase activity, but very high level of ascorbic acid
and high SOD activity. Furthermore, the brain was characterised by comparatively
low levels of Se (0.1870.01 g/g) and moderate levels of Zn (11.670.23 g/g)
and Cu (1.180.06 g/g) (Surai, 2002).
Table 7.2 Antioxidant composition of the brain of a newly hatched chick (Adapted from Surai et al.,
1999)
Antioxidants
-tocopherol, g/g tissue
Ascorbic acid, g/g tissue
Glutathione, nM/mg protein
Mn-SOD, U/mg protein
Cu-Zn-SOD, U/mg protein
Se-dependent GSH-Px,
mU/mg protein
Se-independent GSH-Px,
mU/mg protein
Catalase, U/ mg protein
Cerebrum
Cerebellum
Brain stem
Optic lobes
5.22
889.4
41.12
3.66
6.910
7.12
711.3
38.12
2.837
6.985
6.12
747.72
40.13
3.91
7.52
5.66
850.53
39.56
3.02
8.03
29.83
28.61
33.14
32.6
6.22
1.923
6.60
2.365
5.57
1.966
7.31
1.822
Although the basal levels of MDA in the brain were low (Surai et al., 1996), the
susceptibility of the brain homogenates to lipid peroxidation during incubation in
both the absence and the presence of Fe+ was very high in comparison with other
tissues. Thus the low levels of endogenous vitamin E in the chick embryo brain
may render the tissue homogenates highly susceptible to in vitro peroxidation
(Kornbrust and Mavis, 1980). In embryonic brain antioxidant defence is afforded
by high levels of ascorbic acid to effect the recycling of the low concentrations of
vitamin E in order to maintain physiological requirements (Surai et al., 1996).
From our results (Surai, 1999a) it is clear that SOD may be another important
protective element in the embryonic brain.
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LUNG
The lung of the chick is rich in phospholipids and there is a specific accumulation
of phospholipids at about the eighteenth day. The accumulation of
phosphatidylcholine is associated with the synthesis of the desaturated component,
dipalmitoyl phosphatidylcholine (Noble and Cocchi, 1990). At day 18 of the
development the lung contains a high level of iron (Richards et al., 1991). In the
lung GSH-Px activity decreased on day 19 and again increased just before hatching
but glutathione levels were on the same level during the development. In the
embryonic lung SOD activity significantly decreased from day 15 of the
development up to posthatching time. There was a dramatic decrease in CAT
activity between days 15 and 19. At hatching time the activity of this enzyme in
the lung increased again. The increased GSH-Px and CAT activities at hatching
time reflect the adaptation to the increased oxygen consumption and probably
free radical production.
Did you know that there are tissue-specific features in
antioxidant defences in the chicken embryo?
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At hatching time GSH-Px activity in the lung (206.6110.81) was similar to that
in the liver (229.3312.7) and kidney (207.8112.05) but significantly higher
compared to that of the heart, muscle or brain. GSH concentration in the lung was
half that of the liver or kidney and similar to that of the muscle (Surai, 2002). The
lungs are characterised by moderate levels of vitamin E and low levels of
carotenoids (Surai et al., 1996). In this respect GSH-Px and CAT probably play
an important role in the antioxidant defence of the tissue.
In general, comparatively low SOD activity, moderate levels of vitamin E and
carotenoids (Surai et al., 1996), together with high iron concentration (Richards
et al., 1991) and exposure to the oxygen at hatching time place the lung in a risk
situation associated with possible free radical formation, lipid peroxidation and
tissue injury. The importance of antioxidant/prooxidant balance in the lung is
exemplified by its involvement in the development of Pulmonary Hypertension
Syndrome in the chicken (Bottje and Wideman, 1995) and attenuation of PHS
mortality was achieved by vitamin E implanting to birds (Bottje et al., 1995).
HEART
The importance of the heart-vascular system for the posthatch development
determines the strategy of antioxidant defence. Phospholipids and triglycerides
of the heart contain high levels of docosahexaenoic (22:6n-3) and arachidonic
(20:4n-6) fatty acids (Surai et al., 1999; Noble and Cocchi, 1990). At day 18
heart contains a comparatively high level of iron (Richards et al., 1991). In the
heart the GSH-Px activity was half that of the liver or lung and comparatively
stable during embryo development with a slight increase before hatching. The
embryonic heart was characterised by a high SOD activity which significantly
decreased from day 15 of the development up to posthatching time but remains
higher than that in the liver, YSM or lung. In the heart CAT activity was low and
didnt change during the development. The embryonic heart is characterised by
moderate levels of vitamin E and low levels of carotenoids (Surai et al., 1996).
High proportions of arachidonic and docosahexaenoic acids in the heart
phospholipids are associated with moderate antioxidant protection and in stress
conditions the heart will be vulnerable to oxidative damage. For example, the
embryonic heart was characterised by comparatively high susceptibility to Leadinduced lipid peroxidation in vivo (Somashekaraiah et al., 1992).
KIDNEY
In the kidney GSH-Px activity increased during development in a very similar
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way compared to that of the liver reaching its maximum at hatching time. It was
comparable with GSH-Px activity in the liver and lung and higher than that in
other organs studied. GSH concentration significantly increased at hatching time
as well. On the other hand, SOD activity significantly and constantly decreased
during development. Nevertheless it was comparatively high and similar to that
in the brain, lung and heart and higher compared to the liver, muscles and YSM.
During development CAT activity was comparatively high and stable with
significant increase at hatching time.
Vitamin E accumulation in the embryonic kidney took place in the same manner
as in the liver (Surai et al., 1996). In the kidney the ascorbic acid levels were
comparatively high and didnt change during the development up to hatching
when there was a decrease in vitamin C concentration in this tissue (Surai et al.,
1996). Thus the antioxidant system of the embryonic kidney includes vitamins A,
E and C and comparatively high activities of antioxidant enzymes and probably
has an adequate protective potential at hatching to deal with potentially harmful
metabolic end products as a result of their functional activity.
SKELETAL MUSCLE
At day 16 of incubation, muscles contain 2.4% of total lipids. The major lipid
fractions of skeletal muscle of the embryo are phospholipid, triglycerides and
free cholesterol. Phosphatidylcholine and phosphatidylethanolamine, high in C20
and C22 PUFAs, are by far the major components of the phospholipid fraction
(Surai et al., 1999; Noble and Cocchi, 1990).
In the thigh muscle the activity of GSH-Px decreased on day 17 and then
remained on that level up to the end of the development. There were no significant
changes in the SOD activity throughout the development. In muscle a decrease
in CAT activity lasted from day 15 up to hatching time. Skeletal muscle is
characterised by a comparatively low and stable vitamin E concentration. On the
contrary, the ascorbic acid level significantly decreased during development (Surai
et al., 1996). Thus the antioxidant system of the muscle is not very potent and
imbalance of the natural antioxidants in postnatal development may cause such
diseases as muscle degeneration (Machlin and Shalkop, 1956).
The chicken tissues displayed a considerable variation in the Mn-SOD activity
with the heart having the highest value and lung the lowest. By contrast, the lung
was characterised by high Cu,Zn-SOD activity; in the heart activity of Cu,ZnSOD was comparable to the other tissues. Based on the total SOD activity the
tissues could be placed in the following descending order:
heart>muscle>YSM>kidney>lung>liver. As can be seen the tissues differed
markedly in the GSH-Px activities. In all the tissues Se-dependent GSH-Px was
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the main enzymic form, comprising from 65% (lung) up to 90% (heart) of the
total enzyme activity. The liver and kidney displayed the highest total GSH-Px
activity and the muscle the lowest. As in the case of GSH-Px, catalase activity was
also maximal in the liver and kidney. The liver contained the highest concentrations
of Zn, Cu and Mn. The kidney was also characterised by comparatively high
levels of the metals. The liver and kidney also contained high concentrations of
Se. Comparison between the tissues (excluding YSM) revealed a highly significant
positive correlation between the concentration of Se and the activity of Sedependent GSH-Px (r=0.95). There was also a positive correlation (r=0.93) between
Fe content and catalase activity in all the tissues other than YSM (Surai, 2002).
Antioxidant enzymes are a major cell defence against acute oxygen toxicity
(Harris, 1992) and they are responsible for the detoxification of reactive oxygen
species and preventing lipid peroxidation (Gille and Sigler, 1995). The expression
of their activities is regulated at the gene level (Tsan, 1997; Weiss et al., 1997)
with tissue-specific features (Bermano et al., 1995). They are considered as
important embryoprotective enzymes (Ozolins et al., 1996; Fantel, 1996) during
organogenesis when increased exposure to oxidant-derived free radicals or
inadequate systems for antioxidant defence could alter cellular response at critical
points in development (Fantel, 1996; Allen and Venkatraj, 1992).
The results of our studies (Surai, 1999a; Surai et al., 1999) indicate that different
tissues of the embryo display distinct development strategies with regard to the
acquisition of antioxidant capacity. A low oxygen pressure in the environment
surrounding embryos during development seems to have been retained in the
course of evolution to protect the vulnerable developing tissues from the damage
caused by the action of reactive oxygen species (Ar and Mover, 1994) as far as
the rate of free radical generation in cells is influenced by ambient oxygen
concentration (Turrens et al., 1982). The reactivity of oxygen radicals and their
derivatives implicate them as possible effectors of oxygen-mediated changes in
gene expression (Allen and Venkatraj, 1992).
Did you know that antioxidant enzymes, including a range of
selenoproteins, are considered a major cell defence against
oxygen toxicity?
Embryonic gene expression of antioxidant enzymes develops in proportion to
the degree of exposure to the reactive oxygen species in their environment and in
preparation for the oxygen-rich environment after birth (Ar and Mover, 1994).
Exposure of rat embryos with an immature antioxidant system to a high
concentration of oxygen (20%) during early neurulation significantly increased
the incidence of neural tube defects compared with control embryos exposed to a
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-tocopherol (g/g)
600
Chicken
Turkey
Duck
Goose
500
400
300
200
100
0
10.0
16/20/20/22
21/28/28/30
10
Figure 7.1 Vitamin E transfer from yolk to embryonic liver (Adapted from Surai, 1999).
The samples were collected in approximately equal stages of embryo development for each species. For
example day 20, 26, 26 and 28 for chicken, turkey, duck and goose respectively.
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supplementation of the maternal diet on their transfer to the egg yolk and their
concentrations in the tissues of the newly hatched chick was studied and the
effect of increased Se and vitamin E supply on the activity of Se-GSH-Px in the
chick liver in early postnatal development was determined (Surai, 2000). In this
experiment 90 Cobb broiler breeder hens were divided into nine equal groups
and housed in pens at 25 weeks of age. Each hen received one of the treatment
diets (Table 7.3). Selenium was supplemented in the form of selenium-enriched
yeast (Sel-Plex, Alltech, Inc). After six weeks, the hens were artificially inseminated
once a week. From week 8 eggs were collected and placed in an incubator.
Liver, yolk-sac membrane, brain and blood plasma were collected from chicks
for biochemical analysis.
Table 7.3 Selenium and vitamin E supplementation of the basal dietsa (Adapted from Surai, 2000)
Dietary Group
1
2
3
4
5
6
7
8
9
Diet
Vitamin E (mg/kg))
Semi-synthetic
Commercial (CD)
CD
CD
CD
CD
CD
CD
CD
No
No
No
No
40
100
200
40
100
Se (mg/kg)
No
No
0.2
0.4
No
No
No
0.2
0.4
The level of selenium in the semi-synthetic diet was 44 g/kg and in commercial diet 171
g/kg. Selenium was supplemented in the form of Sel-Plex. Semi-synthetic and commercial
diets contained 4.86 and 10.05 mg/kg -tocopherol. Both, commercial and semi-synthetic
diets were balanced in other nutrients.
Our results (Figure 7.2; Surai, 2000a; Surai and Dvorska, 2001) indicated that the
inclusion of organic Se into the commercial diet significantly increased the Se
concentration in the egg yolk and the albumin. The correlation between dietary
Se and egg yolk Se content was found to be very high. This relationship was
quantified through a regression equation:
y = 179.4 + 1.01x (r2=0.96, P<001),
where y is Se concentration in the egg yolk, ppb; and x is the Se concentration in
the feed, ppb).
Similarly, a significant correlation between Se in the egg white and feed was
found and an equation describing Se concentration in the egg white (y) is as
follows:
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White
1200
1000
800
600
400
200
0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Se in diet (mg/kg)
Figure 7.2 Effect of selenium supplementation on its concentration in egg (Adapted from Surai and
Dvorska, 2001).
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Diet
Basal diet *(B)
B+0.1 Selenite
B+0.2 Selenite
B+0.3 Selenite
B+0.1 Sel-Plex
B+0.2 Sel-Plex
B+0.3 Sel-Plex
Egg, g
2.6
6.6
7.1
6.9
7.1
8.7
11.2
0.10
0.33
0.37
0.38
0.32
0.42
0.48
0.04
0.07
0.07
0.07
0.08
0.13
0.15
Tissue
0.1 Se
Yolk
Albumen
Breast
Liver
183
40
58
199
0.1 Se + FO
194
42
79
282
0.5 Se
0.5 Se + FO
505
209
235
684
496
205
217
698
*/ The diet contained 0.1 or 0.5 ppm organic selenium and also supplemented with fish oil
(FO)
Did you know that main form of selenium in the egg is SeMet
and this can explain why organic selenium is much
more effective than selenite in transferring to the egg?
Results of our experiments indicate that the Se concentration in egg yolk was
significantly higher than that in the albumen in all groups. However, it is interesting
to note that the albumen response to Se supplementation was higher in comparison
to egg yolk. For example, when chickens were fed a semi-synthetic diet, egg yolk
Se content was almost 5-fold higher in comparison to the albumen. When organic
Se was included in the diet at 0.4 ppm, this difference was reduced to 2-fold.
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Similar results were obtained in experiments with quail. For example, two groups
of quail (3 families in each group consisting of 4 females and 1 male) were formed
at the beginning of the reproductive period. They were fed a commercial maizebased diet containing 0.1 ppm feed-derived Se, supplemented with 0.2 ppm selenite
Se (control group) or 0.5 ppm organic selenium (Sel-PlexTM) for 6 months after
which eggs were analyzed and incubated in standard conditions (Karadas et al.,
2004). Selenium concentration in egg yolk and egg white significantly increased
as a result of organic selenium supplementation (Figure 7.3). The highest increase
(8.8-fold) was observed in egg albumin, while Se concentration in egg yolk
increased only 2-fold.
900
Control
800
Sel-Plex
700
600
500
400
300
200
100
0
White
Yolk
Shell
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diet the Se concentration in the liver and breast muscle of the newly hatched
chicks increased by 3.6 and 4.1-fold respectively.
1800
Liver
1600
Brain
Selenium (ng/g)
1400
1200
1000
800
600
400
200
0
1
Dietary groups
Figure 7.4 Selenium concentration in the liver and brain of day old chicks (Adapted from Surai, 2000)
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Table 7.6 Effect of Se in the maternal diet on the Se concentration in quail tissues, ng/g (Adapted from
Karadas et al., 2004).
Group
Liver
Breast
Leg
Brain
Control
Experimental
253.8
885.4
136.3
238.5
Control
Experimental
167.8
326.3
151.9
250.6
Control
Experimental
79.0
149.7
14 day-old quail
37.3
34.4
53.6
52.5
105.5
152.8
*/ Basic diet contained 0.096 ppm Se. Control diet was supplemented with 0.2 ppm Se in the form of
sodium selenite and experimental diet was supplemented with 0.5 ppm Se in the form of Sel-Plex
Group
Liver
Control
Experimental
180
717
Control
Experimental
215
300
Control
Experimental
225
280
Brain
1 day-old chick
125
210
7 day-old chick
124
154
14 day-old chick
126
145
*/ Control chicks were hatched from laying hens fed the diet containing 0.1 ppm selenium and
experimental chicks were hatched from laying hens fed on 0.5 ppm Se. Both control and experimental
chicks were fed diets containing 0.2 ppm Se
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Recently a new experiment was conducted at SAC to address this question (Surai,
Karadas and Pappas, unpublished). Maternal diet was supplemented with 0.4 ppm
organic selenium in the form of Sel-Plex and comparison was made with a basic
diet containing 0.1 ppm feed-derived selenium. As a result of dietary Se
supplementation the Se concentration in the egg yolk, albumin, shell, shell
membrane and perivitelline membrane was significantly increased (Karadas et
al., 2005). The newly hatched chicks were placed on basal diet (0.1 ppm) without
Se supplementation for the next 4 weeks posthatch. After hatching, chickens fed
diets low in Se (0.1 ppm) but originating from parents fed diets high in Se (0.5
ppm) had, up to 4 weeks post-hatch, significantly higher blood Se levels than
those originated from parents fed diets low in Se (0.1 ppm; Pappas et al., 2005a).
Furthermore, our results indicate that maternal diet affected selenium concentration
in the liver up to 3 weeks posthatch and in the breast muscle and up to 4 weeks
posthatch. GSH-Px activity in the liver and muscles was also elevated. Taking
into account recent data presented by Koutsos et al. (2003) showing similar effect
of carotenoids in the maternal diet on the carotenoid concentration in 4-week-old
chickens, it is possible to suggest that Se and probably carotenoids could affect
gene expression during the embryonic development. As a result, Se/carotenoid
assimilation in postnatal development could be affected. Alternatively, antioxidant
system could be affected and less Se/carotenoids being used for metabolic needs
and higher concentrations of these compounds were observed in tissues. Indeed,
this hypothesis needs further clarification, however, it is clear that maternal effect
is seen beyond newly hatched chicks.
Did you know that increased Se concentration in the egg is
responsible for the increased Se levels in tissues of chickens
up to 4 weeks posthatch?
Vitamin E accumulation in the egg yolk reflected its level in the breeder diet and
varied with Se supplementation (Figure 7.5). Dietary organic Se significantly
increased vitamin E level in the yolk, but a combination of selenium and increased
vitamin E supplementation did not further increase vitamin E accumulation in the
egg yolk. Similarly, inclusion of Sel-Plex in fish oil-supplemented diet was
associated with a significant increase in vitamin E concentration in the egg yolk
(Narahari et al., 2004). Vitamin E in the liver (Figure 7.6), yolk sac membrane
(Figure 7.5), plasma and brain of day old chicks also reflected vitamin E levels in
egg yolk. Again there was a positive effect of selenium supplementation of the
maternal diet on the levels of vitamin E in the liver, brain and blood plasma of day
old chicks. Over the first 10 days of age, liver vitamin E rapidly decreased (Figure
7.6). A positive effect of Se and vitamin E supplementation of the maternal diet
was seen at day 5 and day 10 of age when vitamin E concentrations in the liver
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and plasma were significantly elevated compared with those of the control group
(Surai, 2000b).
2500
Egg yolk
YSM
-tocopherol (g/g)
2000
1500
1000
500
0
1
Dietary groups
Figure 7.5 Vitamin E concentration in the egg yolk and yolk sac membrane of day old chicks (Adapted
from Surai, 2000)
1800
Day old
5 day old
10 day old
1600
-tocopherol (g/g)
1400
1200
1000
800
600
400
200
0
1
Dietary groups
Figure 7.6 Vitamin E concentration in the chick liver (Adapted from Surai, 2000)
This study showed that one of the important features of chick postnatal
development is the depletion of vitamin E in the liver. Selenium supplementation
of the maternal diet increased the vitamin E levels in the liver and plasma of day
old chicks; and this difference was maintained through 10 days of postnatal
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GSH (g/g)
1000
5 day old
800
600
400
200
0
1
Dietary groups
Figure 7.7 Effect of maternal diet on reduced glutathione concentration of chick liver (Adapted from
Surai, 2000)
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45
40
35
30
25
20
15
10
5
0
1
Dietary groups
Figure 7.8 Glutathione peroxidase (Se-GSH-Px) activity in the chick liver (Adapted from Surai, 2000)
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chick, Se-dependent GSH-Px is the major form of the enzyme comprising about
61% of total activity (Surai et al., 1999). In the majority of the tissues of the newly
hatched chick there was a highly significant correlation between Se level and the
activity of Se-GSH-Px (Surai et al., 1999). It is interesting to note that in chicken
liver about 28% of GSH-Px activity is represented by the monomeric form of the
enzyme (Miyazaki and Motoi, 1992). It has been suggested that the effect of Se
on the activity of GSH-Px is achieved through pretranslational mechanisms,
including Se-GSH-Px gene expression and cytosolic mRNA stabilisation
(Christinsen and Burgner, 1992). Further, dietary Se can also regulate the level of
GSH-Px mRNA in the post-transcriptional step (Toyoda et al., 1990). Therefore
GSH-Px mRNA is a primary target of the Se regulatory mechanism (Weiss et al.,
1997).
Tissue susceptibility to peroxidation significantly decreased between days 1
and 5 of age (Figure 7.9). MDA accumulation in the liver of day-old and 5-day
old chicks from antioxidant-supplemented hens was significantly reduced.
Therefore, liver susceptibility to lipid peroxidation substantially decreased in
postnatal development despite decreasing vitamin E and carotenoid concentrations.
30
Day old
5 day old
25
MDA (g/g)
10 day old
20
15
10
5
0
1
Dietary groups
Figure 7.9 Malondialdehyde (MDA) accumulation in the chick liver (Adapted from Surai, 2000)
This can be explained as a result of increased glutathione (Figure 7.7) and GSHPx activity (Figure 7.8) as well as of lipid composition changes (Noble and Cocchi,
1990). In fact, MDA accumulation in the chick liver in groups 3-6 was similar
and significantly lower than the controls (commercial diet). This means that
antioxidant protection afforded by increased GSH-Px activity is equal to dietary
inclusion of 40-100 mg/kg vitamin E. Similarly, in another experiment inclusion
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of 0.5 ppm Se or its combination with vitamin E for five weeks increased activities
of GSH-Px and SOD and decreased MDA in tissues, confirming the antioxidant
protective effects of Se (Huang et al., 1998). An inverse linear correlation was
found between lipid peroxide concentration and Se-GSH-Px activities in various
tissues of rats (Gromadzinska et al., 1988).
Did you know that increased Se concentration and activity of
GSH-Px in the embryonic tissues are associated with
decreased tissue susceptibility to lipid peroxidation?
The benefit of organic selenium use in breeder diets lies in its efficient absorption,
transport and accumulation in egg and embryonic tissues. This results in improved
antioxidant status of the newly hatched chick. As the levels of major natural
antioxidants (vitamin E and carotenoids) in chicken tissues progressively declined
after hatch, the antioxidant enzymes become a critical arm of antioxidant defence.
Therefore, enhanced GSH-Px activity in tissues as a result of organic Se
supplementation of the maternal diet may have a positive impact on chick viability
in the first few weeks post-hatch. An improved antioxidant system of the chick
may also enhance immune system function, which is extremely important at this
point in physiological development. The sparing effect of organic Se on vitamin
E also presents the possibility of further improvement of antioxidant defence of
the chick. Indeed, the source and level of Se has a large influence on the amount
of Se transferred to the developing embryo. It would also appear that the embryo
absorbs greater amounts of Se during days 10 to 15 of incubation than during
other periods. Improving the transfer of Se from the hens diet by using a Se-yeast
instead of inorganic sodium selenite is a useful strategy to improve the nutritional
status of the embryo as well as that of the newly hatched chick (Cantor et al.,
2003).
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is developing (Siddons, 1969) and by 4 days after hatch the ability to digest
starch can reach 85% complete (Noy and Sklan, 1995). In contrast pancreatic
lipase activity increases until 16 days after hatching (Vieira and Moran, 1999).
Ontogenic changes after hatching include increased levels of pancreatic and
intestinal enzymes, increases in gut surface area for absorption and changes in
nutrient transporters (Dibner, 2000). The intestine reaches a maximum growth
between 3 and 7 days posthatch (Murakami et al., 1992). In addition, early access
to food stimulates the growth of the intestine and its absorptive capacity (Moran,
1985). On the other hand, a delay in access to food and absence of stimulation
from food intake causes shortening and thinning of the villi (Michael and Hodges,
1973; Vieira and Moran, 1999) which would result in decreasing absorptive
efficiency of the intestine. Indeed, under commercial conditions, chicks may be
delayed access to feed for a considerable time after hatch and this increases the
likelihood of ketosis and dehydration (Vieira and Moran, 1999). The delays
between emergence and access to food and water resulted from the time spent in
the hatchery and transportation to the farm and could be considered as substantial
stress conditions. Chicks hatching early may be held 36 hours longer than those
hatching late. Because of the asynchrony of chick emergence eggs from older
breeders and smaller eggs tend to hatch earlier (Vieira and Moran, 1999). This
could result in dehydration and a shortage of available energy and lead to
subsequent reduction in the rate of nutrient absorption, growth rate and increase
early mortality. Immune system development is also compromised due to this
stress (Wyatt et al., 1986; Casteel et al., 1994).
Did you know that improved Se status of the chicks in early
postnatal development could improve chicken immunity and
the development of important physiological functions?
The delay in food and water intake is associated with a delay in the maturation of
the enzymatic systems that control metabolism (Decuypere et al., 2001), therefore,
holding chickens without feed (in commercial conditions many birds would have
an access to feed only 36-48 hours after hatching) leads to decreased body weight
and a decrease in the performance of the broiler (Noy and Sklan, 1999; Decuypere
et al., 2001). It is necessary to underline that any birds with restricted nutrition
early after hatching are not able to recover completely and do not reach the same
weight gain as those that are fed early (Vieira and Moran, 1999). Therefore, delayed
availability of feed and water is associated with an increased early mortality, lower
placement weight, poor four week and long term performance (Dibner, 2000). In
addition, these stress conditions put antioxidant defences under the pressure and
optimal dietary antioxidant supplementation of the maternal diet can help to
maintain high chicken viability.
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It is generally accepted that the ability to absorb dietary lipids is not well
developed in the newly hatched chick but improves with age and it is recommended
to avoid long chain saturated fatty acids and to use unsaturated fat during at least
the first week posthatch (Vieira and Moran, 1999; Noy and Sklan, 1995). It is
obvious that vitamin E is poorly assimilated from the diet during this period (at
least during first 5 days posthatch) when it is extremely important as a natural
antioxidant and as a result the chicken actively use tocopherol reserves accumulated
in the liver during embryonic development. At this period of the development
selenoproteins seem to provide essential antioxidant defences.
In the newly hatched chick, the immune system is immature and it is not
completely functional. At the time when a chicken is placed in poultry house it is
poorly protected from various infections, since its immune system is based mainly
on maternal antibodies (IgG and IgA) transferred from breeders via the egg. This
is a major immunological protection in the first week of chicken life. However,
the immune system is actively developing in postnatal life and this process involves
accumulation of PUFAs and increased susceptibility to lipid peroxidation. As a
result, natural antioxidants are considered to be absolutely essential for the
successful development of chicken immunity.
The first week of chicken life is a period of rapid expansion of leukocyte
populations, seeding of lymphoid organs, and educational events forming the
unique clones of lymphocytes mediating immunity at later stages of the growth
and development (Klasing, 1998). For example, the heterophils (the avian
counterpart to the mammalian neutrophil) of chickens are functionally immature
for the first 4-5 days posthatch and day old chicks are not able to activate them
(Kogut et al., 1998). Peripheral blood counts demonstrated no differences in the
percentages of heterophils during the first week post-hatch and the phagocytic
index of the heterophils did not change on day 1 or day 4, but doubled by day 7
(Wells et al., 1998). When heterophils (H) and lymphocytes (L) were measured in
embryos that had pipped into the air cell and through the shell, and in chicks from
1-d-old to 8 d of age, numbers of L in the perinatal stages were very low with H:L
ratios greater than 5.0. Posthatching H:L ratios decreased in a quadratic manner
from 1.76 at hatch to 0.39 on day 8 (Zulkifli and Siegel, 1994).
It is important to mention that in poultry, the heterophils are the primary cells
in the innate immune response to early bacterial invasion. Moreover, independently
of the efficiency of vaccination, at least 7-10 days are required for the stimulation
of the acquired immune system for protection (Kogut et al., 1998). In chickens
the bursa, producing B-cells, reaches maximum size and activity at about 2 weeks
after hatching. At the beginning the bursa is able to express only IgM and later
development of this lymphoid organ results in the appearance of circulating IgA
and IgG.
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Furthermore immune cells use free radicals as an effective weapons to kill pathogen
(Kettle and Winterbourn, 1997) and as a result the surrounding tissues could be
damaged if antioxidant protection is not appropriate (Schwarz, 1996). As described
in details in chapter 4, an immune response requires extensive communications
between wide variety of cell types and once cell receptors are damaged by free radical
attack communications are disrupted and immune competence compromised. In stress
conditions the requirement for antioxidant defence substantially increases and the
antioxidant system could be the crucial factor in chicken health. In absence of growth
promoters in chicken feed, the role of natural antioxidants in immune system
modulation is difficult to overestimate. It has been recently appreciated that nutrition
plays a vital role in reinforcing the innate immune system resulting in lower early
mortality and less variation in seven-day body weight and physiological development
(Postmouth, 2001). Therefore a strong immune system becomes a major player in
terms of health maintenance and disease prevention. At the same time it has long
been acknowledged that the first seven days growth determines a body weight at
marketing (Postmouth, 2001).
The generalised scheme of Se involvement in chick embryo development is shown
in Figure 7.10.
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Se in the feed
H2O2+GSH-Px = H2O
Biosynthesis of
selenoproteins
and stabilisation
of their mRNA
Lipid peroxidation
Tissue membrane damages, enzyme inactivation etc.
Hormone, prostaglandin, enzyme synthesis is disrupted and their
levels are compromised
Growth and development are compromised
Viability in early postnatal life decreased
Figure 7.10 Relationship between selenium and embryonic postnatal development (Adapted from Surai,
2000)
Table 7.8 Selenium requirement of birds, mg/kg feed (Adapted from NRC, 1994)
White egg-laying
strains, 0-6 weeks
White egg-laying
strains, 6 weeksfirst egg
Brown egg-laying
strains, 0-6 weeksfirst egg
Brown egg-laying
strains, 6 weeksfirst egg
0.15
0.10
0.14
0.10
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noted when breeders where younger (Pappas et al., 2005b). Therefore, there was an
indication of depletion over time for the breeder hens that were fed NRC recommended
Se levels. On the other hand, hens fed the high Se diets (0.5 ppm total Se) were
supplied with enough Se to maintain their requirements as well as build Se reserves in
the tissues.
Since the process of Se transfer from feed to egg yolk, and subsequently to
embryonic tissues, has received limited attention (Cantor, 1997; Paton et al., 2002),
there is no clear answer as to which level of Se supplementation is optimal for broiler
breeders. However, an analysis of published research and commercial data indicates
that 0.3 ppm organic selenium in the form of Sel-Plex would be a recommended dose
of Se dietary supplementation for breeders. For example, at 21 weeks of age Hubbard
Ultra-Yield broiler breeders (11,600 pullets and 1530 roosters) were placed in each
of two floor barns on two separate farms (Sefton and Edens, 2004). They were reared
on Selenite until placement and after that fed on Selenite or Sel-Plex (0.3 ppm) for 33
weeks of production. Sel-Plex supplementation was associated with improvement of
fertility (by 0.4-4.5%), hatchability of fertile eggs (by 1-6%). As a result, the number
of settable eggs were greater in Sel-Plex group showing an advantage of over 67,000
on one farm and over 8,500 on the second farm at 27 and 30 weeks of production
respectively. An average 4.5 extra chicks per hen capitalized on Sel-Plex treatment
was realized during the field trial. When data were combined for the whole production
period (41 weeks on Farm 1 and 43 weeks on Farm 2) it was concluded that Sel-Plex
in broiler breeder diets was very beneficial from both production and economic
viewpoints (Edens and Sefton, 2003; Sefton and Edens, 2004c). Indeed, 5.63 extra
chicks per hen housed were obtained as a result of Sel-Plex supplementation and it
was calculated that the improved performance resulted in an increased potential revenue
of +$1.17 per hen housed.
Did you know that a great body of evidence indicates that Se
requirement of breeders is 0.3 ppm in the form of Sel-Plex?
From photostimulation (22 wk) Ross 508 pullets were fed a Se-free laying ration (Se), a standard Selenite-supplemented ration (0.3 ppm, Selenite) or Sel-Plexsupplemented (0.3 ppm) ration (Renema and Sefton, 2004). The egg production was
similar, with 175, 173 and 178 eggs produced by 58 wk of age. However, the rate of
lay was affected after 48 wk of age, when hen-housed production was 68% in SelPlex group and 60 and 61% in Se and Selenite groups respectively. In the Sel-Plex
supplemented group settable egg production from 40 weeks (87.4) was higher than
in Se (80.6) or Selenite (83.7) birds. Prior to 34 wk, hatchability averaged 88% in
Sel-Plex eggs compared to 80% in Selenite group and 77% in Se group. Sel-Plex
also reduced shell defects (Renema and Sefton, 2004a; Renema. 2003).
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Two experiments with broiler breeders have been recently conducted in Brazil. In
the first study, conducted at an integrated farm in the state Sao Paolo 42,000 Cobb
breeders were divided in two groups and allocated in four farms (Rutz et al., 2003).
The control group was fed on a corn-soybean meal basal diet containing 0.5 ppm Se
as sodium selenite, while in the experimental diet sodium selenite was replaced by
0.3 ppm Se from Sel-Plex and birds were on these diets throughout the laying period.
In the second trial, conducted in the state of Parana 16270 Cobb breeders were used.
The control diet were supplemented with 0.2 ppm Se in the form of sodium selenite
and the experimental diet sodium selenite was supplemented with 0.3 ppm selenium
as Sel-Plex in addition to 0.2 ppm Se as sodium selenite. The major results of the
experiments are shown in Table 7.9 (Rutz et al., 2003). As can be seen from the
results, replacement of sodium selenite by Sel-Plex gave 1-2 extra chicks per hen
housed. Similar results were reported by Renema (2004) showing increased egg
production at 49-58 weeks (Table 7.10) and chick produced per hen housed (Table
7.11). as a result of replacement of 0.3 ppm sodium selenite by the same amount of
Sel-Plex (Renema, 2004). Recently, it has been shown that embryonic mortality in
eggs laid by 23 wk old broiler breeders was higher in the first and last week of
incubation and significantly reduced as the age of the flock increased. In the 27 wk
old breeders mortality in week 3 of egg incubation was 3.53%, and 10.6% in the soya
oil and fish oils supplemented breeders (Pappas et al., 2005c). Inclusion of Sel-Plex
(0.4 ppm) in the diets decreased mortality to 3.06 and 6.22% respectively.
Table 7.9 Effect of selenium on broiler breeders (Adapted from Rutz et al., 2003)
Trail 1
Selenium
source
Selenite
Sel-Plex
Trail 2
Egg
Chicks/hen
production, % housed
82.28
83.52
81.66
82.61
Egg
Hatchability,
production, %
%
69.75
70.39
Chicks/hen
housed
84.19
84.22
71.09
73.04
Table 7.10 The effect of dietary selenium level and source on egg production traits of broiler breeders
(Adapted from Ranema, 2004)
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75.6
73.1
75.5
403
60.2
60.1
67.7
174.5
172.7
177.7
168.5
168.6
174.6
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3.49
2.37
1.9
404
Table 7.11 The effect of dietary selenium level and source on reproduction traits of broiler breeders
(Adapted from Ranema, 2004)
No Se
Selenite
Sel-Plex
Day 1-14,
%
Day 15hatch, %
5.33
3.72
3.52
3.66
3.85
3.14
Reproduction parameters
Fertility, Hatchability,
%
%
86.9
90.1
90.1
77.9
82.5
83.5
Hatch of
fertile, %
Chick
production, No
88.6
91.5
92.5
131.3
139.1
145.3
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into the chicken diet for 11 days at 0.5 ppm and comparison with the same amount
of sodium selenite was made (Dobrzanski et al., 2003). There was no difference
in Se availability from both sources and the Se concentration in the egg content
only slightly (by 10.5%) increased. Similarly selenium-malt produced in laboratory
conditions in China and clamed to be organic selenium and fed to laying hens at
0.51 ppm for 24 days was not different from that of sodium selenite in terms of
affecting Se content of the egg (Jiakui and Xialong, 2004). Similarly, inorganic
selenium supplementation in the feed showed no effect on its concentration in the
egg (Salobir et al., 2003).
160
3 weeks
140
4 weeks
120
100
80
60
40
20
0
Basal (B)
B+0.2SN
B+0.2SP
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Pre-stress
160
Post-stress
140
120
100
80
60
40
20
0
Basal (B)
B+0.2SN
B+0.2SP
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improved feather score and decreased 24 h drip-loss and showed markedly higher
deposition rate in breast muscle and a lower excretion rate in the excreta compared
to the sodium selenite (Choct and Naylor, 2004).
Table 7.12 Results of field trials of Sel-Plex efficiency (Edens and Gowdy, 2003)
Se, ppm
Body weight, kg
0
Selenite, 0.2
Sel-Plex, 0.2
Selenite+Sel-Plex (0.1+0.1)
FCR
Mortality, %
North Carolina field trial (Arbor Acre male broiler for 42 days)
2.38
1.93
2.3
2.43
1.87
2.5
2.45
1.84
2.3
2.45
1.82
2.3
Selenite, 0.3
Sel-Plex, 0.3
Selenite
Sel-Plex
Selenite, 0.15
Sel-Plex, 0.15
Selenite 0.15 + Sel-Plex, 0.15
1.89
1.94
1.92
5.63
6.72
6.94
Table 7.13 Field results of replacement of Selenite by Sel-Plex (Edens and Gowdy, 2004)
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Country
Body weight, g
USA
Brazil
Mexico
Thailand
India
+30
+126
+198
+50
+59
-0.03
-0.093
-0.089
-0.120
-0.061
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Body weight, g
No Selenium, no E. coli
Sel-Plex, no E. coli
No selenium + E. Coli
Sel-Plex + E. coli
2047
2283
1896
2098
FCR, g/g
1.95
1.82
2.09
1.93
Mortality, %
16.7
5.0
36.7
15.0
Recently, it has been shown that Se supplementation at 0.4 mg/kg for White
Leghorn type chickens reduced death or lesions from E. coli or sheep erythrocyte
antigen challenge from 86 to 21% and dietary additions of Se between 0.1 and
0.8 mg/kg resulted in a substantial (77%) antibody titre increase in chickens (Larsen
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Post-stress
4.5
4.0
3.5
3.0
Basal (B)
B+0.2SN
B+0.2SP
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Breast
4.0
Thigh
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Chicken
Turkey
Duck
Se content in the feed, ppm: chicken: 0.42; turkey: 0.49; duck: 0.44
Figure 7.14 GSH-Px activity in muscles, U/g (Adapted from Daun and Akesson, 2004)
Species
Breast muscle
Thigh muscle
Chicken1
Turkey2
Duck3
109.1
67.4
148.5
116.6
109.5
139.1
1,2,3
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/selenium in the diet was 0.42, 0.49 and 0.44 ppm respectively
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16
Breast M
14
12
10
8
6
4
2
0
Basal (B)
B+0.2SN
B+0.2SP
B+0.7SP
Figure 7.15 Effect of dietary sodium selenite (SN) and Sel-Plex (SP) on selenium content in blood (mol/l)
and breast muscle of 7 week old chickens, mol/kg DM (Adapted from Kirikova et al., 2003)
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supplemented with either 200 ppm vitamin E or 100 ppm vitamin E in combination
with 0.4 ppm organic Se.
Table 7.16 Effect of selenium and vitamin E on breast muscle quality during storage (n=5) (Adapted
from Surai and Dvorska, 2002)
Dietary supplementation
Dietary Diet
group
1
2
3
4
5
6
7
8
9
Vit. E,
mg/kg
Semi- synthetic
Commercial (CD)
CD
CD
CD
40
CD
100
CD
200
CD
40
CD
100
Se,
mg/kg
0.2
0.4
0.2
0.4
Vit.E,
g/g
1.34 a
1.66 a
1.88 a
2.14 b
2.55 bd
4.88 c
6.11 c
3.22 d
5.88 c
Se-GSHPx, %
63.5 a
100 b
226.4 c
257.2cd
109.3b
122.2b
119.4b
265.4cd
305.9ed
3.96 a 10.22a
2.49 b
7.66b
c
1.44
3.28 c
df
0.81
2.99 c
ed
1.05
2.93 c
edf
0.72
2.06 ce
gf
0.66
1.01 d
gf
0.62
1.66 de
gf
0.51
0.96 fd
Values are means S.E.M; values in a column which do not have a common superscript are significantly
(P<0.05) different
a
The level of selenium in the semi-synthetic diet was 44 g/kg and in commercial diet 171 g/kg.
Selenium was supplemented in the form of Sel-Plex. Semi-synthetic and commercial diets contained 4.86
and 10.05 mg/kg -tocopherol. Both, commercial and semi-synthetic diets were balanced in other
nutrients.
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1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-E-Se
-E+Se
+E-Se
+E+Se
Figure 7.16 Effect of dietary selenium and vitamin E on lipid peroxidation in chicken muscle
mitochondria, nmol TBARS/mg protein (Adapted from Avanzo et al., 2001)
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in the breeders diet could improve hatchability of stored eggs (Edens and Sefton,
2004; Sefton and Edens, 2004b). Furthermore, during storage at 20C lipid
peroxidation was significantly decreased in egg yolk enriched with Se, which
had increased GSH-Px activity (Yaroshenko et al., 2003). When yolk was incubated
at 37C, lipid peroxidation was enhanced and the protective effect of increased
dietary Se was significant (Dvorska et al., 2003). Therefore, inclusion of organic
Se in the diet of laying hens can be used to maintain quality during storage.
Furthermore, inclusion of organic selenium in the chicken diet can also increase
Se concentration in the perivitelline membrane. Indeed, as can be seen from the
Figure 7.17, inclusion of Sel-Plex in the commercial diet significantly increased
the Se level in the perivitelline membrane (Surai et al., 2004c). Therefore, this
could be an additional mechanism of a positive effect of Sel-Plex on egg freshness
during storage. The effect of Se on egg freshness probably depends on age of
hens, composition of diet and conditions of egg storage. For example, Paton and
Cantor (2000) were not able to show an effect of dietary Se form on the Haugh
units.
600
500
400
300
200
100
0
Semi-synthetic
Commercial (C)
C+0.2 Sel-Plex
C+0.4 Sel-Plex
Figure 7.17 Selenium in perivitelline membrane, ng/g (Adapted from Surai et al., 2004c)
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and albumin (Rutz et al., 2003). Sel-Plex increased yolk and white weights relative
to control and trends toward improved egg production, weight and FCR were
noted with Sel-Plex addition (Pan and Rutz, 2003). Similarly, replacement of 50%
selenite (total supplemental Se was 0.4 ppm) in the laying hen diet by Sel-Plex
was associated with a significant increase in egg shell weight and thickness (Klecker
et al., 1997; 2001). Inclusion into the quail diet of high selenium (5.7 ppm) wheat
at the level of 60% was associated with increased Se content in tissues by 3-13
fold and eggshell thickness was significantly increased (Stoewsand et al., 1978).
Replacement of sodium selenite (0.2 ppm) in the diet by the same amount of
organic selenium as Sel-Plex significantly increased egg specific gravity after 9
weeks (Renema, 2004). In an experiment conducted in Brazil, ISA Brown laying
hens at age of 77 weeks were supplemented with Bio-PlexTM, a combination of
organic minerals, including Zn, Mn and Se (Xavier et al., 2004). Egg production
and feed conversion were improved. Furthermore, improvement in egg shell quality
(weight, thickness and specific gravity), Haugh units and increased yolk and
albumin weight were also observed.
Selenium was found in all parts of the egg, including shell and membranes
(Figure 7.18).
200
180
160
140
120
100
80
60
40
20
0
Albumin
Egg yolk
Perivitelline
membrane
Shell
membrane
Shell
Figure 18. Selenium distribution in egg, ng/g (Adapted from Surai et al., 2004)
In fact, the highest Se concentration was detected in the shell membrane and Se
concentration in the shell was comparable to that in the albumin. Selenium
concentration in quail shell ranged from 122.5 to 186.5 ng/g, which represented
about 12% of total egg Se (Surai et al., 2004). In comparison, shells of eggs of
roseate terns (Sterna dougallii) and herring gulls (Larus argentatus) in wild contained
about 1-5% of total Se of the egg (Burger, 1994). Inclusion of Sel-Plex in the quail
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diet (0.5 ppm) significantly increased Se concentration in the shell (up to 231.2458.3 ng/g, Figure 7.3). Therefore Se concentration in shells from control birds
comprised 152.6 11.4 ng/g and was almost doubled in the Sel-Plex group, comprising
307.640.5 ng/g. Furthermore, in Se-enriched eggs the shell contained about 9.2%
of the total egg selenium (Surai et al., 2004a; 2004b). This change could possibly
affect shell structure and could be potentially an additional source of Se for the
developing embryo. It has been recently appreciated that organic matrix of the egg
shell is responsible for crystal formation and ultimately determines shell quality.
However, selenium excess could be detrimental for egg production and shell quality.
For example, in Japanese quail hens 7 ppm of dietary Se depressed egg weights with
no effect on eggshell thickness (Stoewsand et al., 1978a).
Did you know that organic selenium could improve
shell quality?
Furthermore, it is possible that medullar bone in laying hens could accumulate Se
to some extent and use it for shell formation. It is interesting that both Se deficiency
and Se excess significantly decreased the biomechanical strength of rabbit bones
while the bones belonging to the Se-adequate control group always had the largest
modulus of elasticity (Turan et al., 1997). Molecular mechanisms of the Se effect on
shell/bone formation are not known at present. However, Se presence in bones has
been shown. For example in humans about 16% of total body Se was found in bones
(Zachara et al., 2001).
Recently a relationship between active vitamin D metabolites and the Se-dependent
enzyme thioredoxin reductase was established (Schutze et al., 1998; 1999). In fact
TrxR was identified as a 1,25(OH)2D3-responsive gene, providing a link between the
induction of a differentiation program by 1,25(OH)2D3 and the expression of the Seresponsive TrxR system in human osteoblasts. Similarly, the expression of TrxR and
its regulation by 1,25(OH)2D3 and selenite in monocytes might be important for
induction of differentiation and maintenance of function. An effect of TrxR activity
on 1,25(OH)2D3 would be expected, and this could be an important link between Se
and bone metabolism. On the other hand, antioxidant properties of various
selenoproteins could also be of importance to maintain antioxidant protection of the
oviduct during egg shell formation.
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metabolising the organic form of Se (Surai and Dvorska, 2001). Indeed feed
ingredients contain Se only in organic form, mainly as SeMet. This means that the
digestive system is ill-prepared for inorganic Se (selenite or selenate). This fact is
underscored by the fundamental differences in absorption and metabolism of inorganic
and organic Se sources. Organic Se (SeMet) is actively absorbed in the intestine as an
amino acid employing similar processes as methionine. In contrast, inorganic Se is
passively absorbed. The chemical similarity between SeMet and methionine allows
the body to use them interchangeably in protein synthesis. This makes it possible to
build Se reserves in the body (mainly in muscle). Our data with chicken (Table 7.17)
and quail (Table 7.18) clearly indicate increased Se concentration in tissues of birds
fed organic selenium in the form of Sel-Plex. Since SeMet is not synthesised in animals
and only plants, bacteria and marine algae can synthesise this amino acid (Schrauzer,
2000), there is little if any Se reserve in the body when inorganic Se is used.
Table 7.17 Effect of various levels of organic selenium (0.1 vs 0.5 ppm) in the diet on the Se
concentration in tissues of laying hens, ng/g (Adapted from Pappas et al., 2004; 2004a; 2005a)
Tissue
0.1 Selenium
0.5 Selenium
Increased, fold
23 weeks
Liver
Kidney
Lung
Breast Muscle
Brain
152
250
120
94
135
Liver
Kidney
Lung
Breast Muscle
Brain
122
256
72
66
136
421
521
251
191
186
2.8
2.1
2.1
2.0
1.4
434
528
257
216
223
3.6
2.1
3.6
3.3
1.6
27 weeks
Table 7.18 Effect of various levels of selenium (0.3 vs 0.6 ppm) in the diet on Se concentration in tissues
of adult quail, ng/g (Adapted from Karadas et al., 2004b)
Tissue
0.3 Selenium*
Liver
Kidney
Leg Muscle
Breast Muscle
326
663
130
133
0.6 Selenium**
Increased, fold
821
1256
357.4
438.3
2.5
1.9
2.8
3.3
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This difference in tissue reserves of Se can explain why organic Se is more effective
than inorganic sources, especially in stress conditions. Consider two different
scenarios for Se supplementation of poultry. The first and most common scenario
is addition of inorganic Se to diets containing low levels of naturally occurring
organic Se (Figure 7.19). When overproduction of free radicals occurs as a result
of stress conditions, the body responds to by attempting to mobilise antioxidant
reserves; and more importantly, by synthesising additional selenoproteins. In this
scenario, the main limitation is absence of Se reserves and a restricted ability to
synthesise additional selenoproteins. Therefore in this scenario we would expect
a decrease in productive and reproductive performance of poultry as well as
compromised immunity defence.
Overproduction of free
radicals and lipid
peroxidation
Selenium reserves are much higher in the scenario where organic Se sources
provide dietary Se (Figure 7.20). The major benefit would come from Se reserves
accumulated in the form of SeMet in muscles. Protein catabolism during stress
conditions releases SeMet to the free amino acid pool, thereby supplying Se needed
for the synthesis of additional selenoproteins to prevent damaging effects of free
radical overproduction. It must be emphasised that SeMet and SeCys are major
forms of Se in the body. However, cells do not contain free pools of these
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Se reserves in
tissues
Maintenance of high
productive and reproductive
performances, resistance to
diseases
Figure 7.20 Selenium and stress conditions-2 (Adapted from Surai, 2002)
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Conclusions
It is quite clear that the roles of Se in avian nutrition and reproduction need new
consideration in light of better understanding of molecular mechanisms of Se action
at the cellular and sub-cellular levels. In particular, discovery and characterisation of
a range of new selenoproteins, better understanding of relationships between different
antioxidants as important parts of integrated antioxidant system with possibilities for
antioxidant recycling in vivo gave a new insight in this matter.
The most fascinating part of Se-related research is coming from understanding a
principal difference between various Se sources in the diet. The digestive system of
animals, including birds, adapted to metabolise organic Se from plant-based feedstuffs
during evolution. Therefore, inclusion of selenite or selenate in the diet is not the
natural situation and differences in assimilation, distribution and accumulation of
Se in tissues depend on source of Se. Furthermore, main Se compound of selenized
yeast, SeMet itself, possesses antioxidant properties, which could be beneficial during
digestion (Surai et al., 2003; 2004d). In contrast, selenite is pro-oxidant and in
combination with iron and zinc potentially could stimulate lipid peroxidation and
cause damages to enterocytes and as a result decrease absorption efficiency of various
nutrients, including antioxidants. In addition, the natural form of Se, selenomethionine,
contributes to Se tissue reserves thereby providing a better chance for animals to
adequately respond to stress conditions by synthesising additional selenoproteins.
However, most of Se-related research in food animals was until recently conducted
using inorganic Se. Therefore much of the data related to effects of Se on various
physiological processes and on the productive and reproductive performance of
animals need to be re-evaluated using natural sources of Se. The data presented
above and summarised in Table 7.19 indicate that replacement of sodium selenite by
organic selenium in form of Sel-Plex in the chicken diet improves antioxidant defences
and has a range of commercial advantages. Indeed, in breeders main advantages
include:
improved fertility
improved hatchability
increased early chick viability
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improved FCR
decreased mortality
decreased drip loss
decreased lipid peroxidation during meat storage
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Parameter
Effect of organic
vs inorganic
selenium
References
Improved
Improved
Improved
Improved
Increased
Decreased
Improved
Improved
Improved
Chicken growth
Improved
Improved
Decreased
Decreased
Improved
Edens, 2001
Decreased
Decreased
Improved
Improved
Improved
Chicks/hen housed
Increased
Effective
Effective
Less toxic at
high doses
Selenium was detected in egg shell and Sel-Plex can increase Se concentration in
all parts of the egg, including egg shell, shell membrane and perivitelline
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improve FCR
improve shell quality
improve egg freshness during storage
decrease lipid peroxidation during storage
References
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in pig nutrition. Pakistan Journal of Nutrition 1: 25-30
Allen, R.G. and Venkatraj, V.S. (1992). Oxidants and antioxidants in development
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Anciuti, M.A., Rutz, F., Da Silva L.A., Cosenza, R.C. and Da Silva, R.G. (2004).
Effect of replacement of dietary inorganic by organic selenium (Sel-Plex) on
performance of broilers. Nutritional Biotechnology in the Feed and Food
Industry. Proceedings of the 20th Annual Symposium (Suppl. 1), May 22-26,
2004, Lexington, Kentucky, USA, p. 14
Ar, A. and Mover, H. (1994). Oxygen tensions in developing embryos - system
inefficiency or system requirement?. Israel Journal of Zoology 40: 307-326
Arai, T., Sugawara, M., Sako, N., Motoyoshi, S., Shimura, T., Tsutsui, N. and Konno,
T. (1994). Glutathione peroxidase activity in tissues of chicken supplemented
with dietary selenium. Comparative Biochemistry and Physiology 107A: 245248
Arruda, J.S., Rutz, F. and Pan, E.A. (2004). Influence of replacing dietary inorganic
with organic selenium (Sel-Plex) on performance of broilers. Nutritional
Biotechnology in the Feed and Food Industry. Proceedings of the 20th Annual
Symposium (Suppl. 1), May 22-26, 2004, Lexington, Kentucky, USA, p. 13
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Biotechnology in the Feed and Food Industry. Proceedings of the 20th Annual
Symposium (Suppl. 1), May 22-26, 2004, Lexington, Kentucky, USA, p. 19
Yaroshenko, F.A., Dvorska, J.E., Surai, P.F. and Sparks, N.H.C. (2003). Selenium/
vitamin E enriched eggs: nutritional quality and stability during storage. Poster
presented at Alltechs 19th Annual Symposium on Nutritional Biotechnology in
the Feed and Food Industries, Lexington, Ky, May 12-14, 2003, CD-ROM.
Yaroshenko, F.A., Surai, P.F., Yaroshenko, Y.F., Karadas, F. and Sparks, N.H.C. (2004).
Theoretical background and commercial application of production of Seenriched chicken. Book of Abstracts XXII Worlds Poultry Congress, 8-13 June,
2004, Istanbul, Turkey, p.410
Yaroshenko, F.A., Dvorska, J.E., Surai, P.F. and Sparks, N.H.C. (2003). Seleniumenriched eggs as a source of selenium for human consumption. Applied
Biotechnology, Food Science and Policy 1: 13-23
Yoshida, M. and Hoshi, H. (1977). Preventive effect of selenium, methionine and
antioxidants against encephalomalacia of chicks induced by dilauryl succinate.
The Journal of Nutrition 107: 35-41
Yu, B.P. (1994). Cellular defences against damage from reactive oxygen species.
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Yuan, H., Kaneko, T., Kaji, K., Kondo, H. & Matsuo, M. (1995). Species difference
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Introduction
Among farm animals pigs have a special place providing tasty meat and many
different pork-related products. World pig meat production reached 95.8 million
metric tons in 2003 being higher than poultry meat (72.5 million tons) or beef
(61.9 million tons) production (Best, 2004). Indeed, pork is the most popular
type of meat in almost every country in the European Union. The only exception
is the United Kingdom, where poultry meat is slightly more popular than pork. In
2002, the US production of red meat was 47.3 billion pounds, including 19.7
billion pounds of pork and 27.2 billion pounds of beef and pork consumption per
capita was 51.5 pounds (American Meat Institute, 2004). Furthermore, in 2004
pork production in the US is expected to be 20.3 billion pounds, on a slaughter of
more than 102 million head of hogs. Recent achievements in pig breeding and
substantial increase in growth rate and percentage of lean meat is associated with
higher demand for balanced diet providing all necessary macro- and
micronutrients, including vitamins and minerals. In particular, role of natural
antioxidants in pig production is a topic of recent research all over the world.
Did you know that world pig meat production reached
95.8 million metric tons in 2003 being higher than poultry
meat (72.5 million tons) or beef (61.9 million tons) production
Indeed, oxidative stress, related to overproduction of free radicals and toxic
products of their metabolism, is responsible for decreased immunocompetence,
compromised reproduction, poor growth and development of farm animals
including pigs. During last few years deeper understanding of the role of maternal
diet in the health status and development of the progeny has been greatly
appreciated. Among different antioxidant compounds selenium has a special place
been responsible for regulation of antioxidant defences. Restricted transfer of
selenium and vitamin E via placenta makes newly born piglets vulnerable to
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Se-deficiency
Clinical Se deficiency is a rear event in commercial pig production; however,
sub-clinical Se deficiency could be responsible for decreased productive and
reproductive performance of pigs. Indeed, the incidence of selenium deficiency
is seen most frequently in the neonatal, postnatal and post-weaning periods (Close,
2003). It is well known that most feed ingredients in the USA contain much higher
Se concentrations than the same feedstuff growing in Europe. However, most
production of maize and soya in the USA is concentrated in the Midwest region
with poor Se level in the soils. Therefore, very often the corn and soybean meals
used in American pig diets are Se-deficient.
There have been several important changes in swine industry for the last
20-30 years that have contributed to Se-related problems (Mahan, 1991):
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Reduced pig performance (growth rate, feed intake, feed utilization and
the apparent digestion for dry matter, ether extract and nitrogen; Glinke and
Ewan, 1977). For example, selenium-deficient pigs (Se in the feed was 0.019
ppm) had a significantly lower daily food intake and daily weight gain than
control pigs (0.25 ppm Se), whereas, food conversion was identical within
both groups. Selenium concentrations in liver, muscle and serum, as well as
activity of GSH-Px in serum, was significantly lower in selenium-deficient
pigs (Kirchgessner et al., 1995).
Hepatic necrosis (hepatosis dietetica); an acute type with liver failure and
sudden death or a subacute type with ascites and jaundice with edema and
cardiomyopathy. The disease occurs most frequently in young pigs up to 3
months of age. Pigs often dying without clinical signs of deficiency (Jenkins
and Hidiroglou, 1972). Massive necrosis can be seen in the liver of affected
pigs and selenium concentration in the liver substantially decreased (Figure
8.1). Selenium inclusion into the feed or injection can prevent the disease.
The disease can occur in combination with nutritional muscular distrophy
and mulberry heart disease.
0.30
Healthy
NMD
HD
0.25
0.20
0.15
0.10
0.05
0.00
Liver
Heart
Figure 8.1 Se concentrations in tissues of normal pigs and pigs with Mulberry heart disease (MHD) and
Hepatosis diabetica (HD), mg/kg wet weight (Adapted from Pedersen and Simensen, 1977)
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2.0
Healthy
1.8
NMD
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Liver
Skeletal muscle
Heart
Pancreas
Figure 8.2 Se concentrations in tissues of normal pigs and pigs with nutritional muscular
distrophy (NMD), mg/kg DM (Adapted from Lindberg, 1968)
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atria and ventricles bilaterally. Selenium concentration in the liver and heart
is reduced (Figure 8.1).
Impaired reproduction. Total number and number of liveborn piglets and
number of piglets alive at weaning were significantly lower for sows in the
Se-deficient group in comparison to the sow group supplemented with 0.1
ppm Se. Average litter weights at birth and weaning were also lower in the
Se-deficient group (Mihailovic et al., 1989). Adding sodium selenite to Sedeficient diet during gestation (from 60 days before farrowing) and lactation
(28 days) improved weight gain by 7.5-12% and feed utilization by 1420% (Bonomi, 2001). Decreased semen quality (lower motility and increased
percentage of abnormal sperm, lower fertilizing rate of oocytes, MarinGuzman, 1997; decreased number of spermatozoal reserves and Sertoli cells,
Marin-Guzman et al., 2000) and reduced the conception rate of gilts to first
service (Edwards et al., 1977) were characteristic of Se deficiency.
Furthermore, decreased litter size (Mahan et al., 1974) and increased piglet
mortality (Nielsen et al., 1979) were observed as a result of Se deficiency.
Progeny from Se and vitamin E-deficient sows are weak at birth with a
reduced desire to nurse the dam (Mahan, 1991) and smaller litter size was
related to low Se-diet (Mahan and Peters, 2004).
Increased incidences of mastitis, metritis and agalactia and reduced milk
production. These are post-parturient diseases in sows with lactation failure
(Mahan, 1991a; 1994; 1999).
Impaired prolonged farrowing due to poor muscle tone with increased
piglet mortality, lethargic and weak piglets (Acda and Chae, 2002; Close,
2003) and reduced muscle tone and strength with an increased incidence of
stillborn piglets (Close, 1998)
Impaired immune response and increased susceptibility to infection (see
chapter 4)
Reduced tolerance to parenterally administered iron and iron toxicity in
baby pigs
Tissue damage (elevated activities of lactate dehydrogenase, glutamicoxaloacetic transaminase and glutamic-piruvic transaminase in serum)
It seems likely that Se deficiency can affect fatty acid profile of some tissues. For
example, Se-deficient pigs were characterised by increased levels of total and n6 PUFAs in liver neutral lipids and lower levels of total saturated fatty acids in
muscle total lipids than control pigs, whereas the fatty acid composition of serum,
erythrocytes and back fat was not affected by selenium deficiency. Increased
lipid unsaturation, simultaneously with compromised antioxidant defences in Se
deficient pigs could trigger a chain of events related to lipid peroxidation and
leading to various lesions described in literature.
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The changes in arterioles depended on the severity of damage. From the one
hand, in mildly injured arterioles, increased endothelial permeability was associated
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452
at birth than the normal piglets. Serum creatine phosphokinase activities were
found to be increased in association with selenium deficiency (Fontaine et al.,
1977b) and it reflects the occurrence of sub-clinical muscular dystrophy.
It is possible that disturbances in antioxidant defences are responsible for the
development of microangiopathy (MAP, mulberry heart disease) in pigs. However,
there is some controversy among data related to this disease and it is not clear at
present what role Se deficiency plays in the development of this disease. As
mentioned above, the mulberry heart disease of the pig represents a disease with
major manifestation at the heart, primarily affecting young animals with vascular
and myocytic lesions. The vascular lesions include fibrinoid necrosis in
intramyocardial amall arteries and arterioles with fibrin microtrombi in the
myocardial capillaries (Jonsson, 1993). However, it seems likely that apart from
the characteristic haemorrhagic appearance of the heart, the brain can also be
affected. In particular, histopathological changes include marked alteration of the
small cerebral blood vessels with fibrinoid degeneration, necrosis or infiltration
with pleomorphic cells (Stierstorfe et al., 2001). Furthermore, the vessel lumina
are frequently obliterated by thrombi, and as a consequence large areas of malacia
are found in brain parenchyma. It was reported that three 5-week-old pigs died of
the cardiac form MAP of selenium-vitamin E deficiency. It was observed that the
hearts had prominent hydropericardium and extensive serosal and myocardial
hemorrhage (Van Vleet and Ferrans, 1977). Histopathologic observations
demonstrated marked myocardial edema, congestion, and hemorrhage, with
numerous capillary hyaline microthrombi that stained red with the periodic-acid
Schiff rocedure. Ultrastructurally, these thrombi were composed of elongated,
dense masses of intertwined fibrin strands with occasional entrapped erythrocytes.
It was also shown that MAP persisted among young pigs in Denmark although
the diets of sows and pigs were supplemented with selenium and vitamin E (Nielsen
et al., 1989). Furthermore, the concentrations of selenium and vitamin E in the
liver and heart tissues of young pigs, which had died suddenly, and had the
characteristic lesions of mulberry heart disease post mortem, were not significantly
different from the concentrations found in pigs of the same age, which had died
suddenly for other reasons. Similarly, swine with dietetic MAP had lower heart
and liver -tocopherol concentrations than did control pigs. However, heart and
kidney Se concentrations and heart and liver PUFA concentrations were similar
in pigs of either group (Rice and Kennedy, 1989). Indeed, in spite of apparently
adequate amount of dietary -tocopherol, pigs with MAP had lower tissue tocopherol concentration than did control pigs. The authors concluded that
spontaneous MAP is associated with altered -tocopherol metabolism, but is
unrelated to alterations in tissue Se and PUFA concentrations and GSH-Px activity.
In another study, the significance of selenium deficiency was also investigated in
pigs that died suddenly of MAP. It was shown that hepatic selenium concentration
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Collection
period
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Se injection,
mg Se/kg BW
Kidney
Liver
Longissimus
muscle
Serum
Initial
Control
1.24
0.43
0.24
24 days
postinjection
Control
0.275
0.55
1.10
2.62
4.06
5.17
5.30
0.43
0.72
0.91
1.05
0.17
0.32
0.37
0.31
0.025
0.053
0.078
0.093
60 days
post-injection
Control
0.275
0.55
1.10
3.97
5.99
6.99
6.54
0.38
0.54
0.60
0.71
0.17
0.20
0.28
0.32
0.030
0.047
0.062
0.060
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Se requirement
It has been suggested that most, if not all, aspects of swine production are affected
by feed levels of Se and vitamin E (Jenkins and Hidiroglou, 1972). After research
work showing that many regions in the USA are deficient in Se, great efforts have
been made to gain FDA approval for Se supplementation of animal diets (Ullrey,
1992). For example, results of a cooperative survey of the dietary selenium fed
growing swine in 13 states (Ku et al., 1972) showed that the highest Se
concentration was in South Dakota (0.493 ppm) and lowest in Virginia (0.027
ppm) being more than 15 fold lower (Table 8.2). In 1974 U.S. FDA approved
the addition of 0.1 ppm of selenium to all swine diets. The approval of selenite for
use in animal diets followed in other countries as well. For example, addition of
selenium to swine feed (max. 0.1 ppm) was legalized in Denmark in 1975
(Pedersen and Simesen, 1977). In 1991 the Denmark recommendation was
changed to 0.4 ppm for pre-starter diets and 0.22 ppm for both growing and
breeding pigs (Poulsen, 1993).
Did you know that in 1974 U.S. FDA approved the addition
of 0.1 ppm of selenium to all swine diets for the first time?
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State
South Dakota
North Dakota
Nebraska
Iowa
Wisconsin
Wyoming
Arkansas
Idaho
Indiana
Michigan
New York
Illinois
Virginia
Dietary Selenium,
ppm
Se in longissimus muscle,
ppm (wet weight)
0.493
0.412
0.330
0.235
0.178
0.158
0.152
0.086
0.052
0.040
0.036
0.036
0.027
0.521
0.386
0.313
0.278
0.125
0.311
0.212
0.111
0.063
0.052
0.046
0.059
0.034
The current maximum accepted total dietary Se level in Denmark is 0.5 ppm.
Indeed, the inability to supplement selenium-deficient diets prior to FDA approval
has cost hundreds of millions of dollars. For example, annual losses to the beef
cattle, dairy cattle and sheep industry were estimated in 1975 at nearly $545
million (Ullrey, 1980). Later, in 1982, FDA reconsidered a level of Se
supplementation and allowed the addition of 0.3 ppm of selenium to diets for
pigs up to 20 kg (Nutrient Requirements of Swine, 1998). Ultimately, in 1987,
levels of supplemental Se in diets for chickens, turkeys, ducks, swine, sheep, and
cattle were approved at 0.3 ppm (Ullrey, 1992). Therefore, currently 0.3 ppm of
selenium in the diet is allowed for all pigs. This dietary Se allowance for pigs was
based on tissue Se concentrations, Se balance and the activity response of cellular
GSH-Px. In addition to dietary Se supplementation, other solutions of Se deficiency
are also available. For example, inclusion of selenite into fertilizers are shown to
be effective in increasing Se level in grains, animal diets and animal tissues (Table
8.3). However, this application found its commercial use only in Finland and
New Zealand. Indeed, environmental concerns of potential Se pollution from animal
production re-opened a question whether the Se supplementation was in excess of
dietary needs (Ullrey, 1992), but there was no sufficient evidence to change Se
supplementation and the level of 0.3 ppm has been maintained.
There is a great deal of difficulties to establish an optimal Se requirement of
pigs:
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Table 8.3 Selenium concentrations (mg/kg wet weight) in tissues of pigs and cattle in Finland (Adapted
from Venalainen et al., 1997).
Muscle
Liver
Year
pig
cattle
pig
cattle
1974
1981
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
0.11
0.08
0.08
0.12
0.23
0.26
0.30
0.29
0.26
0.19
0.19
0.13
0.15
0.04
0.07
0.10
0.16
0.16
0.21
0.19
0.16
0.16
0.14
0.09
0.10
0.45
0.56
0.49
0.56
0.64
0.7
0.73
0.68
0.62
0.57
0.56
0.50
0.52
0.10
0.16
0.28
0.34
0.42
0.45
0.51
0.46
0.40
0.37
0.35
0.32
0.28
Se was introduced to all multi-nutrient fertilizers used in Finland after July 1984 at 16 mg/
kg fertilizers for cereal crops and 6 mg/kg for grassland crops. In 1990 the level of Se
inclusion to the all fertilizers was decreased to 6 mg/kg.
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mg/day
0.30
0.30
0.25
0.15
0.15
0.15
0.15
0.15
0.15
0.08
0.15
0.25
0.28
0.39
0.46
0.30
0.80
0.30
*/Recent recommendations by Close and Cole (2000) are 0.3 mg Se/kg diet
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changes are different between GSH-Px1 and GSH-Px4 in pigs (Lei et al. 1997). In
particular, it was found that 0.1 mg Se/kg was insufficient and 0.3 mg Se/kg was
required for young pigs to express the full activity of GSH-Px1 in liver. However,
the authors did not test Se level at 0.2 mg Se/kg in their study. In an experiment
conducted by Lei et al. (1998) eight different tissues were collected from Seadequate male pigs aged 1, 28 and 180 days, and PH-GSH-Px, and GSH-Px
activities were assayed. Both mRNA and activity expression of PH-GSH-Px in
most assayed tissues was increased with age, but increases in PH-GSH-Px mRNA
levels between ages did not fully account for all changes in activity (Lei et al.,
1998). Expression of GPX activity was increased more than that of PH-GSH-Px
between day 1 and day 28. In this experiment, there was no difference in GSH-Px
activities between pigs fed 0.2 and 0.3 or 0.5 mg Se/kg. Therefore, it was
concluded that full activity expression of GSH-Px1 and GSH-Px4 in various tissues
of weanling pigs requires 0.20 mg Se/kg in the corn-soy-based diets (Lei et al.,
1998).
Did you know that current recommendations on Se requirement
of swine are 0.15-0.30 ppm, but in commercial conditions this
should be increased depending on level of stresses?
As mentioned above, pig Se requirement depends on the experimental conditions,
especially on amounts of stresses caused by environmental and technological
factors and in experimental laboratory conditions it seems to be lower than in
practical conditions of pig producing units. Indeed, in physiological conditions
Se requirements of the growing pigs are quite low. For example, using serum
GSH-Px activity as the measurement criterion, Mahan et al. (1999) showed that
the supplemental dietary Se requirement did not exceed 0.10 and 0.05 mg Se/kg
diet for the growing and finishing phases, respectively, when added to a basal
diet containing 0.06 mg Se/kg. Grower pigs fed sodium selenite had serum GSHPx activity that reached a plateau at 0.1 ppm Se and 0.3 ppm when the Se-enriched
yeast source was fed, but the interaction response was not significant. On the
other hand, during the finisher period, serum GSH-Px activity reached a plateau
at 0.1 ppm Se for both Se sources (Mahan and Parrett, 1996). In earlier work Se
requirement to maintain an optimal GSH-Px activity was shown to be higher. In a
trial with sows different dietary selenium levels (0.1; 0.3 and 0.5 mg/kg) were fed
and GSH-Px activity in plasma was determined at day 60, 90 and 110 of gestation,
day 5, 15 and 25 of lactation and day 7 post weaning. It was shown that GSH-Px
activities dropped within the treatments, independent of dietary selenium, from
day 60 of gestation to a minimum at day 5 and 15, respectively, of lactation and
increased again after weaning (Neumann et al, 1989). Highest GSH-Px activities
were observed at the 0.5 ppm Se level in the diet. However, reproductive
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Gestation
Parturition and lactation
First few days after piglet birth
Weaning period
Maintenance of semen quality in boars
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homogenates from gilts fed the low-Se diet, even though there was no difference
in GSH-Px activity. It could mean that other than GSH-Px selenoproteins were
affected and resistance of fetus to oxidative stress decreased.
Did you know that the most important periods of pig
development where Se is of great importance include:
gestation, parturition and lactation, first few days after birth,
weanling period, maintenance of semen quality in boars?
The activity of the selenium-dependent enzyme GSH-Px in the serum of piglets
was very low during the first week of piglet life despite the fact that the sows feed
had been supplemented with 0.35 mg inorganic selenium/kg (Pehrson et al., 2001).
The authors concluded that the selenium status of newborn piglets might be more
critical for their health than their vitamin E status. Similar conclusions were made
by Loudenslager et al. (1986) showing that plasma biological antioxidant state
(tocopherol content and GSH-Px activity) of pigs at birth was very low predisposing
piglets to oxidative stress. Indeed, mean concentrations of vitamin E (1.77 mg/
litre) and GSH-Px (682 U/litre) were low in the blood plasma of the piglets at
birth, but a large increase was observed on the next sampling occasion at the age
of 3 days (vitamin E: 5.31 mg/litre; GSH-Px: 996 U/litre) reflecting transfer of
these antioxidant compounds via colostrum (Hakansson et al, 2001). The authors
showed that the highest concentrations of vitamin E (17.9 mg/kg) and Se (200
g/litre) were found in colostrum, but with a large variation between sows. One
week postpartum the concentrations of vitamin E and Se in sow milk had
decreased, on average by 86 and 68%, respectively. Furthermore, sow milk
selenium and vitamin E declines with advancing parity (Mahan, 1991, 1994).
This means that adult sows need more vitamin E and Se to maintain their reserves
in the body and to transfer them to progeny. Furthermore, piglets born to older
sows could be at greater risk of oxidative stress at birth as well as during early
postnatal development.
In the experiment conducted by Mahan (2000) six dietary treatments were
used in a 2 x 2 factorial arrangement with two additional treatments. Inorganic
(sodium selenite) or organic (Sel-Plex) Se sources were added to the diet at 0.15
or 0.30 ppm Se. A non-Se-fortified corn-soybean meal basal diet served as a
negative control, and a sixth group was fed 0.15 ppm Se from both inorganic and
organic Se sources. A total of 43 sows were fed their treatment diets at 2.2 kg/d
from 6 d pre-partum to parturition and at full feed through a 14-d lactation period.
The major results can be summarised as follows:
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Similarly, Se dietary supplementation of pigs for 105 days in the form of selenized
yeast was associated with increased Se concentration in whole blood and liver in
comparison to pigs supplemented with sodium selenite (Ortman and Perhson,
1998). Surprisely, there was no difference in Se concentration in the blood or
liver between pigs supplemented with 0.1 or 0.3 ppm of organic selenium.
Did you know that piglets are born with low antioxidant
defences and their postnatal development and health depends
on antioxidants transferred via colostrum and milk?
When diet for Crossbred barrows was supplemented with 0.1; 0.3 or 0.5 ppm
selenium in the form of selenite or Sel-Plex, selenium retention increased as dietary
Se levels increased, particularly when the Sel-Plex was provided (Mahan and
Parrett, 1996; Table 8.5). Similar results were reported when selenite or organic
selenium in the form of selenized yeast (Sel-Plex) was fed to grower and finisher
swine (Table 8.6; Mahan, 1999). Increasing selenite concentration in the diet from
0.05 to 0.3 ppm only slightly affected Se concentration in the loin and pancreas,
but substantially increased Se concentration in the liver. In contrast, organic Se in
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Table 8.5 Effect of dietary selenium on selenium level in tissues of grower and finisher swine, ppm
(Adapted from Mahan and Parrett, 1996)
Item
Basal
Serum
Serum
Loin
Liver
Pancreas
Kidney
Serum
Loin
Liver
Pancreas
Kidney
Inorganic selenium
0.1
0.3
0.5
Organic selenium
0.1
0.3
0.5
0.159
0.300
0.679
0.645
1.870
0.170
0.362
0.820
0.604
1.756
0.168
0.212
0.737
0.614
2.990
0.187
0.262
0.901
0.725
3.040
Table 8.6 Effect of dietary selenium on tissue selenium concentration, ppm (Adapted from Mahan et al.,
1999)
Item
Basal
Inorganic selenium
0.05
0.1
0.2
Loin
Liver
Pancreas
Kidney
0.087
0.195
0.314
1.305
0.091
0.425
0.327
1.716
0.103
0.464
0.360
1.771
55 kg body
0.113
0.526
0.400
1.76
Loin
Liver
Pancreas
Kidney
0.085
0.258
0.290
1.811
0.106
0.452
0.355
2.283
0.114
0.436
0.391
2.333
Organic selenium
0.3
0.05
0.1
0.2
0.3
weight
0.120
0.522
0.368
1.655
0.113
0.319
0.335
1.597
0.143
0.398
0.396
1.734
0.184
0.577
0.529
1.973
0.206
0.513
0.529
1.972
0.134
0.469
0.395
2.282
0.17
0.516
0.426
2.333
0.249
0.627
0.483
2.473
0.332
0.717
0.573
2.677
the swine diet was much more effective in transferring to loin and pancreas. It is
interesting to note, that as dietary Se levels increased, urinary Se excretion was
substantially increased when pigs were fed sodium selenite, whereas excess of
selenium in the form of Sel-Plex was mainly excreted via fecal route. Indeed, loin
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Item
0.039
0.236
Neonate
0.055
0.250
0.068
0.283
0.085
0.310
0.062
0.101
0.352
Weaning, 21 d
0.075
0.121
0.388
0.082
0.129
0.535
0.102
0.244
0.509
0.1
Se-yeast, ppm
0.3
It is interesting that by increasing sodium selenite level in the sows feed by 9fold, it was possible to increase Se concentration in blood and colostrum only
moderately. For example, in sows, average whole blood Se concentrations near
parturition were 144.1 and 174.3 g/litre for sows given Se 0.1 and 0.9 mg/kg,
respectively. Average whole blood Se concentrations were 59.2 g/litre in piglets
from sows given Se 0.1 mg/kg diet and 79.9 g /litre in piglets from sows given
Se 0.9 mg/kg diet. Sows given Se 0.9 and 0.1 mg/kg had average concentrations
of Se in colostrum of 148.0 and 86.0 g/litre (Blodgett et al., 1989).
In general, it was shown that increased from 0.1 to 0.3 ppm Se supplementation
with both Se sources was related to increased Se concentration in neonatal loin,
milk and weaning pig loin with organic selenium to be more efficient. On the
other hand, gilt serum GSH-Px activity was generally similar at the 0.1 and 0.3
ppm Se level for either Se source, whereas serum Se was consistently higher
when the dietary Se level was 0.3 ppm. Furthermore weanling pig serum GSH-Px
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activity was similar regardless of the Se level or Se source fed to the dam, but
serum Se increased when the 0.3 ppm Se level and the Se-yeast was fed to the gilt
(Mahan and Kim, 1996). As parities progressed, sow milk Se concentration
decreased when the diet was supplemented with sodium selenite (Mahan and
Peters, 2004; Mahan et al., 1991; 1994) and the authors suggested that:
Basal
Item
Sow serum Se, mg/l
70 d
110 d
Weaning
Colostrum Se, mg/l
Milk Se, mg/l
Sow hair, Se, mg/kg
Litter, weaning serum
Se, mg/l
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Organic Se
Inorganic Se
Combination
0.15
0.30
0.15
0.30
0.30
0.114
0.088
0.086
0.075
0.040
0.271
0.146
0.130
0.144
0.151
0.067
0.482
0.169
0.159
0.179
0.168
0.101
0.763
0.143
0.125
0.160
0.094
0.055
0.303
0.135
0.124
0.162
0.095
0.056
0.326
0.162
0.152
0.173
0.148
0.064
0.507
0.056
0.078
0.092
0.067
0.062
0.077
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Basal
Organic Se
Inorganic Se
Combination
Item
0.15
0.30
0.15
0.30
0.30
0.298
2.23
0.071
0.300
0.583
2.40
0.203
0.487
0.781
2.50
0.329
0.510
0.450
2.68
0.088
0.334
0.571
2.94
0.092
0.369
0.565
2.91
0.217
0.467
0.197
0.037
0.075
0.348
0.066
0.123
0.424
0.107
0.200
0.238
0.046
0.079
0.279
0.046
0.100
0.344
0.074
0.116
35
30
25
20
15
10
Basal
0.15 Sel-Plex
0.30 Sel-Plex
0.30 Selenite
Figure 8.3 Effect of selenium in sows diet on splay leg (% live pigs) in progeny (Adapted from Mahan
and Peters, 2004)
Indeed, the progeny from sows between the 4th and 7th parity often are deficient
in selenium and their mortality rates can be increased. Furthermore, the higher
the level of productivity, the greater the loss of Se from the sows stores (Close,
2003). As mentioned above, if Se in the milk exists in the form of SeMet, which is
not synthesized in sows body, therefore, only when Se is supplemented in organic
form it can be effectively transferred to colostrum and milk. It could well be, that
SeMet reserves in sows body are depleted and this caused Se declining in milk
with advanced parity. Therefore, even high inclusion rate of selenite cannot
provide a form of selenium which is transferred to colostrum and milk and the
only solution for this problem is to use organic selenium, providing SeMet which
is effectively transferred to the colostrum and milk.
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Figure 8.4 Effect of selenium in sows diet on number of stillborn piglets (No/litter) (Adapted from Mahan
and Peters, 2004).
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20
33
82.5
20
35
87.5
386
11.7
330
10
1.2
420
12
385
11
1.1
1480
7530
15.5
1530
7930
10.6
Recently, sows have been fed organic selenium instead of selenite at 0.15 or 0.3
ppm from the time when sows entered the breeding area and continued during
gestation and lactation period (Pineda et al., 2004). The advantages of organic
selenium can be summarised as follows:
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468
Effect of selenium (Se) source on litter performance was evaluated in a side-byside comparison of Se-enriched yeast (Sel-Plex) with sodium selenite at 0.3 ppm
Se supplementation (Lampe et al., 2005). The trial encompassed 6 weeks of
farrowings within a 3,400 sow farrow to wean facility. Litter data were analyzed
from 380 sows per treatment. Females remained on their respective treatment
through weaning. Milk Se concentrations were significantly greater in Sel-Plex
group than in sodium selenite group (0.111 + .003 ppm vs 0.088 + .003 ppm).
Sows receiving Sel-Plex weaned more pigs (9.61 vs 9.26) and had a greater litter
weaning weight (53.15 vs 51.07 kg). Prewean mortality of Sel-Plex treatment
was 9.76% compared to 11.30% for sodium selenite treatment. In another
experiment, one thousand pigs were weaned, 500 from sows receiving Sel-Plex
and 500 from sows receiving sodium selenite (at 0.3 ppm each) as Se source
(Lampe et al., 2005a). The weaned pigs were placed in a commercial nursery and
penned in groups of 25 head. Average weight of pigs at entry to the nursery was
5.31 + 0.04 kg and was not different dependent on Se source of the sow. Pigs
were fed a 3-phase nutrition program during the 42 d nursery trial. The results of
the experiment indicated that fewer dead and culls (no value pigs) in the nursery
when pigs were from Sel-Plex fed sows. In fact, total mortality and culls in the
nursery was 3.2% for those pigs weaned from sows receiving Sel-Plex compared
to 5.4% for those pigs weaned from sows receiving sodium selenite. Therefore,
the authors concluded that in commercial production prewean piglet survivability
and piglet survivability in the nursery can be enhanced when Sel-Plex replaces
sodium selenite as a dietary selenium source of the sow.
It seems likely that feeding to sows a premix where all trace minerals (Cr, Cu,
Fe, Mn, Zn and Se) were replaced by organic minerals (BioplexesTM) can increased
number of pigs born and greater pigs gain during the nursing period (Peters and
Mahan, 2004). This could be the next step in the pig nutrition: to use only organic
minerals in the swine diets. The additional benefit from a mixture of organic
minerals can be related to prevention of pro-oxidant properties of inorganic iron
and copper in the digestive tract (Surai et al., 2003; 2004), prevention of some
mineral interactions and better assimilation efficiency of trace minerals.
Summarising data presented above it is necessary to underline that the selenium
status of the developing fetus and weanling pig are dependent on the Se status of
the mother. This is extremely important because about 90% of all pre-weaning
death occurs within the first 7 days (Varley, 1995). Indeed, organic selenium in
the maternal diet helps building Se reserves in the animal body. For a pregnant
sow it is important to have a sufficient amount of Se for body maintenance and
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This is a period of active pig growth and it is well accepted that growth rate of the
pig is determined by a number of factors and selenium is involved in most of
them (Huo et al., 2003):
Genotype
Genetic potential of pigs is constantly improving and therefore their ability
to utilise nutrients is also improved. However, the price for this improvement
is an increased sensitivity to various stresses and therefore antioxidant
defences are an integral part of the body to prevent damaging effects of
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470
Nutrition
To meet increased demands for nutrient of fast growing pigs is an important
challenge for the nutritionist. Since digestive system of the piglet at birth is
not mature and actively developing for the first few weeks, colostrum and
milk are extremely important sources of essential nutrients. Antioxidants
compounds such as vitamin E and selenium are transferred from mother to
progeny providing essentials for building antioxidant defences of fastgrowing piglet.
Environment
Temperature is the most important environmental factor affecting growth
and development of piglets. Indeed, thermoregulation of piglets is dependent
on thyroid hormone function. Selenium as part of iodothironine deiodinase
is involved in activation of thyroid hormone and in thermoregulation of the
piglets. Since the fat reserves in the piglet body at birth is limited (about 1%
of body mass), its efficient usage is a priority and aforementioned
selenoenzymes are involved in this process. Furthermore, environmental
pollutants such as ammonia, carbon monoxide, sulphated hydrogen cause
oxidative stress in piglets and need for additional antioxidant protection,
provided by selenoproteins is increased.
Health status
Immune system of the newly born piglets is very weak and therefore they
are extremely sensitive to various pathogens. Indeed colostrum and milk
provide immunological protection, but a building of immune system of
piglets usually takes several weeks. During this period of time antioxidants
plays an important immmunoregulating role (see Chapter 4).
Management
The decisions about time of weaning, choice of diets and supplements and
some other management factors affect growth and development of piglets.
In particular, a choice of feed supplements could have a positive or
detrimental effect on growth performance of pigs. Indeed, the daily and
long-term management decisions made by swine personnel will greatly affect
the efficiency of pig production.
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Table 8.11 Daily Se balance in pigs supplemented with sodium selenite (Adapted from Groce et al.,
1971)
Item
Basal
Basal+0.1 ppm Se
0.042
18.9
10.3
8.4
1.9
8.6
45.5
Basal+0.5 Se
0.147
66.2
25.3
18.4
6.9
40.9
61.7
0.543
244.4
207.3
57.7
149.6
37.1
15.2
Table 8.12 Effect of supplemental Se on balance measurements in grower swine (Mahan and Parret,
1996)
Selenite, ppm Se
Se balance, mg/d
Intake
Urine
Feces
Retention
Apparent absorption, %
Retention, % of intake
Retention, % of absorbed
Se-yeast, ppm Se
0.1
0.3
0.5
0.1
0.3
0.5
0.271
0.076
0.070
0.125
0.576
0.233
0.146
0.198
0.941
0.442
0.185
0.314
0.299
0.036
0.087
0.176
0.670
0.139
0.166
0.365
1.006
0.238
0.270
0.499
73.9
46.1
62.2
75.1
34.4
46.0
80.5
33.4
41.5
70.8
58.9
87.1
75.2
54.4
72.4
73.3
49.6
67.8
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could be responsible for these detrimental effects on meat quality. Indeed, selenite
can produce free radicals when reacting with glutathione or some other substances
in the gastro-intestinal tract (Surai et al., 2003) and this could ultimately lead to
the compromised antioxidant system in the digestive tract and in tissues.
Table 8.13 Effect of dietary selenium on carcass characteristics of pigs (Adapted from Mahan et al.,
1999)
Item
Basal
0
24- 72 h, 3C 3.35
72-120 h, 2C 2.33
72-120 h, -10C 2.53
24-120 h, 3C 5.68
24-120 h,
3C/-10C
5.88
24-120 h,
average
5.78
Hunter L
value
46.48
Inorganic selenium
0.05
3.95
2.52
2.67
6.47
0.1
0.2
Organic selenium
0.3
0.05
0.1
0.2
0.3
3.19
2.11
2.25
5.30
3.51
2.46
3.00
5.97
2.96
2.56
3.04
5.52
2.95
2.40
2.47
5.35
6.62
7.66
6.09
6.55
5.44
5.46
6.00
5.42
6.55
6.66
6.01
6.54
5.37
5.72
5.76
5.39
46.93
47.68
48.55
49.82
46.57
48.64
46.48
47.36
Selenium-vitamin E combinations
It is well known that Se and vitamin E are working together in concert providing
an effective antioxidant defence (see chapter 2). There is a great body of evidence
to show that a combination of these two antioxidants is more effective in
comparison to their individual use (Surai, 2003). For example, four groups of 12
pregnant sows of the same genetic background, with similar litter size, living
under the same housing conditions and having the same hygienic and nutritional
standards, were used in an experiment conducted by Mavromatis et al. (1999).
In control animals, a basic feed was provided, which contained 20 mg/kg tocopherol and 0.45 mg/kg selenium. Sows in the second group received the
same basic feed but additionally supplemented with 30 mg -tocopherol per kg.
The third group was fed on basic feed but additionally they were injected with 30
mg Se (sodium selenite) on days 30, 60 and 90 of pregnancy. The sows in the
fourth group was fed vitamin E-supplemented feed as in the group 2 and injected
with selenite as in group 3. The experiment started on day 30 of pregnancy and
lasted until weaning day (28 days post-farrowing). It was shown that piglets born
from sows supplemented with vitamin E and injected with selenium (group 4)
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pigs was decreased (Loudenslager et al., 1986). It has been shown that injectable
iron is toxic to pigs from Se and vitamin E-deficient dams (Nutrient Requirements
of Swine, 1998; Mahan, 1991). Consequently, vitamin E was found to be an
effective means in preventing toxic detrimental effects of iron consumption or
injection (Tollerz, 1973; Hill et al., 1999). However, injection of iron in combination
with sodium selenite was associated with depressed hemoglobin concentration
and decreased growth rate of piglets (Hill et al., 1999). Indeed, pro-oxidant
properties of selenite could be responsible for these detrimental effects of a
combination of iron and sodium selenite. It is interesting to note that recently in
the most intensive swine producing region of Columbia, a sudden 80% mortality
outbreak has occurred on one day old piglets from a two site farrow-to-finish
production system of 270 sows as a result of iron toxicity (Velasquez and Aranzazu,
2004). This happened due to attempts of cutting down the feed costs by lowering
vitamin E supplementation and changing from an organic to a cheaper inorganic
form of selenium supplementation. As the authors stated, several months after
such changes newborn piglets were affected by a deadly hemorrhagic clinical
entity developed after a normal iron dextran injection. An outbreak of acute
selenium poisoning among suckling pigs was reported by Sivertsen et al. (2003)
when 92 piglets were found dead or moribund without preceding symptoms.
Necropsy revealed acute congestion of liver and small intestine. It is interesting
that the source of toxicity was a powdered iron supplement contaminated by
sodium selenite. It is likely that pro-oxidant properties of selenite, especially in
combination with iron, are responsible for free radical production consequently
causing toxicity.
Conclusions
From the data presented above it is clear that Se plays an important role in swine
nutrition. The requirement of swine in selenium varies depending on many
environmental and other conditions and in general is considered to be 0.15-0.30 mg/
kg feed. It seems likely that reproducing sows and boars are especially sensitive to Se
deficiency (Figure 8.5) and to meet their requirement in this important element is a
great challenge for pig nutritionists. Indeed, in many countries in the world there are
legal limits of how much Se can be included into the diet (Table 8.14) and this restricts
flexibility of a nutritionist in terms of addressing Se needs of the developing and
reproducing swine. For example, immunomodulating properties of Se are usually
seen at doses substantially higher than those needed to maintain pig growth and
development. Indeed, organic selenium in the form of selenized yeast is proven to be
an important natural solution for pig industry. The advantages of organic selenium in
comparison to selenite (Table 8.15) are obvious and include better Se transfer from
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Semen
Fertilization rate
Se
Embryo survival
Uterine capacity
Se
Newborn piglets
Se
Sow fertility
Se
Weaned piglets
Figure 8.5 Potential role of selenium in sow reproduction (Adapted from Close, 1999; Close and Cole,
2000).
Table 8.14 Approved levels of selenium supplementation in feed (Adapted from Mahan and Kim, 2001)
Countrya
Canada
Denmark
Finland
Norway
Sweden
United Kingdom
United States
Complete feed
Concentrate
supplement
Salt/mineral
mixes
0.1
0.2
0.3
0.2b
0.3
0.5
0.3
0.7
-c
25
20
30
90 d
mother to the progeny via placenta, colostrum and milk, which could be translated
into better immune system development in piglets resulting in improved piglet
viability and decreased pre-weaning mortality, improved their growth and
development and improved meat quality. Indeed, in stress conditions, when
requirement for antioxidants substantially increased, but feed consumption is
usually decreased Se reserves, accumulated in tissues, could
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Parameter
Effect of organic vs
inorganic selenium
References
Increased
Less toxic
Increased
Decreased
Improved
Increased
Increased
Increased
Increased
Improved
Increased
Increased
Increased
Decreased
Decreased
Increased
Increased
Increased
Increased
Reduced
Increased
Decreased
Decreased
Increased
FCR
Improved
be a key for the resistance of swine to detrimental effects of free radicals and
toxic products of their metabolism. Environmental concerns about selenium as a
potential pollutants lead to legal restrictions of levels of Se, which can be included
into animal diets. Therefore, better assimilation of selenium from organic sources
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Introduction
Despite substantial achievements in dairy and beef cattle industries for the last 20
years there are several problems which need to be solved. It seems likely that
three weakest links include:
immunity
high somatic cell counts
mastitis
reproduction
low conception rate
retained placenta
metritis
cystic ovaries
viability in early postnatal life (calf death loss at birth or between birth and
weaning is a significant source of reduced reproductive rate
487
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Table 9.1 Selenium responsive diseases in ruminants (Adapted from Gates and Johnson, 1995; Pehrson,
1993; NRC, 1996)
Disease
NMD
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The disease mainly affects both the skeletal and cardiac muscles. However, rib
muscles and diaphragm may also be affected (McDowell et al., 2002). When the
skeletal muscles are affected, symptoms vary from mild stiffness to obvious pain
upon walking, to an inability to stand. Muscular tremor may take place and the
affected muscles tend to be swollen and firmer than normal. The diseased animals
have a stiff gait, arched backs and are unwilling to move. Lambs/kids may tremble
in pain when held in a standing position. Indeed, affected young animals are
characterised by difficulty walking and may be unable to rise and nurse. They
gradually loose strength and become weak with decreased foraging with a possible
death from starvation. Therefore the main clinical symptoms of calves with WMD
were motor disturbances including recumbency and stiffness and weakness
(Hoshino et al., 1989). In addition, heart failure, paralysis of the hind legs and a
dystrophic tongue may be evident. Mortality of diseased animals can be as high
as 30% (Telford and Einarson, 1971). Because of weakening immune system
secondary infections can also be found in affected animals. In fact, some animals
show signs of respiratory distress and may be treated for pneumonia by antibiotics
without success and they can die as a result of fluid accumulation in the lung.
This disorder has been observed in all farm animal species, especially in rapidly
growing lambs and calves in areas where the feeds consumed contain between
20 and 30 ppb selenium in dry matter. Two etiologic forms of the disease have
been described, congenital and juvenile. The congenital form usually results in
abortion, stillbirths, or death shortly after birth with degenerative changes of the
myocardium (Andrews et al., 1968; Hartley and Grant, 1961; Hidiroglow, 1980).
For example, a 13-hour-old calf was involuntary recumbent since birth. Congenital
nutritional muscular dystrophy was diagnosed based on clinical findings, increased
serum creatine kinase and decreased serum vitamin E and selenium levels
(Abutarbush and Radostis, 2003). Recovery followed after supportive therapy
and parenteral vitamin E and selenium (Table 9.2). Similar case was described
with 18-hour-old recumbent calf developing dyspnea (Gawley and Bradley, 1979).
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Table 9.2 Effect of Se and vitamin E treatment on a beef calf with congenital muscular distrophy
(Adapted from Abutarbush and Radostitsm 2003)
Reference range
Day 2 (before treatment)
Day 3
Day 4
Day 5
Day 6
Day 7
35-280
29280
6200
966
267
119
166
78-132
299
198
145
97
78
Therefore, cows receiving a Se-deficient diet during gestation may give birth to
offspring suffering from NMD. Furthermore, the calves may be born dead or
extremely weak and die during the first few days of life (Hansen et al., 1993).
An outbreak of nutritional muscular dystrophy which caused the perinatal death
of 11 calves in a herd in the Las Colonias region of Santa Fe Province was
described (Rodriguez et al., 1998). Post-mortem examination of the calves showed
skeletal muscle degeneration as well as heart, liver, kidney and spleen lesions.
Furthermore, CPK and AST levels were higher than normal in the sick animals.
After parenteral treatment of calves and pregnant cows with Se and vitamin E,
blood chemical values returned to normal. Indeed, selenium deficiency in bovine
fetuses may be responsible for myocardial necrosis and heart failure (Orr and
Blakley, 1997). In general, the juvenile or delayed form of the disease most
commonly affect calves 3 to 8 weeks old with predominant sign in muscular
weakness (Campbell et al., 1990).
Muscular dystrophy in adult selenium-deficient dairy cows due to increased
locomotor activity and stress associated with the change in housing conditions
was described (Pavlata et al., 2001). In particular, the investigations were conducted
in a herd of Bohemian Red Pied cattle at the time of a transfer from a stanchion
barn into loose boxes. Clinical muscular dystrophy, manifested by general
weakness, subcutaneous oedema and downer syndrome, accompanied by marked
increases in creatine kinase and lactate dehydrogenase activities was observed in
dairy cows of the transferred group.
Similar to calves, there are two major critical ages for NMD in lambs. Firstly,
during first days of life, lambs born to Se-deficient sheeps were weak and died
within a few hours after various stresses, such as physical exercise. Secondly, Sedeficient lambs between three and six weeks were also very sensitive to NMD
(Pehrson, 1993). Furthermore, lambs and calves three months old are also affected
(Shamberger, 1983). In general, lambs are more affected by NMD than calves.
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Group
No of lambs
ALT
AST
LDH
CPK
20
20
18
4.9
17.2
6.8
26.8
238.8
42.7
684.8
1815.7
642.5
43.4
1168.9
153.3
Control
NMD
NMD after treatment
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Protective effect of selenium and vitamin E was shown in sheep and in cattle
(Shamberger, 1983, Pehrson, 1993). Indeed, early cases of NMD can be treated
with parenteral injection of vitamin E and selenium. As can be seen from Table
9.2, elevated activities of muscle-related enzymes in plasma are normalised in 56 day period. The major preventive measure is optimal dietary supplementation
of selenium (preferentially in organic form) and vitamin E. It has been shown that
maternal nutrition is the most important factor in preventing their progeny from
early development NMD (Hidiroglou et al., 1972; Jenkins and Hidiroglou, 1972).
However, in spite of sodium selenite dietary supplementation of the cows, many
practitioners report that NMD still is a common disease among fast-growing calves,
particularly during their first month of life. It seems likely that this is a result of
poor Se transfer to the colostrum and milk from sodium selenite associated with
inadequate Se status of calves. Therefore NMD in nursing calves can be prevented
if the diet of the cows are supplemented with organic selenium (Pehrson, 2004).
Did you know that the major preventive measure against NMD
is optimal dietary supplementation of selenium
(preferentially in organic form) and vitamin E?
As a result of degenerative myopathy from Se deficiency a disease condition
called shoulder lameness or flying scapular was described (Buergelt et al.,
1996). It is characterised by bilateral dorsal scapular displacement which occurs
suddenly and leads to stiff gait and reluctance to move (Gunning and Walters,
1994).
adaptation of the rumen to lactation diets that are high in energy density
maintenance of normocalcemia
maintenance of a strong immune system
It is well accepted that, the incidence of both metabolic and infectious diseases is
greatly increased when one or more of these physiological functions are impaired
First two weeks of lactation are considered to be an important stress-period for
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cows. This is a most sensitive part of transition period, which lasts from 3 weeks
before parturition to 2-3 weeks after parturition (Drackley, 1999). This is a
transition from non-lactating pregnant status to non-pregnant lactation status, a
period critically important to health, production and profitability of dairy cows.
Indeed, most infectious diseases and metabolic disorders including retained
placenta, metritis and mastitis take place during this time. It is well established
that a significant suppression of immune function takes place in the peripartum
period. The physical and metabolic stresses of pregnancy, calving, and lactation
may contribute to this decrease in host resistance and the subsequent increase in
disease incidence (Goff and Horst, 1997; Mallard et al., 1998). Indeed, substantial
evidence indicates that innate and acquired defense mechanisms are lowest from
3 wk precalving to 3 wk postcalving. This leads to the increased incidence of
peripartum disease.
Did you know that retained placenta has remained a
problem for dairy industry affecting about 9% of all
calvings in US ?
Retained placenta (RP) is a reproductive abnormality unique to the cow and water
bufallo among domestic ruminants with the incidence varying from 3 to 39% of
parturition (Kimura et al., 2002). Indeed RP has remained a problem for dairy
industry affecting about 9% of all calvings in US (Kellogg et al., 2001). In fact,
RP is responsible for decreased milk production (Lucey et al., 1986). In
accordance with various estimations total cost associated with RP ranges from
about $100 to $280/case (Joosten et al., 1988; Kelton et al., 1998; Weiss, 2003).
In particular, substantial economic losses in dairy industry were related to RP
with the cost estimated per case to be $285 (Guard, 1999).
It has been shown that, major reproductive disorders (dystocia, retained placenta
and metritis) are interrelated and can affect the length of calving interval, the
number of days open, and the reproductive efficiency in general (Rajala and
Grohn, 1998). In general, cows with RP are those which are at high risk of
development of metritis and low fertility (Coleman et al., 1985). Furthermore,
dystocia and stillbirth increased the risk of retained placenta, while dystocia and
retained placenta increased the risk of metritis (Emanuelson et al., 1993; Table
9.4). On the one hand, a cow with a retained placenta was 4.4 times more likely to
develop early metritis and 2.5 times more likely to develop late metritis than one
without. On the other hand, a cow with endometritis was 1.5 times more likely to
develop cystic ovaries and silent heat. The involution of the uterus is prolonged,
the resumption of ovarian function is delayed, fertility is impaired and the period
between two calvings extended (Kudlac, 1991). Cows with retained placenta had
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Risk factors
Dystocia Retained
placenta
Dystocia
Abortion
Reteined placenta
Early metritis
Late metritis
Silent heat
Ovarian cysts
Other infertility
Ovarian
cysts
4.1
19.8
Early
metritis
3.2
3.7
4.4
1.5
2.9
1.5
5.4
2.8
Late
metritis
Silent
heat
Other
infertility
3.5
2.5
1.6
2.1
1.3
1.5
3.0
1.7
2.4
2.2
3.6
1.6
2.6
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Table 9.5 Reported positive effects of vitamin E and Se on the incidence of retained placenta (Adapted
from Allison and Laven, 2000).
Percentage
incidence
Control
Treatment
28.3
8.9
16
37.7
20.2
20.6
25.4
51.2
16.3
14.9
8.8
37
Treatment
Year
1985
1984
1980
1981
1976
1969
35
30
25
20
15
10
5
0
Control
Vitamin E
Selenium
Se + vit. E
Figure 9.1 Effect of Se and vitamin E on incidence (%) of retained placenta (13 studies; Adapted from
Harrison and Hancock, 1999)
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500
(Lewis, 1997) and the cost to producers for each lactating dairy cow with a uterine
infection (i.e. metritis) was $106. Therefore, metritis has a major impact on the
profitability of dairy operation.
It was shown (LeBlanc, 2002) that of 1865 cows examined, 316 cows (16.9%)
were classified as having clinical endometritis. Furthermore, cows with endometritis
had a longer time to pregnancy and therefore affected cows had a 30% relative reduction
in the probability of pregnancy at first insemination. Furthermore, cows with clinical
endometritis were also less likely to become pregnant at all, and correspondingly
70% more likely to be culled for reproductive failure. It took approximately 32 d
longer for half the cows with endometritis to become pregnant than it did for unaffected
cows, adjusted for herd, parity, ovarian structures, and treatment (LeBlanc, 2002).
It seems likely that Se-deficiency is a factor related to metritis development and Se
supplementation could decrease incidence of this disorder. For example, a 104-cow
herd was divided into 2 groups, each given daily 150 g of a mineral supplement
containing either 0.5 or 19 mg of Se per kg of supplement for 15 months. For the first
3 months there was little difference between the groups, but subsequently the Sedeficient cows had a higher incidence of mastitis (22 versus 12 cases), endometritis
(17 vs. 8 cases) and embryonic mortality (5 vs. 0 cases) (Klawonn et al., 1996).
Decreased incidence of metritis in Se-treated dairy cows provides an example of an
association between Se deficiency and decreased disease resistance (Suttle and Jones,
1989). Injection of sodium selenite (50 mg) at 21 days prepartum was associated
with a significant reduction of metritis insidence (from 83% to 57%) in cows (Harrison
et al., 1984). In the same study the incidence of cystic ovaries decreased from 47 to
19% (Figure 9.2). Furthermore Se supplementation of cows with metritis resulted in
more rapid uterine involution (Harrison et al., 1986).
60
50
ab
Se (ppm)
a
40
30
b
20
10
Control
Selenium
Vitamin E
Se + E
Figure 9.2 Incidence (%) of cystic ovary depending on Se and vitamin E status of cows (IM Se of 50 mg
21 days prepartum, 740 m g Vit E/day orally; Adapted from Harrison et al., 1984)
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Physiological factors:
stage of lactation
number of lactations
milk yield
Environmental factors:
climate
season
time of day
Production factors:
technological environment
feeding
milking
It was shown that SCC increases immediately after calving and the elevated count
is seen for 5 days to 2 weeks. Subsequently, SCC drops to the normal value,
though it tends to increase during late lactation. SCC also increases with lactation
number or age (Baltay and Bedo, 2000). A significant negative correlation between
milk yield and SCC during lactation has been also described (Kehrli and Shuster,
1994). Indeed, increased SCC is associated with milk loss (Table 9.6). It is
necessary to take into account that high-yielding cows are especially susceptible
to stress, therefore they need additional attention to maintain low SCC.
Table 9.6 Estimated differences in lactation milk production associated with an increase in SCC score
(adapted from Harmon, 1994; Raubertas and Shook, 1982).
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1
2
3
4
5
6
7
12.5
25
50
100
200
400
800
1600
502
-91
-181
-272
-363
-454
-181
-363
-544
-726
-907
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breeding
husbandry
hygiene
milking technique
nutrition
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during the subsequent lactation, but the effect on mastitis and intramammary
infections was not significant (Morgante et al., 1999). This was associated with a
redistribution of various cell counts in the milk. Similar results were reported in
goats (Atroshi et al., 1985). Indeed, Se supplementation was associated with a
decrease in the output of milk somatic cells in cows (Malbe et al., 1995). This was
confirmed by Hemingway (1999) using Se boluses and showing 2-fold decrease
in SCC. A significant negative correlation between the activity of GSH-Px in whole
blood and the bulk milk somatic cell counts in the herds with a low incidence of
mastitis was shown (Ndiweni et al., 1991). The authors suggested an existence of
an association between the incidence of subclinical mastitis or inflammation and
the selenium status of these herds. The herds with the low SCC (less than or equal
to 150000 cells/ml) had higher mean blood GSH-Px activity (35.6 2.95 mU/mg
of haemoglobin) than did the herds with the high SCC (20.2 2.38 mU/mg of
Hb). Whole blood concentrations of Se were higher in herds with low SCC (0.133
0.01 mg/l of blood) than in herds with high SCC (0.074 0.007 mg/l of blood).
Furthermore significant negative correlations were found between the prevalence
of intramammary infection with major pathogens and mean herd activity of GSHPx and mean herd concentrations of Se (Erskine et al., 1987).
Table 9.7 Effect of selenium and vitamin E on ewes (Adapted from Morgante et al., 1999).
Control
Experimental*
Blood
Lysozyme, g/ml
GSH-Px, U/ml of packed cell volume
White blood cells, x 106/ml
Lymphocytes, x 106/ml
4.3
86.3
10.8
6.3
4.8
139.5
9.0
4.9
Milk
SCC, x 103/ml
SCC, log10
2733
6.0
1272
5.4
38.4
50.7
7.6
0.8
1.4
48.8
40.1
8.4
1.0
0.7
*Two subcutaneous injections of Se (0.1 mg/kg of BW) and vitamin E (5 mg/kg BW) at 2
weeks intervals from about 1 month before lambing
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508
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Trial A
Sedeficient
Ewes to ram
Se in blood of
At joining flock to rams
ewes, ng/ml
A month before start of lambing
No. of barren ewes
No. of lambs born
No of ewes with twins
Wt. gain, kg
One month before joining to
one month before lambing
Ewe mortalities
Between joining and lamb
docking
During late pregnancy
Wt. of single
lambs at birth, kg
Trial B
Sedosed
Sedeficient
Sedosed
108
9.1
9.8
45
46
4
-5.6
108
48.0
23.0
15
96
15
-0.4
200
8.0
8.6
79
139
28
200
55.0
25.0
14
249
68
22
12
10
4.1
2
4.6
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supplementation improved the live body weight of calves. The overall mean of
weight gain and relative growth rate of calves per week were significantly increased
(4.24 vs. 3.64 kg and 8.42 vs. 7.84%, respectively; Youssef et al., 2000).
In experiment conducted in Italy selenium was added to the calve diets during
weaning to increase their selenium content from 0.06 to 0.1, 0.15 and 0.2 ppm
for 120 days (Bonomi et al., 2001). There was an improvement in calves weight
gain by 7.0, 12.5 and 17.0% respectively. Selenium supplementation improved
the feed utilisation efficiency of the calves by 5, 9 and 12% respectively.
Furthermore, selenium supplementation, though limited to doses 0.15 and 0.2
ppm, improved the reproductive efficiency of the heifers: the number of services
per conception was 1.8 in the control group and 1.4 and 1.2 in experimental
groups respectively (Bonomi et al., 2001a). In another experiment selenium was
added to the milk replacer at a dose of 0.3 or 0.4 ppm and this improved the
weight gain of the calves (by 9 and 12%, respectively) and feed efficiency (by 9
and 14%; Bonomi et al, 2001b). Furthermore, it improved the carcass yield (by
4.5 and 8.0%), meat digestibility and meat tenderness. Selenium supplementation
also positively affected the health status of the calves, with a strengthening of the
organic resistance of the animals to respiratory and gastro-intestinal diseases.
Similar positive results were reported for young bulls fed from 300-650 kg
liveweight a selenium deficient diet (0.06 ppm Se) or the same diet supplemented
with selenium to give Se level 0.3-0.4 ppm (Bonomi, 2001).
There are also other detrimental consequences of Se deficiency in cattle.
For example, swelling of the hocks with lameness developed in 80 heifers of a
herd, commencing just after calving in Germany. It responded to sodium selenite
injection and a daily supplement of 5 mg Se for cows, or 2-3 mg Se for calves
(Eicken et al., 1992).
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Parameter
Control
Se-injection
Se + Vitamin E
injection
Ewes in oestrus, %
76
100
Ewes lambing, %
68
100
Number of lambs born of ewes lambing
100
112
Lamb live weight at birth, kg
4.2
4.4
No. lambs reared to 28 days
15
25
Lamb live weight at day 28, kg
9.4
10.9
Daily live weight gain for 28 days, g
197.7
241.8
.
* Four weeks before the mating season and 4 weeks before lambing
92
76
105.3
4.3
19
10.0
216.1
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endometrites and ovarian disorders. Furthermore, days to first service and service
period were reduced. Cows (296) from a Se-deficient herd, with blood serum Se
concentration <25 ng/ml, were divided randomly into two groups and injected
i.m. with 0 or 20 mg Se as sodium selenite at about 3 weeks after parturition. The
frequency of ovarian cysts, conception rate and service period in treated cows in
one herd were 20.8%, 55.8% and 97.6 days, compared with 39.6%, 40.2% and
121.4 days in controls (Jaskowski and Rogoziewicz, 1990). At the beginning of
the dry period and 30 days later, cows were injected with 50 mg sodium selenite
+ 600 g alpha-tocopherol. Treatment reduced the incidence of retained placenta
by 7% and involution time of the uterus by 23%, and reduced the service period
by 16.3 days compared with these traits in untreated cows (Kolchina et al., 1991).
Three selenium supplementation trials, conducted from 1986 to 1991, at the
University of Arkansas Southwest Research and Extension Center. The results
showed that Se supplementation increased fertility of cattle from 55 to 80%, and
increased weaning weight by 41 lb (Phillips et al., 1992). The authors admitted
that when cattle are deficient or low in Se, it takes 60 to 90 days for body stores of
Se to accumulate for production levels to exceed maintenance requirements.
Table 9.10 Effect of Se-supplementation (0.1 ppm) of Se-deficient cows in two herds (Adapted from
Cortese, 1988).
1st herd
Control
Number of services per conception
Days open
Calving interval, days
Blood selenium concentration
immediately after calving, mg/L
Blood selenium concentration at peak
of lactation, mg/L
2nd herd
SeControl
Sesupplemented
supplemented
2.34
136.5
421.5
0.038
2.10
116.1
401.1
0.081
2.82
167.6
452.6
0.021
1.81
98.0
383.0
0.073
0.053
0.088
0.047
0.076
Table 9.11 Effect of Se injection on cow reproduction (Adapted from Jaskowski, 1990)
Retained placenta,%
Stillborn calves, %
Endometritis, %
Ovarian disorders
Days to first service
Service period, days
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Control
50 mg Se, 21 d
before parturition
17.9
5.1
30.8
35.9
79.2
128
10.2
0
33.4
25.6
57.4
86.9
50 mg Se, 10 d
before parturition
10.2
2.6
18.0
23.2
59.9
106
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Control
Se/vitamin E
63.3
46.1
52.1
2.0
98.1
61.7
45.4
69.8
1.7
84.6
*A single intramuscular injection of selenium (50 mg) and vitamin E (500mg) at day 303
postpartum
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ewes, increased proportion of ewes with multiple births and some others. Selenium
prepartum injections also resulted in higher colostrum and milk concentrations of
Se (Cuesta et al., 1995).
It seems likely that ROS may function as intracellular regulators of
steroidogenesis in the corpus luteum (Carlson et al., 1993). Therefore, luteal cells
can employ reactive oxygen species at specific sites in controlling the production
of progesterone over the course of the reproductive cycle and in inhibiting its
synthesis during regression at the end of the cycle. Se supplementation of cows
(0.5 ppm) did not affect the length of the oestrous cycle, but it did significantly
increase the concentration of plasma progesterone during the oestrous cycle
(Kamada and Hodate, 1998).
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Supplementation
No supplementation
West
Central
South
east
West
Central
South
east
4.4
8.0
20.3
67.3
0
3.6
30.9
65.5
16.7
23.3
40.9
19.1
4.9
4.4
13.1
77.6
7.9
9.2
36.4
45.6
29.1
32.0
30.8
8.1
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(Palvata et al. 2001a). In Poland, a total of 262 animals were evaluated: 151
horses, 60 cattle and 51 sheep. In 70.1% of the animals, Se levels were either low
or very low (<10 ng/ml, Ramisz et al., 2001). When 11 dairy herds from the
central regions of Estonia were studied, it was concluded that the selenium status
of cows was deficient, similarly to that in Sweden, Norway and Finland (Pehrson
et al., 1997). When 18 selenium unsupplemented or irregularly supplemented
herds in Slovenia were evaluated based on selenium concentration in the liver,
34.3% of animals were classified as adequate, 37.2% as marginal and 28.5% as
Se-deficient (Zust and Hrovatin, 1994). Similarly, in late lactation and the dry
period when Se was given as a mineral mixture, about 60% of the cows had
marginal blood Se values (Zust et al., 1995). Blood selenium was estimated in a
total of 172 calves, 163 heifers and 399 cows examined in a cattle clinic in Giessen,
Germany, from May 1990 to May 1991. Average values were Se 0.046 mg/litre,
compared with literature values of <0.04, 0.04-0.07 and >0.07 mg/litre, identified
as Se-deficient, marginal supply and adequate supply, respectively. Indeed, 50%
of individual cattle were deficient, 25% marginal and only 25% were sufficient in
Se (Grunder and Auer, 1995). In Ireland in 1993 soil Se total content ranged from
0.2 to 6.0 mg/kg DM with over 90% of herbage samples being deficient in Se.
Furthermore, low or very low blood Se status was found in 17% of herds (Mee et
al., 1996). Selenium status of 50 dairy herds in the Irish Republic were studied
during the spring and autumn of 1991. Only dry cows were sampled in spring
and only lactating cows were sampled in the autumn. Herds were classified as
very low, low, marginal, normal, high or very high Se status. In the spring 18
herds were very low in Se with a further 9 herds having low Se status, while in the
autumn, 20 herds were low or very low in Se (Mee et al., 1993). Similarly, out of
the 205 cattle slaughtered in the area of Thessaloniki, Greece, 78% presented
deficient concentration of Se in liver (0,110-0,600 g/g DM), 17% marginally
deficient concentration (0,601-0,900 g/g DM) and only 5% normal concentration
(0,901-1,512 g/g DM; Christodoulopoulos et al., 2000a). Plasma selenium was
studied in dairy cows within the region of Thessalonica, Greece. The survey
included 65 dairy farms from which samples of blood were collected from 10
cows from each farm. Out of the total 650 cows examined, 40% presented deficient
concentration of Se in blood (<0.08 mg/L), 29% presented marginally deficient
concentration (0.08-0.12 mg/L) and only 31% had a normal concentration (>0.12
mg/L; Christodoulopoulos et al., 2000). Regional and seasonal selenium
nutritional status was studied in cattle and sheep, in 5 regions of Turkey, using
neutron activation analysis of blood. Blood Se was 51.0 to 75.0 ng/ml. In particular,
in the Bursa region cattle on a state farm were severely Se deficient (33.0 ng/ml,
Oncuer et al., 1996).
Many Latin America countries are also Se deficient. For example, the content
of Se in forage samples and the blood activity of GSH-Px of lactating cows and
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Table 9.14 Effect of Se supplementation on Se status of cows (Adapted from Hemigway, 1999)
Treatment
Results
References
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1987
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Treatment
Results
References
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chemical forms of Se
level of Se in blood and tissues before supplementation
presence of various minerals and amino acids in the diet
concentration of Se in the diet
Did you know that inorganic Se assimilation in ruminants is quite
low: rumen bacteria reduce it to unavailable metallic Se and Se
incorporated into bacteria proteins is characterised by low
availability?
In a Se-balance trial with cows in the late pregnancy and lactation period, feeding of
diets containing Se 0.106 mg/kg resulted in daily Se intake of 973.3 g and daily Se
accumulation of 136.9 g (Kamada et al., 1998). Therefore only 14% Se consumed
was accumulated in the body. Estimates of total body selenium in cows are extremely
limited; in dry dairy cows fed diets with 0.03 mg Se/kg DM estimated total body
selenium was 44 mg (Koenig et al., 1991a).
The metabolism of intravenously dosed 75Se was studied in 10 Holstein bull calves
fed ad libitum on a commercial diet. The total endogenous 75Se in the faeces over the
4-day collection period was about 4.14% of the dose. About 97% of the 75Se dose
disappeared from the calfs blood within 6 h after dosing (Neathery et al., 1990). The
principal route of Se excretion in ruminants is the faeces (Wichetel, 1998; Podoll et
al., 1992), but selenium is also expelled from the body via the lung, especially in the
case of high Se consumption. When Se is administered by intravenous or subcutaneous
injection to ruminants, urine is the major route of excretion (Wright and Bell, 1966).
Urinary Se excretion in sheep was shown to account for 23-33% with the remainder
voided via feces (Koenig et al., 1997).
Urinary excretion of Se in lambs and goats was higher when sodium selenite was
used as a dietary supplement in comparison to SeMet (Ehlig et al, 1967; Aspila,
1991). A significantly higher faecal excretion, lower apparent absorption and lower
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Period I
Period II
Selenite
SeMet
Selenite
SeMet
Se intake, ug/day
60.4
55.5
56.0
55.8
Faeces
Se output, ug/day
Apparent absorption, %
34.0
43.9
22.8
59.0
31.1
44.4
24.2
56.8
Urine
Se output, ug/day
Output/Intake,%
Se retention, % intake
3.6
6.0
38.3
2.5
4.4
54.5
5.4
9.7
34.7
5.2
9.4
47.4
Table 9.16 Selenium balance in lactating cows (Adapted from Ivancic and Weiss, 2001)
Parameter
Dietary Se supplementation
0.1 ppm Se
0.3 ppm Se
Se intake, mg/day
Se apparent digestibility, %
Se output, mg/day
2.50
38.6
4.82
46.9
Fecal
Urinary
Milk
Total
Absorbed Se, mg/day
Se balance, mg/day
1.46
0.52
0.48
2.46
1.04
0.04
2.58
1.40
0.39
4.36
2.24
0.46
When dose of Se was increased to 4.82 mg/day fecal, urinary and milk Se output
was 2.58, 1.4 and 0.39 mg/day. Therefore, increasing selenite dose was associated
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with lower transfer Se to the milk. The authors showed that milk Se concentrations
were related with plasma Se:
Milk Se (g/ml) = 037 x plasma Se (g/ml) 4.2
The amount of Se excreted per day via urine was also related with plasma Se
concentrations:
Urinary Se (g/day)= 0.031 x plasma Se (g/ml) 0.91
It is well accepted that absorption of selenium (sodium selenite) in ruminants is
much lower than in monogastric animals, while the bioavailability of selenium
from selenite and selenate is similar in ruminants (Table 9.17; Podoll et al., 1992;
Ortman and Pehrson, 1999). Indeed, absorption of orally administered 75Se was
85% in swine and only 34 % in sheep (Wright and Bell, 1966). Similarly, in the
rat, about 86% of labelled selenium ingested orally either in the form of selenite
or selenate was absorbed (Mason and Weaver, 1986).
Table 9.17 Effect of selenium supplementation (0.3 ppm) on farm animals (Adapted from Podoll et al.,
1992)
Species
Dairy cattle
Sheep
Horses
Dairy cattle
Sheep
Horses
Se form
0
Days of supplementation
14
42
Selenate
Selenite
Selenate
Selenite
Selenate
Selenite
Se, mg/l
0.047
0.050
0.084
0.076
0.093
0.101
0.079
0.075
0.114
0.122
0.157
0.165
0.087
0.078
0.131
0.132
0.190
0.199
Selenate
Selenite
Selenate
Selenite
Selenate
Selenite
GSH-Px, U/ml
0.135
0.165
0.089
0.141
0.677
0.674
0.113
0.115
0.123
0.129
0.505
0.606
0.087
0.112
0.152
0.185
0.559
0.610
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and 0.4 mg Se/kg DM. In particular, the production of short chain fatty acids was
optimal at 0.2 and 0.4 mg Se/kg DM but decreased significantly for concentrations
greater than 12.8 mg Se/kg DM (von Brehm, 2001). These data are consistent
with previous observations indicating that Se supplementation can influence rumen
microbial fermentation and that Se compounds differ in this regard (Kim et al.,
1997a)
age
physiological stage
rate of productivity
health status
amount of stresses
balance of nutrients (sulphur, lipids, vitamin E, proteins, amino acids and
several microelements)
species
In particular, increases in the growth rate, milk yield, and reproductive performance
are associated with increase in the Se requirement. It has been shown that increasing
dietary sulphur linearly reduced apparent and estimated true Se digestibility
(Ivancic and Weiss, 2001). Therefore, dietary S from sulfate can reduce Se balance
especially when cows were fed diets with less than 0.3 mg of Se/kg of diet dry
matter. Similarly nitrates in water may tie up selenium and prevent absorption and
high dietary calcium may decrease intestinal absorption of selenium (Harrison
and Conrad, 1984b; Valle, 2001). Excessive consumption of copper, zinc or iron
also reduced Se availability (Smith et al., 1998).
It is important to mention that Se requirements of growing cattle, growing pigs
and the recommendations for children (on the basis of daily intake per kg body
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Table 9.18 Prevalence of infection in groups of cows with low and high Se (adapted from Jukola et al.,
1996)
All pathogens
Cows infected
CNS
Cows infected
SA
Cows infected
APC
Cows infected
no.
no.
no.
no.
Se<150 ng/ml
S>150 ng/ml
108
700
53
345
Se <150 ng/ml
Se>200 ng/ml
108
289
Se <180 ng/ml
Se >200 ng/ml
310
289
28.3
20.8
-27%
28.3
19.7
-31%
28.3
19.3
-32%
24.6
19.3
-21%
11
29
108
498
38.1
29.1
-24%
38.1
30
-21%
38.1
29.5
-23%
30.2
29.5
-2%
34
221
Se<150 ng/ml
Se>180 ng/ml
55.7
45.7
-18%
55.7
45.9
-18%
55.7
45.8
-18%
47.7
45.8
-4%
11.3
3.3
-71%
11.3
3.5
-70%
11.3
3.9
-65%
5.3
3.9
-26%
53
251
53
143
147
143
34
731
34
82
111
82
11
21
11
14
19
14
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reduced prevalence of retained fetal membranes (estimated cost of $100120/case; Harrison et al., 1984; Smith et al., 1998),
reduced severity and prevalence of clinical mastitis (estimated cost $120/
case; Smith et al., 1997; 1998),
reduced milk somatic cell counts (potential quality bonuses for milk; Weiss
et al., 1990; Smith et al., 1998),
reduced calf mortality (Spears et al., 1986).
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Se concentration in serum
Se concentration in whole blood
Se concentration in liver
GSH-Px activity in erythrocytes
GSH-Px activity in liver
mRNA levels for GSH-Px
PH-GSH-Px activity in tissues
Se concentration in muscles
Se concentration in milk and colostrum
Selenoprotein P concentration and expression in various tissues
Deiodinase activity in tissues
TR activity in various tissues
It is necessary to mention that each method mentioned above has its advantages
and disadvantages and data obtained with various methods are interrelated but
are not the same. For example, the concentration of Se in blood plasma was
positively correlated to concentration of Se in the diet when the data set contained
just dry cows (Table 9.19; Weiss et al., 1990):
Plasma Se = 0.059 + 0.044 Feed Se (r2=0.39)
However, no relationship existed between dietary Se and plasma Se in lactating cows.
It is necessary to mention that Se concentration in whole blood is approximately 3fold higher than that in serum (Scholz and Hutchinson, 1979; Smith et al., 1998).
The ratio of whole blood Se to serum Se is approximately 4 to 1 in sheep with low Se
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Table 9.19 Regression equations for predicting blood values from dietary data (Adapted from Weiss et
al., 1990; Knowles et al., 1999)
Intercept
Slope
r2
Se intake, mg/d
Se intake, mg/d
Se intake, mg/d
0.033
0.144
45.67
0.014
0.019
4.49
0.81
0.53
0.40
0.031
0.142
45.46
0.215
0.295
66.25
0.84
0.54
0.39
0.13
1.33
6.7 x 10-5
6.1
0.24
0.63
0.69
0.73
Dependent
Independent
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Status
Se in whole
blood, ng/ml
Severely deficient
<60
Marginally deficient 60-200
Adequate
210-1200
High adequate
>1200
Se in liver
of adults,
ng/mg DM
Se in livers
of newborns,
ng/mg DM
Se in milk,
ng/ml
0.1-0.5
0.6-1.25
1.25-2.5
>2.5
<1.1
1.1-2.2
2.3-8.0
-
<5.5
5.5-10.3*
>10.3
-
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dietary calcium
dietary sulphur
trace elements
genetics
Table 9.21 Selenium reference ranges for liver and serum to diagnose Se deficiency in cattle (Adapted
from Thompson et al., 1998)
Liver Se,
nmol/kg fresh tissue
<130
130-250
250-2000
<600
600-850
>850
<85
85-140
>140
Responsive
Marginal
Adequate
In young sheep liver Se concentrations <250 nmol/kg fresh tissue are considered
deficient, 250-450 nmol/kg fresh tissue, marginal and >450 nmol/kg fresh tissue
are considered adequate (Andrews et al., 1976). Indeed, blood and liver Se
concentrations are highly correlated in sheep (R2=0.97; Sheppard et al., 1984).
Similarly concentrations of Se in whole blood of the lambs were positively
correlated to the Se concentrations in liver (r=0.77; Rock et al., 2001).
Selenium concentration in milk can also be used for Se status assessment.
Indeed, Givens et al. (2004) calculated linear regressions for the relationships
between milk and dietary selenium concentrations:
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Milk Se
Whole blood Se
Erythrocyte GSH-Px
Serum Se
Milk Se
Whole blood Se
Erythrocyte GSH-Px
Serum Se
1.00
0.78
0.70
0.71
0.78
1.00
0.96
0.85
0.70
0.96
1.00
0.76
0.71
0.85
0.76
1.00
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Depending on the level of stress the cow can adapt without detrimental
consequences for productive and reproductive traits or, if stress exceeds adaptive
ability of the animal, the cow can loose its productive potential. In general oxidative
stress was observed in cows during transition period (Miller et al., 1993;
Formingtoni et al., 1997; Ronchi et al., 2000; Bernabucci et al., 2002), which
could be an important contributing factors to various disorders and metabolic
diseases. In particular, a hot environment is responsible for oxidative stress which
worsens already weak antioxidant defences in transition cows (Bernabucci et al.,
2002).
Did you know that depending on the level of stress the cow can
adapt without detrimental consequences for productive and
reproductive traits or, if stress exceeds adaptive ability of the
animal, the cow can loose its productive potential?
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Component
Uterine tissues
Caruncles
Cotyledons
Fetal membranes
Fetus
Entire conceptus
Equation
Linear functions
y= -0.286 + 0.0039t
y= - 0.409 + 0.0038t
y= - 0.152 + 0.0015t
y= - 0.072 + 0.0006t
y= - 7.278 + 0.0414t
y= - 8.839 + 0.0552t
0,75
0,75
0.59
0.75
0.97
0.98
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and calves (Table 9.24; Donald et al., 1994; Awadeh et al., 1998b). Similarly,
pregnant ewes, which were fed additional Se had higher concentrations of triiodothyronine (T3) and thyroxine (T4), however, differences in concentrations of
these hormones in progeny (in lambs at 12 h of age) were not significant and only
trend (P<0.1) for higher T3 level was admitted (Rock et al., 2001). It seems likely
that effect of Se supplementation on the thyroid metabolism is dependent on the
Se status of the animals and when the status is not deficient there is no additional
benefit of selenium. Furthermore, activation of BAT causes large increases in
oxygen consumption and consequently causes increases in free radical production
(Hatfield et al., 1999) and such selenoproteins as GSH-Px, TR and MSRB could
be involved in maintenance of antioxidant defences. As mentioned above, when
Se and vitamin E were injected into cows and calves which were fed diets
marginally deficient in Se calf death loses compared to controls from birth to
weaning reduced from 15.3% to 4.2% (Spears et al., 1986). Indeed a proper Se
supplementation of the dry cow prior to calving and ingestion of colostrum by
calves is critical to providing sufficient Se to neonatal calves and maintain their
antioxidant defences.
Table 9.24 Effect of maternal intakes of selenium on selenium status and thyroid hormones in day old
calves (Adapted from Awadeh et al., 1998)
0.12
0.6
3.1
0.14
0.8
3.9
0.14
0.8
3.1
Year 2
Se in blood, g/ml
GSH-Px in blood, EU
T3, ng/ml
T4, ng/ml
Ratio T3:T4
0.12
0.8
3.4
85.3
0.032
0.14
0.8
3.0
99.2
0.033
0.15
0.7
4.3
70.1
0.053
0.17
0.9
3.4
0.16
0.7
5.3
116.4
0.04
It has been shown that there are some species-specific differences in Se transfer
from the dam to the fetus. For example, in ewes maternal tissues had higher
concentration of labelled Se than the corresponding fetal tissues (Hidiroglou et
al., 1969). Furthermore in sheep the concentrations of labelled selenium in the
maternal plasma was 11.7-fold higher than that in fetal plasma (Wright and
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Alfalfa hay+
soybean+ Se in
mineral mix
Cows
Se in whole blood at time of parturition,
mg Se/kg of blood
GSH-Px in whole blood at time of parturition,
mU/mg of hemoglobin
Alfalfa hay+
soybean
Alfalfa hay
0.250
0.162
0.052
144
80
30
0.175
113
0.081
50
Calves
Se in whole blood, mg Se/kg of blood
0.242
GSH-Px in whole blood, mU/mg of hemoglobin 154
It has been shown that plasma concentrations of Se of calves at birth were correlated
positively with that of their dams at calving (Hidiroglou et al., 1994). Similarly
the RBC GSH-Px activities of 15-day-old calves (U/g of Hb) were significantly
correlated with values of their dams (Enjalbert et al., 1999):
Calf RBC GSH-Px = 59.8 + 1.26 Maternal RBC GSH-Px (r2=0.52; P<0.001)
It is interesting to note that in the aforementioned study using high Se
supplementation (13-45.5 mg of Se/day for 15 days) the RBC GSH-Px activity in
cows and their calves were also significantly correlated 77 to 115 days after calving
(r2=0.44, P<0.001). A significant correlation of Se concentration of the calf at
birth with that of the dam at 2 days after calving (r=0.5) was also reported by
Hidiroglou et al. (1994).
Campbell et al. (1990) suggested several possible mechanisms of the active
accumulation of Se in the fetus:
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There are no data available to show in which form Se is transferred to the fetus.
Indeed, from the one hand if SeMet is present in the diet as an integral part of feed
ingredients one could expect SeMet to be transferred to the fetus and nonspecifically incorporated into the body proteins building Se reserves which can
be used in stress conditions when Se requirement increases. On the other hand,
inorganic Se in the form of sodium selenite or sodium selenate will be transferred
to the fetus most likely as SeCys and will be specifically incorporated into
selenoproteins. However, the rate of synthesis of selenoprotein during fetus
development is not known. Furthermore, the role of rumen bacteria in synthesis
of various selenocompounds with their possible transfer via placenta is not
investigated yet.
When fetuses were collected from pregnant primaparous beef cows it was shown
that in fetal liver Se concentration decreased significantly during the final 60 days
of gestation (Abdelrahman and Kincaid, 1993). Therefore Se dietary
supplementation during late gestation is of great value for the developing fetuss.
Furthermore, supplementation with Se during the dry period increased
concentrations of Se in the liver of calves at birth and at 42 days of age
(Abdelrahman and Kincaid, 1995). The authors showed that supplementation of
dams with >3 mg of Se/day was needed for calves to be born with Se concentration
in plasma of 50 ng/ml.
Did you know that there is a limited ability of the cow to
transfer Se into the colostrum and milk when sodium selenite
is used as a supplement?
The transfer of Se to the colostrum and milk is of great importance to the newly
born calf. Selenium concentration in the milk depends on the Se level and form in
the maternal diet. Indeed, a small proportion of added (as sodium selenite) dietary
Se (4.8%) was transferred to the milk with a deficient diet but only 0.9% of the
amount of added selenium was in milk of cows consuming diets adequate in
selenium. In contrast, 19% of the organic selenium furnished in brewers grains
appeared in the milk when the ration was deficient in selenium (Conrad and Moxon,
1979). In more recent study by Givens et al. (2004) for cows fed the Se-yeast the
efficiency of Se transfer to the milk ranged from 9.9 to 12.5%, compared with
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Colostrum
st
Control
1
Mu-Se (selenite)
2
Deposel (selenate)
3
Sel-Plex-1
3
Sel-Plex-2
nd
Milk*
st
Plasma*
nd
st
1 year
2 year
1 year
2 year
1 year
2nd year
0.032
0.056
0.071
0.092
0.092
0.024
0.035
0.049
0.039
0.065
0.01
0.02
0.02
0.03
0.03
0.02
0.01
0.02
0.02
0.03
0.02
0.02
0.02
0.07
0.07
0.02
0.02
0.02
0.06
0.07
*/180 days postpartum; plasma of calves from cows supplemented with selenium
1
/Subcutaneous injection of 5 ml of Mu-Se (5 mg Se per ml as sodium selenite)
2
/Subcutaneous injection of 9 ml of Deposel ( 50 mg Se per ml as barium selenate)
3
/ The free choice mineral mixture containing 54.5 g/kg Sel-Plex resulting in a Se
concentration of 30 mg/kg in the mineral mixture with average daily Se intake to be 2.1 mg
per cow
Selenium distribution in cow milk was studied. Skim milk contained 93% of the
total milk selenium. Irrespective of the separation technique used, skim milk
selenium was mainly found to be associated with the casein fraction (>55%). In
fact selenium was mainly protein bound. Of all the milk proteins, beta-casein
contributed most to the total skim milk selenium (33% of total Se), while 17% of
the total skim milk selenium was associated with whey proteins (Van Dael et al.,
1991). In goat milk also about 80% of Se was associated with casein fraction
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(Allen and Miller, 1981). Technological processes such as milk heating (UHT,
pasteurisation) and the preparation of milk powder do not provoke substantial
losses of Se. During the production of cheese the Se concentration in dry matter
increases by a factor of 1.4 in comparison to the starting milk (Haldimann et al.,
1999). Recently, the selenium distributions in the different milk phases have been
studied in 14 whole milk samples, and the highest selenium levels were found in
milk whey (47.2-73.6%), while the lowest level was found for the fat phase (4.816.2%). A strong correlation was found between the selenium levels in whole
milk and the selenium levels in the milk components (Muniz-Naveiro et al., 2005).
Polyunsaturated fatty acids of milk can be oxidized causing problems with
milk rancidity. Predisposing factors associated with the development of rancid
flavours in milk include (Duval, 1995):
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and continued this trend throughout the test period (Duarte et al., 2004). Similarly,
inclusion of selenium (0.2 ppm) in the form of Sel-Plex (0.2 ppm) into the cows
diet providing 2.2 mg Se/day in the form of sodium selenite for 8 weeks was
associated with an increased Se level in the milk (0.138 mg/l vs 0.048 mg/l) and
decreased SSC (174,500 vs 229,300 cells/ml; Foltys et al., 2004). Field trial results
with Sel-Plex (Alltech, Inc.) confirm earlier university work indicating that a 20
to 30% increase in whole blood Se and an 80 to 100% increase in milk Se levels
can be associated with selenium yeast supplementation (Eliott et al., 2005).
In a 2 x 2 factorial design experiment, 20 early-lactation Holstein-Friesian
cows, that had been receiving a diet containing 0.33 ppm supplemental Se as
sodium selenite (SS), were fed Se supplement in the form of selenium-enriched
yeast (SY) or SS, each at levels that provided 0.15 and 0.33 ppm supplemental
Se, for a period of 10 weeks following a 2-week period with no supplemental Se
(Hemken et al., 1998). Blood Se levels were significantly higher in cows fed SY
than SS, indicating that the bioavailability of Se in SY is about twice that in SS.
The possibility of improving Se status of pregnant dairy cows and their calves by
supplementing dairy cows with inorganic or organic forms of Se was studied. A
Se-poor diet (0.03 ppm) for 30 dry cows was supplemented with Se. Two months
before calving, cows were divided into 3 groups and supplemented with inorganic
(0.23 ppm) or organic (0.105 ppm) selenium until calving. Se source and
supplementation influenced GSH-Px activity in whole blood of dams and their
calves. Indeed, usage of Se from yeast product for GSH-Px synthesis by cows
was 3.5-fold higher than that of Se from the mineral mixture (Ling and Ploom,
1999). In an experiment conducted at Agricultural Grasslands Research Centre
(New Zealand) Se-enriched yeast or sodium selenate was administered per os
(equivalent of 2 or 4 mg Se/day) three times a week for 133 days to previously
unsupplemented cows that were grazing low-Se pastures. Control cows were not
supplemented with selenium (Knowles et al., 1999). Selenized yeast was 2-3times more effective than was sodium selenite in increasing Se level in blood. In
the first 100 days of lactation cows fed selenium-supplemented rations had
significantly elevated serum selenium concentrations: 21.4 g/l (no
supplementation), 45.3 g/l (sodium selenite, 4 mg/cow/day), 65.4 g/l (selenized
yeast, 4 mg/cow/day) and 57.3 g/l (selenized yeast, 2 mg/cow/day; Falkowska
et al., 2000). Indeed, selenium from yeast was utilized better than sodium selenite.
Supplemental organic Se gave higher blood Se and GSH-Px activity than inorganic
Se. After 4 months of Se supplementation (1 mg/day) both Se concentration in
whole blood of beef calf and GSH-Px activity were higher for calves given Seenriched yeast those given inorganic Se (Nicholson et al., 1991a,b). Se
supplementation with Se-enriched yeast 5 mg/day increased Se in milk from 13.2
to 34.7 g/litre (Charmley et al., 1993). It is interesting that efficiency of Se transfer
from feed to the milk was highest (up to 24.6%) when organic selenium in the
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At the end of the trial, the mean whole blood Se concentrations increased
from 104 g/l (control group) up to 138, 141, 165 g/l in the selenite,
selenate, and yeast groups, respectively.
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These data were confirmed by McIntosh and Royle (2002) showing that inclusion
of Sel-Plex in the cows diet to provide 2 or 6 mg Se/day was associated with
increased Se concentration in the milk from 6.9 up to 15.0 and 25.2 ng/ml
respectively (Figure 9.3). Similarly, in commercial conditions of Florida dairy a
replacement of sodium selenite with Sel-Plex was associated with a significant
increase in Se concentration in blood and colostrum (Figure 9.4). Furthermore,
increase Se supplementation in the form of Sel-Plex from 2.67 to 6 mg/day was
associated with an increase in Se concentration in the colostrum for the first two
milkings by 42% and for the first 8 milkings by 35% (Lewis, 2004).
30
25
Se (ng/ml)
20
15
10
5
0
0mg Se/day
2mg Se/day
6mg Se/day
Figure 9.3 Effect of Se-Yeast on milk Se status (Adapted from McIntosh and Royle, 2002)
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Blood Se
Colostrum Se
abc
Se (ppb)
170
120
b
70
a
20
Pre-Sel-Plex
1 month on
2 months on
Date sampled
Figure 9.4 Blood and colostrum Se at calving at Florida dairy (Adapted from Eliott et al., 2005)
and blood measurements. Cows had ad libitum access to one of three free-choice
minerals: 1st: no supplemental Se, 2nd: 26 mg of supplemental Se from sodium
selenite/kg, and 3d : 26 mg of supplemental Se from seleno-yeast/kg (designed
intake = 113 g/cow daily). At the beginning of the calving and breeding seasons,
cows supplemented with Se were characterised by greater whole blood Se
concentrations and GSH-Px activities in comparison to cows receiving no
supplemental Se; cows fed selenoyeast had greater whole blood Se concentrations
than cows fed sodium selenite, but GSH-Px did not differ between the two sources.
At birth and near peak lactation, calves from cows supplemented with Se had
greater whole blood Se concentrations than calves from cows fed no Se and calves
from cows fed seleno-yeast had greater whole blood Se concentrations and GSHPx activities than calves from cows fed sodium selenite (Tables 9.27-9.28, Gunter
et al., 2003). Furthermore immunity of weaned beef cattle was improved. Indeed,
lymphocyte proliferation (non-stimulated) and macrophage phagocytosis was
significantly improved by Se-Yeast in comparison to sodium selenite or nonsupplemented animals (Table 9.29).
A field trial was conducted at a large Florida dairy to evaluate the response in
whole blood and colostrum selenium to organic selenium supplementation (SelPlexTM). In the trial Sel-Plex replaced 100% of the sodium selenite in the dry cow
supplement and provided 0.3 ppm Se. Whole blood samples were taken from 16
cows as they entered the dry lot (pre-Sel-Plex). Samples were also taken 1 mo
and 2 mo on Sel-Plex from the same cows. An additional set of blood and colostrum
samples were collected from 4 randomly selected cows after calving (Harrison et
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Table 9.27 Whole blood Se concentration in beef cows depending on selenium supplementation
(Adapted from Gunter et al., 2003)
Sel-PlexTM**
No Se
Sodium selenite*
Cows
Beginning of mineral feeding
Start of calving season
Initiation of breeding season
101
108
93
111
142
150
122
174
187
Calves
Birth
Peak of lactation
105
51
134
66
203
122
No Se
Sel-PlexTM**
Sodium selenite*
Cows
Beginning of mineral feeding
Start of calving season
Initiation of breeding season
57
74
58
46
101
150
52
106
187
Calves
Birth
Peak of lactation
56
96
62
121
115
190
Item
No Se
Sodium selenite**
Sel-PlexTM***
54.7
40.2
105.0
119.1
172.0
143.9
1,426
65,586
12,286
13,441
5.0
867
77,658
18,414
10,077
4.2
1,408
92,261
24,066
16,238
9.2
*22 days post-weaning; the weaning supplement was calculated to supply 0.24 ppm Se (DM
basis).
**/26 mg of supplemental Se/kg of free choice mineral from sodium selenite for cows
***/26 mg of supplemental Se/kg of free choice mineral from Sel-Plex for cows
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3mg
selenite
0.75mg 0.75mg
Se yeast Se yeast
3mg Se
yeast
3mg Se
yeast
Figure 9.5 Selenium concentration (ng/g wet weight) in skeletal muscle of cows supplemented with
selenium (Adapted from Ortman and Pehrson, 1997)
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Serum fraction
20 mg/kg
Albumin
GSH-Px
Selenoprotein P
29.3
14.6
53.6
60 mg/kg
120 mg/kg
% of Se in protein fraction
20.1
17.1
73.0
60 mg/kg (SeY)
11.8
6.0
73.9
15.6
9.3
75.2
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Growing beef and dairy cattle were supplemented with sodium selenite or selenized
yeast to supply 1 mg Se/kg supplement and a comparison was made with
unsupplemented cows (Nicholson et al., 1991a). Blood Se concentration was
significantly (P<0.01) higher for the organic (141 ng/ml) than for the inorganic (102
ng/ml) or control (45 ng/ml) groups. Similarly, the cows receiving Se from fertilized
alfalfa silage were characterised by increased Se concentration in the milk (58 ng/ml)
or plasma (111 ng/ml) in comparison to sodium supplemented or unsupplemented
animals (Nicholson et al., 1991b). It is interesting, that increased Se concentration in
beef as a result of Se-yeast dietary supplementation is associated with improvement
of beef quality, in particular with decreased drip loss (Table 9.31).
Table 9.31 Effect of organic selenium (Sel-Plex, per os 4 mg/heard/day) on beef quality (Adapted from
Simek et al., 2002)
Se in Longissimus
dorsi muscle, mg/kg
Control
7 day supplementation
30 day supplementation
0.107
0.168
0.223
Drip loss, %
Vacuum-stored for
four days, %
0.92
1.17
0.50
0.29
0.45
0.03
Therefore, it has been proven that organic selenium in the form of Selenized yeast
(Sel-PlexTM) is more effectively increases Se concentration in blood and milk in
comparison to selenite (Ortman and Pehrson, 1999; Knowles et al., 1999). In lambs
organic selenium is also more effectively accumulated in skeletal muscles and other
tissues (Ehlig et al., 1967). Furthermore, 90 ppm Se as selenized yeast (Sel-PlexTM) or
sodium selenite in the trace mineralised salts was fed to sheep from Day 56 of the
experiment until lambing and providing Se intake equivalent to about 0.2 mg Se/kg
of diet DM (Rock et al., 2001). The results of the experiment are summarised in Table
9.32 and indicate increased (in comparison to selenite) Se concentrations in the whole
blood and liver as well as in colostrum as a result of organic selenium supplementation.
Table 9.32 Effect of supplemental Se during gestation on concentration of Se in colostrum and liver and
blood and activity of GSH-Px in newborn lambs (Adapted from Rock et al., 2001).
Control
Selenitea
SeYb
0.101
145
0.63
0.012
0.234
414
1.34
0.132
0.434
640
1.80
0.226
/90 ppm Se as sodium selenite in the trace mineralised salts fed from Day 56 of the experiment
until lambing and providing Se intake equivalent to about 0.2 mg Se/kg of diet DM
b
/90 ppm Se as selenized yeast (Sel-PlexTM) in the trace mineralised salts fed from Day 56 of
the experiment until lambing and providing Se intake equivalent to about 0.2 mg Se/kg of
diet DM
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SCC x 103
130
110
90
70
50
0mg Se/day
2mg Se/day
6mg Se/day
Figure 9.6 Effect of Se-Yeast on SCC (Adapted from McIntosh and Royle, 2002)
350
Pre
Post
SSC x 103
300
250
200
150
100
1.0
2.0
3+
All cows
Lactation number
Figure 9.7 Effect of Sel-Plex on SCC in the USA (16 herds, 1607 cows; Adapted from Eliott et al., 2005)
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Conclusions
Selenium nutrition of ruminants has some specific features, which create specific
problem in dairy and beef industries. In particular, in many places in the world Se
level in feed ingredients are not adequate to meet high Se demand of growing,
reproducing and lactating animals. A common practise of dietary Se
supplementation in inorganic form proved to be of low efficiency. That is why in
many cases veterinarians are trying to correct problems of inadequate nutrition
and Se injections are still a common practise in dairy industry. Indeed, part of
selenite consumed is reduced to metallic Se or selenide by rumen bacteria and
both are not available for further metabolism. The second part of selenite is
incorporated into proteins synthesised by rumen bacteria and it seems likely that
Se is also of low availability for animals. The replacement of sodium selenite by
organic Se sources, in particular, by selenized yeast in the form of Sel-Plex, is
proven to be effective means in solving Se problems in dairy, beef and sheep
industries. The data accumulated for the last 10 years clearly indicate advantages
of such replacement (Table 9.33). This includes increased Se concentration in
blood and GSH-Px activity, approximately doubled Se concentration in colostrum
and milk, higher Se transfer via placenta. As a result, cows health is improved
with lower somatic cell counts, decreased mastitis and retained placenta and
improved conception rates. The benefit to the newly born calves is coming from
improvement of their antioxidant defences and thermoregulation leading to better
immunity, viability and lower mortality during first months of the postnatal
development. Similarly to monogastric animals, when organic selenium is used
ruminants can also build Se reserves in their tissues, in particular in muscles, and
those reserves can be effectively used by animals in stress conditions when Se
requirement is increasing while feed consumption is declining.
Indeed, there are still many questions related to Se metabolism in ruminants
which need further clarification and this warrants new studies in this scientific
field.
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560
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Parameter
Effect of organic
vs inorganic
selenium
References
Decreased
Increased
Se in cow milk
Increased
Se in cow colostrum
Se in whole blood of calves
Se in plasma of calves
Se in whole blood of calves
GSH-Px in erythrocytes of calves
Se in cow whole blood
Increased
Increased
Increased
Increased
Increased
Increased
Increased
Increased
Se in cow serum
Se in calve liver
GSH-Px in erythrocytes of yearling
heifers and cows
GSH-Px in whole blood of cows
GSH-Px in erythrocytes of calves
at birth
Se in goat milk, plasma and whole
blood
GSH-Px in whole blood of goats
Casein selenium
Triiodothyronine (T3) in plasma of
calves at birth
IgM in cow serum
Proportion of Se in serun albumin
fraction
Se in skeletal muscles of lambs
Se in skeletal muscles of cows and
calves
Increased
Increased
Increased
Increased
Increased
Increased
Increased
Increased
Increased
Increased
Decreased
Increased
Increased
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Effect of organic
vs inorganic
selenium
References
Decreased
Decreased
Increased
Increased
Decreased
Retained placenta
Reduced
Decreased
Decreased
Decreased
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Ndiweni, N., Field, T. R., Williams, M. R., Booth, J. M. and Finch, J. M. (1991).
Studies on the incidence of clinical mastitis and blood levels of vitamin E and
selenium in dairy herds in England. Veterinary Record 129: 86-88
Neathery, M. W., Varnadoe, J. L., Miller, W. J., Crowe, C. T., Fielding, A. S. and
Blackmon, D. M. (1990). Effects of high dietary lead on the metabolism of
intravenously dosed selenium-75 in dairy calves. Journal of Dairy Science
73: 1107-1112
Nicholson, J. W. G., St-Laurent, A. M., McQueen, R. E. and Charmley, E. (1991b).
The effect of feeding organically bound selenium and alpha-tocopherol to dairy
cows on susceptibility of milk to oxidation. Canadian Journal of Animal Science
71: 135-143
Nicholson, J. W. G., McQueen, R. E. and Bush, R. S. (1991a). Response of growing
cattle to supplementation with organically bound or inorganic sources of
selenium or yeast cultures. Canadian Journal of Animal Science 71: 803- 811
Niekerk, F. E. van, Cloete, S. W. P., Barnard, S. A. and Heine, E. W. P. (1990). Plasma
copper, zinc and blood selenium concentrations of sheep, goats and cattle.
South African Journal of Animal Science 20: 144-147
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10
SELENIUM IN NUTRITION OF OTHER ANIMALS:
HORSES, DOGS, CATS AND FISH
You can take a horse to the water,
but you cant make him drink
Introduction
Antioxidant-prooxidant balance in horses plays an important role in maintenance
of their health. Indeed, it is generally accepted that mitochondria are the main
source of free radicals in biological systems (see chapter 1). It has been shown
that increased exercise is related to higher oxygen consumption and in many
cases to overproduction of free radicals. In fact, exercise-induced oxidative stress
can contribute to accelerated muscle fatigue and muscle fibre damage, leading to
exercise intolerance and poor performance (Sen and Packer, 2000). It seems likely
that horses can adapt to regular exercise and racing by increasing activities of the
antioxidant enzymes. However, a dietary provision of minerals, which are essential
part of GSH-Px (Se) and SOD (Zn, Cu and Mn) in optimal amounts is a challenging
task for horse nutritionist.
Se deficiency
Selenium deficiency in horses is related to muscle degeneration. Indeed, nutritional
myopathy (white muscle disease, WMD) involves skeletal and cardiac muscles
and results in (NRC, 1989):
weakness
impaired locomotion
difficulty in suckling and swallowing
respiratory distress
impaired cardiac function
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fats or unaccustomed exercise. WMD has been observed in foals from birth to 1
year of age, particularly those foals born to dams fed selenium-deficient diets,
during gestation (Lofstedt, 1997).
Did you know that white muscle disease of foals is caused by a
dietary deficiency of selenium and vitamin E, usually in
association with predisposing factors such as a high intake of
dietary unsaturated fats or unaccustomed exercise?
There are two major forms of the disease in foals: an acute, fulminant syndrome,
which is rapidly fatal, or a subacute syndrome characterized by profound muscular
weakness. The disease frequently has complications in the form of aspiration
pneumonia and stunting. On the biochemical level, markedly increased muscle
enzyme and low GSH-Px activities are common findings in affected foals. If
affected foals with the subacute form of the disease are supplemented with Se at
early stages of the disease they may survive; however, mortality rates remain
high (30%-45%; Lofstedt, 1997).
In foal affected by WMD serum selenium levels were below 65 ppb, showing
deficient levels. The mares of affected foals had significantly lower Se levels than
other mares (Higuchi et al., 1989). There was a good correlation between serum
Se concentration and blood GSH-Px activity (r = 0.81). Selenium levels in the
liver of affected foals were lower than in the foals, which succumbed with other
diseases. In acute disease vitamin E and Se should be given parenterally (5 mg
Se/horse) or by oral application of sodium selenite (46% selenium, 20 mg/horse),
in addition to symptomatical treatment (Zentek, 1991).
In Germany, post mortem studies on 143 foals over three years showed a total
of 46 (32.2%) cases of muscular dystrophy (Sandersleben and von Schlotke, 1977).
Histological examination of tissue from foals with subacute muscular dystrophy
showed hyaline skeletal-muscle fibre disintegration with calcification. Grossly,
the muscular lesions were not apparent even in the advanced stages. Most cases
were diagnosed within one week of birth. Vitamin E/selenium deficiency has been
implicated in the aetiology of the disease, and administration of this combination
to the mare during the lactation period is recommended (Sandersleben and von
Schlotke, 1977).
Se excess
Similar to other animals, Se access for horses can be toxic. It is interesting that,
explorer Marco Polo in the 13 th century was the first to describe selenosis in
horses which caused drop off of the hoofs of the animals as a result of consumption
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of specific plants known as selenium accumulators (see Chapter 3). Acute selenium
toxicity (blind staggers) in horses is characterized by (Rosenfeld and Beath, 1964,
NRC, 1989):
blindness
head pressing
perspiration
abdominal pain
colic
diarrhea
increased heart and respiration rates
and lethargy
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Se requirement
In accordance with NRC (1989) physiological requirement of horses is 0.1 mg/
kg of diet, however, it seems likely that horses at high work intensities may have
higher Se requirement (Shelle et al. 1985). Furthermore, foals, pregnant or lactating
mares, and racing horses have increased Se requirements (Zentek, 1991). In fact
recently it has been suggested that the appropriate Se concentration in the total
horse diet to be 0.3 ppm (Stowe, 1998) and calculation conducted by Pagan et al.
(1999) indicated that the grain mix for horses should contain about 0.6 ppm Se.
Did you know that recently it has been suggested
that the appropriate Se concentration in the
total horse diet to be 0.3 ppm?
In general, when sodium selenite was added to the diet, plasma Se concentration
of mature horses reached a plateau at about 140 ng/ml in horses fed 0.14 mg Se/
kg of diet (Shellow et al., 1985). It has been shown that in Bavaria 52% of horses
received Se at 1.25 g/kg BW, which is only half of the recommended daily Se
intake for horses (Wichert et al., 2002). At the same time Se level in plasma (g/
l) was <50 in 25% horses, >50-75 in 27%, >75-100 in 19%, >100-150 in 27%.
Taking the reference values for horses at 100-250 (g/l) it is clear that more than
70% of horses were Se deficient.
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Sodium selenite
Sel-Plex
3.76
1.16
1.85
0.75
51.1
20.4
39.3
3.72
1.10
1.58
1.04
57.3
27.8
48.6
205.1
216.8
209.2
202.8
201.0
224.5
222.1
198.8
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that using organic selenium supplements in the horse diet would be a practical
way to maintain their optimal Se status.
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Field studies have shown that horses during training undergo significant changes
of several blood antioxidant markers and that oral antioxidant supplementation
might help maintaining antioxidant defences. In fact, an antioxidant imbalance
was observed after three months under field conditions in the trained thoroughbred
horses, reflected by a significant decrease in GSH, SOD, GSH-Px, Se and a
significant increase in GSSG (Table 10.2; de Moffarts et al., 2005). The antioxidant
supplement prevented GSH-Px and Se decrease and significantly increased plasma
antioxidant capacity, -tocopherol and -carotene. Similarly, effects of an
antioxidative mixture, containing selenium and vitamin E (Se-E), on the response
of antioxidant enzyme activities following the exercise were examined. For this
purpose seven horses were injected intramuscularly with Se-E (Se: 25 mg, vitamin
E: 54.8 mg) and received the same vigorous exercise. It was shown that the Se-E
treatment decreased plasma lipid peroxide levels before the exercise. However,
SOD, GSH-Px, and catalase activities were not affected. Furthermore, the Se-E
treatment only slightly prevented the decrease in GSH-Px activity and the increase
in plasma peroxide level after the exercise, having no effect on SOD and catalase
activities (Ono et al., 1990).
Table 10.2 Blood antioxidant concentration of healthy trained thoroughbred horses before (T0), after 6
(T6) or 12 (T12) weeks of oral placebo (C) or antioxidant (E) supplement* (Adapted from de Moffarts et
al., 2005)
Marker
Group
TO
T6
T12
C
E
C
E
C
E
C
E
C
E
18.6
19.6
1654
1584
17.8
25.7
3.03
2.64
108
101
37.6
49.9
1512
1485
40.4
39.0
3.30
7.10
82
156
21.1
21.4
1400
1378
41.3
1378
3.30
7.40
73
153
*The supplement provided the following daily quantities of vitamins and trace elements:
ascorbic acid: 11.5 g, -tocopherol acetate: 7 g, -carotene: 0.5 g, copper: 187 mg, zinc:
769 mg and Se: 7 mg
In addition, it has been shown that the controlled training and diet supplements
were able to significantly increase horse antioxidant defences in both the
extracellular fluids and blood cells of horses, thus decreasing peroxidative
phenomena following physical exercise (Avellini et al., 1999). In an experiment
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birth are essential procedures to increase blood selenium levels in foals to optimal
levels. The serum selenium of foals from selenium-adequate mares is typically
much lower than their dams and ranges from 70 to 80 ng of selenium per ml of
serum (Stowe, 1967). According to Blackmore et al. (1982), serum selenium values
below 65 ng/ml are indicative of deficiency.
Did you know that antioxidants are poorly transferred via
placenta and by using organic Se in the maternal diet it
is possible to improve Se status of foals?
Se status of 123 mares and 87 foals during pregnancy, lactation and rearing was
investigated at four thoroughbred breeding farms in different regions in the
southwest of Germany (Schonthaler, 1998). Mean plasma Se concentration in the
different feedlots related to Se intake, however, within the herds with equal Se
intake, plasma Se concentration showed great individual differences. In particular,
it was shown that plasma Se concentration of unweaned foals up to 4 months of
age was significantly lower than that in the mares. The ratio between plasma Se
concentration of mare and foal was roughly 2:1. A decrease of plasma Se
concentration in mares that were in an advanced state of pregnancy (week 40-48)
was also shown. After weaning, plasma Se concentration increased significantly.
The Se concentration in plasma and whole blood correlated closely, the ratio of
the two being roughly 1.4:1 (Schonthaler, 1998). The relationship between blood
Se and GSH-Px activity was higher in mares than in foals at parturition. The Se
status of foals was also significantly correlated with that of the respective mares at
parturition under practical management conditions where wide variations in dietary
Se during gestation were present (Lee et al., 1995). Concentrations of selenium in
the blood plasma of 12 mares and their foals and in maternal milk were measured
over several days. At parturition, the selenium concentrations in plasma samples
from mares were almost twice as high as those in plasma samples from foals
(Hospes et al., 1996). Within a few days, the plasma selenium levels in mares
decreased and the plasma concentrations in foals increased steadily. In milk, the
selenium concentrations decreased to below the detection limit within 12 h. In
fact, selenium concentration of milk was less than 25% of that in colostrum within
one day after foaling and was considered a minor source of Se for the foals
regardless of Se status of the mares during gestation (Lee et al., 1995).
Fetal development, parturition and lactation strain maternal trace mineral
reserves critical; to postpartum health of both mare and foal. It has been shown
that organic selenium in the form of Sel-Plex in the gestation diet improves mare
post-partum selenium status as well as that of her foal. In an experiment mares
received 1 or 3 mg Se/day as selenite or 3 mg Se/day as Sel-Plex beginning 55
days pre-foaling to 56 days post-foaling. It was shown that (Janicki et al., 2001):
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Se and immunity
Immunomodulating properties of Se are characterised in previous chapters and
horses are not exception from the rule. For example, fifteen Shetland ponies were
used in a 7-wk trial to study the effect of supplemental Se on humoral antibody
production. Ponies were depleted of Se before being assigned randomly to either
a low Se (0.02 ppm) or higher Se (0.22 ppm) diet (Knight and Tyznik. 1990). It
was shown that Se supplementation was associated with a significant increase in
GSH-Px activity and blood Se concentrations during the later weeks of the
experiment. An enhanced primary response was also observed in Se-supplemented
ponies as evidenced by increased hemagglutination titers. Furthermore,
significantly higher IgG concentrations were observed in the Se-supplemented
group. In another experiment, fifteen horses used for serum production were
maintained on low vitamin E and selenium diets.
Did you know that dietary Se supplementation can improve
immunocompetence of horses?
They were divided into four groups receiving no supplements, vitamin E, selenium
or a combination of vitamin E and selenium. The humoral immune response to
novel antigens, such as tetanus toxoid and equine influenza virus, was increased
in groups receiving either vitamin E or selenium/vitamin E (Baalsrud and Overnes,
1986). However, no effects were recorded on the titres against Escherichia coli or
the levels of immunoglobulin G.
Conclusions
From the data presented above it is clear that antioxidant-prooxidant balance in
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Introduction
Domestic cats and dogs both belong to the order Carniivora and have some
specific features of general metabolism and antioxidant defences. In particular,
cats cannot convert beta-carotene into vitamin A and require vitamin A to be
supplied with the diet; they have high protein requirement but poorly metabolised
carbohydrates and for cats taurine and arginine are essential amino acids. Dogs
cannot synthesise vitamin D and are characterised by low synthesis of arginine.
Furthermore, cats require preformed long chain polyunsaturated fatty acids such
as arachidonic acid and docosahexaenoic acid to be delivered with the diet. Since
life expectancy of cats and dogs has increased substantially for the last decades,
a special emphasis should be given to health maintenance of elderly animals
similar to human. In fact, in many cases cats and dogs suffer the same metabolic
disorders and diseases as human does.
Did you know that in many cases cats and dogs suffer the same
metabolic disorders and diseases as human does?
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Food quality
Petfood quality control is very strict and ensure only high quality ingredients to
be included into the diet. However, since environmental pollutants such as heavy
metals or mycotoxins are very widely distributed it is almost impossible to
completely avoid their presence in food ingredients. For example, an analytical
survey of 42 elements in 31 commercial canned pet foods was conducted (Furr et
al., 1976). Arsenic, bromine, cadmium, chromium and mercury were highest in
fish-containing cat foods. Lead was most consistently high in those products
containing chicken. Barium, nickel and tin also appeared high in certain of the
samples. An analytical survey of mutagens, nitrosamines, polychlorinated
biphenyls, toxic elements as well as the toxicologically protective constituents
zinc, selenium, and vitamin C, in 48 pet foods was conducted (Mumma et al.,
1986). High concentrations of fluoride and iodide in some samples and increased
concentrations of mercury and selenium in certain cat foods containing fish were
detected. Polychlorinated biphenyls were only detected in one cat food. The
occurrence of ochratoxin A (OTA) in canned (26 samples) as well as dry pet
foods (17 samples) for cats and dogs was investigated (Razzazi et al., 2001). OTA
was detected in 47% of the pet food samples at the levels of 0.1-0.8 ng/g food.
Only two pet food samples contained higher (3.2 and 13.1 ng/g food) OTA levels.
OTA was also detected in 62% cat kidneys (0.35-1.5 ng/g tissue). When one
hundred samples of pet foods including 35 samples of cats food were analysed,
low levels of aflatoxin B1, OA and fumonisins were detected in some of them
(Scudamore et al., 1997).
Did you know that petfood can be contaminated by variety of
pro-oxidants and therefore Se dietary supplementation is an
important factor in preventing oxidative stress
in companion animals?
Aflatoxin B1 in cat food was higher in comparison to the dog food (Sharma and
Marquez, 2001). Recently mycoflora of 21 dry pet foods (12 belonging to dogs
and 9 to cats) that corresponded to 8 commercial brands made in Argentina and
imported has been analysed (Bueno et al., 2001). Ten genera and fungi classified
as Mycelia sterilia were identified. The predominant genera were Aspergillus
(62%), Rhizopus (48%), and Mucor (38%). The most prevalent among Aspergillus
was Aspergillus flavus followed by Aspergillus niger and Aspergillus terreus which
are potential sources of mycotoxins. There are also other contaminants in cats
food. For example, the concentration of bisphenol A ranged from 13 to 136 ng/g
in canned cat food (Kang and Kondo, 2002).
Lipid peroxides are the major petfood nutritionist concern. Since vitamin E in
the food is usually used in esterified form it does not possess antioxidant activity
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per se and only become active after de-esterification in the intestine. Therefore
various synthetic antioxidants are a choice for prevention of lipid peroxidation
during petfood storage. The effects of three different preservative treatments
(ethoxyquin and butylated hydroxyanisole, EX/BHA; mixed tocopherols, TC/TC
and ascorbyl palmitate and mixed tocopherols ATL/TC) applied to extruded dog
food were studied (Gross et al., 1994). After processing the dog foods were
placed in bags and stored for 16 wk at 48.8 degrees C or for 12 months at 22.2
degrees C. The best results were observed with EX/BHA. It is interesting that in
both the high and ambient temperature tests the dogs consumed more of the foods
with the lowest peroxide value when given a two-bowl choice. It is interesting
that dietary oxidised lipids compromised antioxidant defences and altered some
neutrophil and monocyte functions in dogs (Turek et al. , 2003).
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results in enhanced cytokine production and effects. The anorexia that in many cases
follows infection and injury, may be purposeful to permit release of substrate from
endogenous sources to support and control the inflammatory process (Grimble, 1998).
Therefore, prior as well as concurrent antioxidant intake are of great importance in
determining the outcome of the inflammatory response. It is interesting that anorexia
was one of the major clinical signs of toxicosis in aflatoxin B1-treated calves (Brucato
et al., 1986) and Se was demonstrated to improve feed intake in mycotoxin-treated
animals. On the other hand, vitamin E-selenium deficiency in wild ducks was also
associated with the development of anorexia and diarrhea (Dhillon and Winterfield,
1983). Similarly, silver toxicity in weanling swine was associated with lesions typical
of selenium-vitamin E deficiency including anorexia and diarrhea (Van Vleet et al.,
1976). It seems likely that Se deficiency can cause decrease in food consumption
and administration of SeMet increased voluntary feed consumption of seleniumdeficient chickens within 2-3 hours (Bunk and Combs, 1980). Therefore dietary
antioxidants could be supplements of the choice when dealing with anorexia in cats.
Large intestinal disease, especially colitis, is also a common problem in cats and no
single etiologic agent is responsible for this (Simpson et al., 1998). Furthermore,
renal disease, arthritis and compromised immunity are of great importance for cats as
well (Swanson et al., 2003). It seems likely that natural antioxidants, such as selenium,
could be of great value in prevention and treatment of the diseases.
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subcutaneous edema
anorexia
depression
dyspnea
eventual coma
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Species
Cat
Dog
Pig
Chicken
Humans, 20-60 years
Humans, 60-100 years
Horse
Goat
Calves, 3-9 month old
Cattle, >9 months old
Sheep
Range, mmol/l
Range, gl/l
3.60-10.09
1.90-4.31
1.97-3.32
1.68-4.28
0.78-1.48
0.61-1.73
0.36-1.68
0.14-1.42
0.19-0.65
0.10-0.82
0.09-0.54
285.7- 800.8
250.8- 342.1
156.4- 263.5
133.3- 339.7
61.9- 117.5
48.4- 137.3
28.6 133.3
11.1-112.7
0.19-0.65
0.10-0.82
0.09-0.54
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On the other hand, kitten given the low-Se diet had significantly reduced plasma
Se concentration and GSH-Px activity and compromised thyroid hormone
metabolism. In fact plasma total T4 increased and T3 decreased significantly (Yu
et al., 2002). This could affect kitten thermoregulation, growth and development
and ultimately lead to increased mortality.
Selenium requirement
Kitten requirement in Se has been recently estimated to be 0.15 mg Se/kg diet
(Table 10.4; Wedeking et al., 2003). As pet foods for cats contain a high proportion
of animal protein with a Se bioavailability of 30%, it is recommended that
commercial diets for cats contain 0.5 mg Se/kg DM. Recently Se requirement of
puppies was re-evaluated. Thirty beagle puppies (average = 8.8 weeks old) were
used in a randomized complete block design. Puppies were fed a low Se (0.04
mg Se/kg diet) torula yeast-based diet for 14 days (pre-test period) after which
this same diet was supplemented with five levels of sodium selenite for 21 days
(experimental period) to construct a response curve (0, 0.13, 0.26, 0.39 or 0.52
mg Se/kg diet; Wedekind et al., 2004). Based on the result of the study the
authors suggested Se requirement to be 0.21 mg Se/kg diet.
Table 10.4 Response of kittens fed graded levels of sodium selenite (Adapted from Wedekind et al.,
2003)
Treatment Dietary
Se,mg/kg
1
2
3
4
5
6
GSH-Px,
RBC, GSH-Px,
Plasma, U/ml U/106 cells
0.027
0.073
0.100
0.122
0.210
0.314
0.31
1.03
1.38
1.72
2.32
2.60
1.90
3.85
5.23
5.92
5.65
5.81
Plasma Se,
mM/l
T3,
nmol/l
0.22
0.99
1.56
2.06
4.12
4.61
0.73
0.86
0.92
0.91
0.91
1.27
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some specific features and when dogs and cats fed on the same diet were compared,
Se concentration in cats serum was 50-70% higher than that in dogs (Wedeking
et al., 2003; Todd and Hendrix, 2005). The authors indicated comparatively high
tolerance of cats and dogs to dietary selenium.
antioxidant effects
bile acid conjugation and cholestasis
xenobiotic detoxification
modulation of intracellular calcium levels
participation in retinal development and function
endocrine/metabolic effects
osmoregulation, neuromodulation and stabilization of the membranes
modulation of cell proliferation, inflammation and collagenogenesis
reproduction: preservation of the motility of the spermatozoa, support of
their capacitation, improvement of the chances of success of fertilization
and the early embryonic development
immunomodulation
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another experiment, taurine suppressed erythrocyte hemolysis but did not affect
peroxidation (Nakamura et al., 1993). Taurine was also shown to be a weak
scavenger of peroxynitrite and did not attenuate sodium nitroprusside toxicity to
cells in culture (Mehta and Dawson, 2001).
Did you know that taurine is an essential amino acids for cats?
However, later antioxidant properties of taurine were shown in various model
systems in vivo and in vitro. For example, in Chinese hamster ovary cells cultivated
in vitro, taurine scavenged oxygen species (Cozzi et al., 1995). In non-diabetic
rats high glucose caused a significant increase in levels of MDA and 4hydroxynonenal, which were reduced by taurine (Haber et al., 2003). Wistar male
rats were fed on taurine in addition to high fat diet (11% coconut oil w/w) for 6
months. Taurine significantly reduced aortic cholesterol and TBARS and increased
GSH concentration (Sethupathy et al., 2002). Similarly, taurine supplementation
of rats restored kidney GSH content and GSH-Px activity and reduced MDA
production levels in the kidney tissue following cisplatin treatment (Saad and AlRikabi, 2002). When fuctose-fed rats received taurine in drinking water,
peroxidative damage was minimal in both plasma and liver (Nandhini et al.,
2002). Taurine also decreased TBARS (by 26%) in apoE-deficient mice (Kondo
et al., 2000).
In general, taurine can show antioxidant-related protective effects similar to
other antioxidants. For example, it was shown that oxygen persufflation
significantly enhanced the viability of livers during cold preservation only in
combination with SOD or taurine, which were both equally effective in reducing
lipid peroxidation, enzyme release, and vascular resistance upon post-ischemic
reperfusion (Lauschke et al., 2003). The isolated guinea pig lungs previously
being perfused by oxygenated Krebs-Henseleit solution were put in normothermic
ischemic conditions. Decreased pulmonary artery pressure, tissue perfusate MDA
levels and increased perfusate GSH levels were observed in taurine added group
(Oz et al., 2002). It was shown that 3 intraperitoneal injections of 200 mg/kg of
taurine prevented hypoxia-induced lactate accumulation and lipid peroxidation
(LP) in brain, liver, and heart tissues (Mankovskaya et al., 2000). The authors
suggested that the effect of taurine on LP could be due to its membrane stabilising
activity. Exposure of isolated frog rod outer segments to light (5000 lux) induces
membrane disorganization and swelling. An increase of about 50% in lipid
peroxidation accompanied the light-induced damage. Taurine and hypotaurine
(25 mM) prevented the increase in lipid peroxidation, and provided an entire
protection of rod outer segment structure (Pasantes-Morales and Cruz, 1985).
Antioxidant effect of taurine is not restricted to PUFAs. For example, Ogasawara
et al. (1993; 1994) showed an inhibiting effect of taurine against the modification
of protein, as well as an antioxidative effect through the reactions of taurine with
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diene conjugate levels and significant increases in hepatic GSH glutathione, vitamin
E, and vitamin C levels (Balkan et al., 2002). Taurine seems to be capable of
fortifying cells against lead-induced oxidative attack without decreasing lead levels
(Gurer et al., 2001). Similarly, taurine treatment of rats, together with thioacetamide
administration, diminished the severity of the liver injury by decreasing oxidative
stress (Dogru-Abbasoglu et al., 2001). Taurine treatment of rats after 3nitropropionic acid administration protects the striatum from damage along with
increased survival rates (Rivas-Arancibia et al., 2001). Similarly, taurine treatment
of rats reduced gentamicin-induced increases in serum creatinine, tissue lactate
and TBARS levels (Erdem et al., 2000). The protective effect of taurine against
indomethacin-induced gastric mucosal injury was also due to inhibition of lipid
peroxidation and neutrophil activation (Son et al., 1996). It is interesting that
taurine enhanced the hepatic drug-metabolizing systems, leading to the stimulation
of the ascorbic acid metabolism in rats fed diets containing polychlorinated
biphenyls (Mochizuki et al., 2000). Taurine showed a protective role to reduce
cellular damage associated with both NO and metal-stimulated catecholamine
oxidation (Biasetti and Dawson, 2002) as well as ameliorating chronic streptozocininduced diabetic nephropathy in rats (Trachtman et al., 1995). The beneficial
effect of taurine was related to reduced renal oxidant injury with decreased lipid
peroxidation and less accumulation of advanced glycosylation end products within
the kidney. Taurine prevented galactose-induced cataracts in New Zealand white
rabbits (Malone et al., 1993) and its inverse relationship with lens MDA suggests
this is an antioxidant effect.
Did you know that taurine is an important part
of antioxidant defences?
Protective effects of taurine could be related to other functions. For example, the
protective effects of taurine supplementation against renal damage induced by
salt loading in Dahl salt-sensitive rats was suggested to be attributed to the
suppression of LOX-1, probably through its antioxidant effects (Chiba et al., 2002).
Similarly, it was concluded (el Tahir et al., 1987) that endogenous taurine may
participate in regulation of prostaglandin synthesis and that prostanoids may
contribute to its known actions. It was also suggested that taurine promotes the
bioavailability of the lipid soluble vitamins A, D, E, K, and F, probably by forming
different types of water soluble, easily hydrolyzable complexes (Petrosian and
Haroutounian, 2000).
During pregnancy, taurine accumulates in the maternal tissues and later released
to the fetus via the placenta and to the newborn via the maternal milk and taurine
deficiency in the mother leads to growth retardation of the offspring, and to
impaired perinatal development of the central nervous system and of the endocrine
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et al., 1995). Because respiratory burst activity of PMN was also significantly
reduced in the presence of taurine, the beneficial effect appears to be mediated by
antioxidative properties of taurine. In rats the host inflammatory response induced
by the xenotransplantation of neurons derived from the human teratocarcinoma
cell line was studied and taurine was able to facilitate graft survival and attenuate
the immune response generated by the xenograft (Rivas-Arancibia et al., 2000).
Taurine ameliorates endotoxin-induced leukocyte-endothelial cell interactions
associated with sepsis, thereby suggesting that taurine may have a therapeutic
role in the prevention of endothelial damage in sepsis (Egan et al., 2001).
Taurine at concentrations normally found in cells can inhibit oxidative damage
to DNA (Messina and Dawson, 2000) and anti-apoptotic action of taurine is the
most important part of its immunomodulating properties. For example, neutrophils
were stimulated with Fas monoclonal antibody in the presence or absence of
taurine. Fas receptor ligation resulted in significant neutrophil apoptosis at 18 h.
Apoptosis was inhibited and intracellular calcium levels were maintained in the
presence of taurine (Condron et al., 2003). Taurine attenuates hyperglycemiainduced apoptosis (by 78%) in human tubular cells via an inhibition of oxidative
stress (Verzola et al., 2002) and decreased hyperglycemia-induced human
umbilical vein endothelial cell apoptosis through ROS inhibition and [Ca 2+)]
stabilization (Wu et al., 1999). Taurine was significantly protective against arseniteinduced apoptosis of human polymorphonuclear leukocytes (Watson et al., 1996).
Age-related decline in cats immune system (Harper et al., 2001) are of great
importance and mechanisms of a decrease in total plasma antioxidant capacity of
older cats (Harper and Frith, 1999) await further clarification. It is necessary to
mention that after experimental infection with feline immunodeficiency virus,
aged cats developed more severe disease than young adult cats (George et al.,
1993). It is interesting that vitamin E alone in doses up to 4.3 IU/g was not able to
improve immune status of cats (Hendriks et al., 2002). However, a mixture of
antioxidants including lycopene, -carotene, lutein, vitamin E, taurine and
ascorbate were shown to improve antibody response to vaccination in cats (Harper
et al., 2001). Dogs consuming Se-vitamin E supplement had a significantly greater
serum Se and alpha-tocopherol than un-supplemented animals (Kandil et al.,
2005).
Did you know that Se and vitamin E supplementation
can improve immune response in dogs?
The supplement improved immune response against T. hydatigena as evidenced
by increase production of antibody titer and IgG concentration in comparison
with either vaccinated un-supplemented or control unvaccinated animals.
Moreover, the highest protection level against T. hydatigena infection was observed
in the same Se-vitamin E supplemented dogs. It seems likely that antioxidant
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Conclusions
From results presented above it is clear that in many cases effects of antioxidants
on companion animals are very similar to those described for humans, since they
share common environments, stresses and in some cases diseases. Interactions
between antioxidants and prooxidants starts at the level of stomach and small
intestine as described for humans (Surai, 2002; Surai et al, 2003; 2004). Therefore
dietary supplementation with various antioxidants is an effective strategy to prevent
damaging effects of free radicals and toxic products of their metabolism on the
companion animals. In particular a specific attention should be paid to the form
of antioxidants used. For example, organic selenium (Sel-Plex) can improve
antioxidant system of the digesta and selenite in the same conditions could promote
peroxidation. It should also be taken into account that with the same amount of
organic selenium in the diet more selenium would be accumulating in tissues in
comparison to selenite building selenium reserves which could be useful in stress
conditions. Organic selenium is also effectively transferred to the colostrum and
milk providing important means of antioxidant system maintenance of the newly
born kitten. Furthermore, inorganic copper and iron are the major stimulators of
lipid peroxidation in digestive tract and use of organic forms of those elements
could avoid their possible detrimental actions. Use of various plant-derived
antioxidants, such as flavonoids, could also help to prevent oxidative damage in
the gastro intestinal tract (GIT) due to their antioxidant properties and chelating
ability in relation to iron and copper.
Of particular interest for companion animal nutritionist are data showing
protective effects of natural antioxidants in animal and human exercise as well as
their immunomodulating properties. It is well known that vitamin E and carotenoids
are not toxic for animals even at very high level of supplementation. However,
more research is needed to establish optimal antioxidant supplementation of the
companion animals. For example, significant systemic toxicity has been observed
in kittens treated daily with 50-1,000 mg/kg/day parenteral vitamin E for 3 weeks
(Phelps, 1981). The toxicity was dose-related and resulted in significant mortality
at doses over 100-200 mg/kg daily. Functional food business is quickly developing
all over the world. Indeed functional food enriched with various antioxidants in
particular selenium, vitamin E, carotenoids and flavonoids for cats already found
their way to the supermarket shelves (Swanson et al., 2003).
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Introduction
Aquaculture accounted for 29% of global fisheries production in 2000 and of the
38 million tonnes from aquaculture around 5-10% was farmed intensively using
nutritionally complete feeds as the sole source of nutrition (Carter, 2003). Intensive
farming represents the majority of aquaculture in developed countries.
Considerable advances have been made in understanding the nutrient requirements
of intensively farmed finfish and intensive production offers opportunity for
increasing growth efficiency and controlling product quality through optimal
nutrient supply. Fish nutrition is associated with high levels of fat and in particular
polyunsaturated fatty acids in the diet. Therefore, an oxidative stress and
antioxidant protection are among major issues for fish nutritionist. Taking into
account recent data on fish nutrition it should be mentioned that Se is of great
importance for maintenance of fish health, in particular fish immunity, as well as
for fish growth, development and flesh quality.
Did you know that aquaculture accounted for 29% of global
fisheries production in 2000 and of the 38 million tonnes from
aquaculture around 5-10% was farmed intensively using
nutritionally complete feeds as the sole source of nutrition?
Se deficiency
Se deficiency in fish is associated with oxidative stress and causes similar clinical
signs as in various birds and mammals. In particular increased mortality and
compromised immunity are indicative for this deficiency. For example,
unacceptably high mortalities in rainbow trout fry (Oncorhynchus mykiss) six to
10 weeks after they started to feed were recorded in two spring water trout
hatcheries in Northern Ireland in May 1989 (McLoughlin et al., 1992). Muscle
degeneration and necrosis were consistent with histopathological findings in both
outbreaks, and this myopathy was similar to that previously described in salmonids
and other species associated with vitamin E and selenium deficiency.
Experimentally induced Se deficiency was related to similar changes. The trout
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given the diets without added Se or alpha-tocopherol for about 10 weeks (Oberbach
and Hartfiel, 1987) were characterised by:
lost appetite
pale, swollen gills with shortened gill lids
brown-yellow-coloured livers
anaemia
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Se toxicity
Selenium toxicity in fish is well studied as a result of environmental concern
related to Se contamination of various ponds, lakes and rivers. For example, Belews
Lake, North Carolina (USA) was contaminated by selenium in wastewater from a
coal-fired power plant during the mid-1970s, and toxic impacts to the resident
fish community (20 species) were studied for over two decades. Symptoms of
chronic selenium poisoning in Belews Lake fish included (Lemly, 2002):
Experimentally it was proven that toxic Se doses are much higher than those used
for diet supplementation and they are of the same magnitude as in various farm
animals. In fact, selenium toxicity occurred in rainbow trout and catfish when
dietary selenium exceeded 13 and 15 mg/kg dry feed, respectively (Hilton et al.,
1980; Gatlin and Wilson, 1984; Hilton and Hodson, 1983). The major effects of
Se excess include:
reduced growth
poor feed efficiency
high mortality
renal calcinosis
Indeed, fish food was spiked with SeMe to contain 4.6, 12, and 18 mg/kg (dry
weight) of Se. Fish exposed to SeMe for 90 days exhibited a significant decrease
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in body weight and fork length in the 4.6 and 12 mg/g Se treatments compared
with controls (Vidal et al., 2005). Based on decreased growth after 90 days, a
dietary Se lowest observed-effect concentration (LOEC) value of 4.6 mg/kg and
a Se body burden LOEC of 1.20 g/g (wet weight) were estimated. The chronic
effects of dietary Se exposure in juvenile Sacramento splittail (Pogonichthys
macrolepidotus) were investigated in the laboratory. Purified casein-based diets
were supplemented with selenized yeast for 9 months giving 0.4 (control), 0.7,
1.4, 2.7, 6.6, 12.6, 26.0, and 57.6 mg of Se/kg dry weight. Mortalities occurred
only in the two highest Se treatments and were accounted for 8.3 and 18.3% at 5month and 10.0 and 34.3% at 9-month, respectively (Teh et al. 2004). However,
Se-induced deformities were observed in fish fed > or =2.7 mg of Se/kg diets at 5month and in fish fed > or =0.7 mg of Se/kg diets at 9-month. The authors showed
that chronic exposure to 6.6 mg of Se/kg diet induced deleterious health effects
that can potentially impact survival of juvenile splittail. The toxicity of 2
organoselenium diets (high Se fish meal based diet (FM) or SeMet-supplemented
diet) was evaluated in 90- to 120-day partial life cycle tests with 2 life stages of
chinook salmon (Oncorhynchus tshawytscha). After 90 days of exposure in
freshwater, survival was reduced in fish fed Se more than or equal to 9.6 g/g of
either diet, and growth was reduced in fish fed Se more than or equal to 5.3 g/g
of FM diet or Se more than or equal to 18.2 g/g of the SeMet diet (Hamilton et
al., 1990). After 120 days in brackish-water, survival was unaffected but growth
was reduced in fish fed Se more than or equal to 18.2 g/g of FM diet or Se 35.4
g/g of the SeMet diet. After the 120-day feeding experiment, survival during a
10-day seawater challenge test was reduced in fish fed Se 35.4 g/g of either diet.
Casein-gelatin diets containing graded levels of supplementary selenium (as sodium
selenite) 0 to 15 mg/kg were given to fingerling channel catfish (Ictalurus
punctatus) for 15 weeks. Weight gain values generally indicated that the basal
diet containing Se 0.06 mg/kg diet caused growth depression, and a supplementary
Se level of 15 mg/kg also reduced growth response, which indicated Se toxicity
(Gatlin et al., 1984). Chronic dietary Se toxicity occurred at dietary Se 13 mg/kg
dry feed. Major effects of Se toxicity were reduced growth rate, poor feed efficiency
and a high number of mortalities (Hilton et al., 1980). No histopathological lesions
or significant deviation in blood values or liver somatic index were detected in
trout raised on diets with Se 13 mg/g dry feed. Tissue Se analysis indicated that
trout can maintain homeostasis with dietary Se levels up to 1.25 mg/kg dry feed.
To determine if elevated concentrations of waterborne selenium, caused by
coal mining, in the Elk River in southeastern British Columbia, may be causing
reproductive or teratogenic effects in wild cutthroat trout (Oncorhynchus clarki
lewisi), fertilized eggs from exposed and reference fish were raised in the laboratory
(Kennedy et al., 2000). Selenium concentrations in the eggs was 21.0 g/g (range:
8.7 to 81.3). Despite these elevated egg Se concentrations, there was no significant
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effect on fertilization; time to hatch; percent hatch; or egg, larvae, and fry
deformities or mortalities. The lack of any toxic response in this study the authors
explained as a result of an evolved tolerance to higher tissue Se concentrations in
a population of fish living in a seleniferous river system.
Se requirement
To determine Se requirement of fish is a complex task. For example, there are a
large number of species from different phyla, each species may have several
different life-history stages, time to harvest may be as long as five years, a single
species may be exposed to a wide range of environmental conditions, and
production cohorts are exposed to different patterns of day to day variation in
key environmental parameters (Carter, 2003). Similar to other animal species, the
selenium requirement of fish varies with the form of selenium ingested,
polyunsaturated fatty acid and vitamin E content of the diet, and concentration of
waterborne selenium (NRC, 1993). It seems likely that Se requirement in fish is
similar to those established for various farm animals. In rainbow trout (Salmo
gairdneri) peak plasma GSH-Px activity was obtained at dietary Se value between
0.15 and 0.38 mg/kg dry feed (Hilton et al., 1980).
Did you know that Se requirement of rainbow trout is 0.15-0.38
ppm, and fingerling channel catfish 0.1-0.5 ppm and it is
recommended to provide with a mineral premix for Channel
catfish 0.25-0.3 ppm selenium and for rainbow trout 0.3 ppm?
Liver and plasma Se-GSH-Px activities indicated the Se requirement of fingerling
channel catfish was between 0.1 and 0.5 mg/kg diet. Growth results and liver and
plasma Se-GSH-Px activities indicated that the minimum Se requirement of
fingerling channel catfish fed on adequate vitamin E diet was 0.25 mg/kg dry diet
(Gatlin and Wilson, 1984) and it is recommended to provide with a mineral premix
for Channel catfish 0.25-0.3 ppm selenium and for rainbow trout 0.3 ppm (NRC,
1993). GSH-Px activity in plasma and liver is a useful index of selenium status in
fish (NRC, 1993).
When considering Se requirement and physiological concentrations of Se in
fish tissues it is important to have a comparison with fish in wild. It seems likely
that in most of cases Se concentration in wild fish is several-fold higher than that
in farmed fish. For example, selenium was estimated in whole livers from adult
salmon caught at the entrance to the Strait of San Juan de Fuca, caught in Puget
Sound or its estuaries, fish returning to a hatchery and fish reared in saltwater net
pens. In each case, Se concentrations in wild coho salmon (Oncorhynchus kisutch)
were about twice as high as those in hatchery-reared fish (Felton et al., 1990).
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Tissue levels of zinc, iron, copper and selenium in liver, kidney, gills and gonads
of Atlantic salmon were analysed in fish caught near a spawning ground, in the
open sea or from farmed fish. There were significant differences in liver
concentrations of the 4 trace elements between wild and farmed salmon. In fact,
Se varied 10-fold, Cu 5-fold, Fe 3-fold and Zn 2-fold (Maage et al., 1991).
Concentrations of selenium in liver and serum were higher in wild salmon than in
most domesticated animals (Poppe et al., 1985). Liver samples from 37 wild
salmon, 233 healthy farmed salmon were analysed and it was shown that Se
concentrations were 6 to 7 times higher in wild salmon than in farmed salmon
(Poppe et al., 1986).
It is interesting to note that it is difficult to achieve Se concentration in fish
similar to that in wild when sodium selenite is used. Hatchery-reared coho salmon
(Oncorhynchus kisutch) were fed on increased amounts of sodium selenite to
raise eviscerated body concentrations to those seen in wild counterparts.
Eviscerated body Se concentration, GSH-Px and SOD levels were measured during
and at the end of the 9-month freshwater (FW) feeding trial. Se supplements were
added to a commercial ration to give final concentrations of 1.1, 8.6, 11.1 and
13.6 mg/kg (Felton et al., 1996). The results indicated that a dietary Se concentration
as high as 8.6 mg/kg was necessary for inducing eviscerated body loads similar
to those found in wild fish producing healthy coho. This Se supplemental level is
very close to toxic one. Therefore, by using organic selenium in the fish diet it is
much easier to achieve the same Se status as in wild fish.
GSH-Px activity and Se concentrations in blood and tissues are used for
assessment of Se status of fish. For example, Tilapia [Oreochromis] fingerlings
were assigned to 4 groups and fed for 30 days on basic diets low in Se (0.03 mg/
kg) without vitamin E supplementation, supplemented with Se 0.1 mg/kg feed,
vitamin E (500 mg/kg) or a combination of both. Supplementation with Se and
vitamin E separately or together, increased blood Se from 90 to 199 ng/ml
(Dimanov et al., 1998). GSH-Px activity in the blood of experimental fish increased
from 5 to 14 mU/mg Hb.
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Weight gain, g
Colour, Roche score
Pigment, mg/kg
Texture, kg/cm2
Flesh oil, %
GSH-Px, U/g Hb
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Control
Sel-Plex
1.403
14.9
6.7
11.4
10.1
53.3
1.408
15.2
7.4
12.5
9.4
107.3
Change, % of control
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+0.36
+2.01
+10.45
+9.65
-6.93
+101.3
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followed by Se-Yeast and sodium selenite. Broken-line analysis showed that minimum
supplemental dietary Se requirement as sodium selenite, Se-Met and Se-Yeast was
0.28, 0.09 and 0.11 mg/kg diet for live weight gain, and 0.17, 0.12 and 0.12 mg/kg
diet, for liver GSH-Px activity, respectively. Relative bioavailability values of Se-Met
and Se-Yeast compared with sodium selenite were 336 and 269% for growth, and
147 and 149% for GSH-Px activity, respectively. Se from Se-Met and Se-Yeast showed
significantly higher rates of accumulation in liver and muscle than Se from sodium
selenite (Table 10.6 and 10.7; Lovell and Wang, 1997).
Table 10.6 Selenium requirement and relative bioavailability in channel catfish (Adapted from Lovell and
Wang, 1997)
Criterion
Se source
Weight gain
Selenite
SeMet
Sel-Plex
Plasma GSH-Px Selenite
SeMet
Sel-Plex
Liver GSH-Px Selenite
SeMet
Sel-Plex
Liver Se
Selenite
SeMet
Sel-Plex
Muscle Se
Selenite
SeMet
Sel-Plex
Equation
Se requirement*
Relative
bioavailability, %**
y=10.8+ 34.9x
y=10.6 + 117x
y=10.2 + 93.8x
y=2.0 + 4.4x
y= 2.3 + 5.1x
y=2.0 + 5.0 x
y=22.5 + 600.7x
y=22.5+ 883.7x
y=21.4+ 891.8x
y=0.06 + 10.3x
y= 0.77 + 20.3x
y=0.74 + 19.0x
y= 0.16 + 0.7x
y=0.16 +3.3x
y=0.17 + 3.1x
0.28 0.031
0.09 0/007
0.11 0.012
0.17 0.024
0,12 0.021
0.12 0.020
0.18 0.045
0.09 0.015
0.11 0.025
0.10 0.022
0.10 0.024
100
336
269
100
116
116
100
147
149
100
197
184
100
478
453
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Selenite
SeMet
Sel-Plex
64
80
149
192
365
64
70
213
352
512
64
75
320
554
717
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(sodium selenite 0.2 mg/kg diet). GSH-Px activities in serum and liver and hepatic
Se concentrations were similar in all rats after 12 days. Serum Se concentration
was highest in the group fed on cod fillets (Knudsen et al., 1992). Rats fed on cod
fillets and selenomethionine had significantly higher femur Se concentration than
rats fed on selenite. It was concluded that Se in cod fillets was as biologically
available to rats as SeMet and selenite. The experimental results with Se deficient
rats showed that enrichment of fish fillets with SeMet yields fillets with high Se
bioavailability. In fact, Se bioavailability decreased in the order raw salmon >
cured salmon > selenite (Ornsrud and Lorentzen, 2002). This was supported by
the observations for Se accumulation in femur and muscle and induction of GSHPx activity. However, curing salmon altered the utilisation of Se. In a line with
these results are other experimental data. After 9 weeks of dietary Se repletion,
relative activity of liver GSH-Px from the different dietary groups compared with
control rats (100%) was: flounder 106%, tuna 101%, pork 86%, sodium selenite
81%, SeMet 80%, beef 80%, chicken 77%, veal 77%, and lamb 58% (Wen et al.,
1997). From the data presented above it is clear that fish is a good source of Se
and there is a good opportunity to produce Se-enriched fish by using organic
selenium supplements in the fish diet. Furthermore, comparatively high Se
availability from fish makes the issue of producing Se-fish even more attractive.
Did you know that by inclusion of organic selenium
in fish diet it is possible to produce Se-enriched fish
which could be a good source of Se for human consumption?
Conclusions
Fish nutrition is quickly developing in different countries and in many cases fish
nutritionists can learn from other industries, such as poultry. In fact, fish nutritionists
face the same problems as poultry or pig nutritionists. Indeed, antioxidant
misbalance and oxidative stress are major problems in fish nutrition. In fact,
immunocompetence can be compromised and fish viability substantially decreased
as a result of oxidative stress. Importance of Se in fish nutrition is well recognised.
Similar to other food industries, organic selenium is shown to be more effective
in fish nutrition than sodium selenite. Limited results from wild fish studies indicate
that Se level in wild fishes usually several-fold higher than those in farmed fishes.
Possible stabilizing effect of Se on astaxanthin absorption and metabolism resulting
in better flesh coloration needs further investigation. Production of Se-enriched
fish can help solving global Se deficiency. Using organic selenium in the fish diet
it is possible to achieve desirable Se concentration in the fish flesh.
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11
SELENIUM AND HUMAN HEALTH
Diet cures more than lancet
Introduction
From information on effects of natural antioxidants on animals including birds
presented in previous ten chapters of this book it has become clear that natural
antioxidants have important roles in maintaining not only animal health but could
also be beneficial for human. In fact, many physiological processes in human
body are dependent on antioxidant/prooxidant balance in every cell and in whole
body. In this regard diet provides a wide range of antioxidant compounds and is
able to affect our health.
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Three major areas of concern are improvement of the diet, increase physical
activity and reduction of prevalence of tobacco use the major risk factors for
these diseases. In this chapter relationship of the balanced diet to human health
will be considered with a special emphasis to selenium.
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Mediterranean
Japanese
Hunter-gatherer
High consumption of
soya, seaweeds, raw fish
and rice
Moderate consumption of
milk and dairy products and
alcohol
Moderate consumption
of salt
Country
Life expectancy
Japan
Greece, Hong Kong, Spain, Sweden, Switzerland
Australia, Canada, France, Israel, Italy, Netherlands, Norway
Austria, Belgium, Denmark, Finland, Germany, New Zealand, UK, USA
Ireland, Portugal, Singapore
79
78
77
76
75
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wild game;
fish;
uncultivated plant foods.
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Energy expenditure
Omega-3 PUFA
Complex carbohydrate
Fiber
Fruits and vegetables
Animal protein
Antioxidant
Phytochemicals
Potassium and calcium
Vitamins
(Adapted from Simopoulos, 1998; Eaton and Eaton, 2000 and Haenel, 1989)
The main advantage of the described diet is an adaptation of the human digestive
system to most of those nutrients. This means that high efficiency of assimilation
from the diet and distribution in the body could be a driving force in health
promoting properties of various compounds, including antioxidants and
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selenium;
glutathione;
a balanced ratio of (n-6):(n-3) PUFAs;
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Japanese diet. The traditional Japanese diet has attracted the attention of many
researchers since it is beneficial for preventing ischemic heart disease and certain
type of cancers. It has been shown that this diet, containing little fat but enriched
in complex carbohydrates and n-3 fatty acids of marine origin, may be related to
the low atherogenic index in rural areas in Okayama Prefecture in Japan (Okita et
al., 1995). Main characteristic features of this diet is low level of fat and low
calorie which remained constant at the level of developing countries for the last
40 years (Hoshi, 2000). A wide verity of marine products including seaweeds
and root-vegetables make this diet different from others. Thus, the dietary habits
of the Japanese have made possible a high n-3 PUFA intake within a low-fat
regimen. They are currently consuming, on average, approximately 26% of energy
as fats with ratios of polyunsaturated to saturated fats and n-6 to n-3 fatty acids of
approximately 1.2:1 and 4:1, respectively (Sugano and Hirahara, 2000). For
comparison, in the United States the ratio of n-6 to n-3 fatty acids is approximately
9.8:1 (Kris-Etherton et al., 2000).
Among Japanese, flavonoids and isoflavones originated from soya-related
products are the main components among nonnutrient phytochemicals with
antioxidant potential in the diet and their high consumption may contribute to
low incidence of coronary heart diseases in Japanese women (Arai et al., 2000a).
Isoflavones have a limited distribution in nature, and, for practical purposes,
soyfoods are the only nutritionally relevant dietary source of these phytochemicals
(Messina and Bennink, 1998). Seaweeds in the Japanese diet provide substantial
amounts of various antioxidant compounds. Plants consumed by humans contain
thousands of phenolic compounds. Among them the effects of dietary polyphenols
are of great current interest due to their antioxidative and possible anticarcinogenic
activities. For example, soy isoflavones are capable of inhibiting lipoprotein
oxidation in vitro and suppressing formation of plasma lipid oxidation products
in vivo (Patel et al., 2001). It has been also shown that soy isoflavone
supplementation decreases levels of oxidative DNA damage in humans (Djuric et
al., 2001) and this may be a mechanism behind the cancer-preventive effects of
soy isoflavones. The study of Davis et al. (2001) demonstrated that soy isoflavone
supplementation protect cells from oxidative stress-inducing agents by inhibiting
NF-kappaB activation and decreasing DNA adduct levels. In the study of Exner
et al. (2001) genistein, a flavonoid derived from soy has been shown to prevent
atherogenic modification of LDL by glucose autoxidation radical products. As
the protective effect of genistein on LDL atherogenic modification was found at
glucose/genistein molar ratios which may occur in vivo, the authors suggested a
beneficial action of a soy diet in preventing chronic vascular diseases and early
atherogenic events. Similarly, when soy breakfast cereals (providing 168 mg/d
isoflavones) were used for 3 weeks, the level of oxidized LDL in human was
significantly reduced (Jenkins et al., 2000).
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by 1.5 times and SFA more than 2.5 times and the ratio PUFA/SFA decreased
almost twice (Hirai et al., 1986). Therefore, despite of highest life expectancy, in
Japan, nutrition and diet are considered to play important roles in the emerging
problems of obesity, diabetes mellitus, hypertension etc., because of excessive
energy intake and deficiency or excessive intake of certain nutrients (Matsumura,
2001). Therefore internationalisation of food with a fashion for fast food make it
difficult to preserve the typical Japanese or Mediterranean diet. In fact, today
there is no substantial difference in the incidence of ischemic heart disease between
Greece and the UK or USA (Hoshi, 2000).
The main conclusion which can be made from analysis of ideal diets is that
natural antioxidants are among major players in these diets. Recent research on
phytochemicals has changed a view on dietary factors affecting our health. In
particular it seems likely that there are hundreds and even thousands of such
factors and now we are dealing just with the top of iceberg. However, emerged
information can help us to understand and to some extent explain a beneficial
effect of various vegetables and fruits in our diet.
It seems likely that omega-3 fatty acids, vitamin E, carotenoids and selenium
are nutrients, which are commonly present in all three ideal diets and are lacking
in our everyday diets. Vitamin E and carotenoids will be considered in the chapter
13.
Omega-3 PUFAs
Major advances have been made in recent years in our understanding the molecular
mechanisms whereby dietary fatty acids influence the bodys metabolism. It has
become clear that in addition to well defined roles in energy metabolism and as
constituents of biological membranes, PUFAs also have specific regulatory
functions. For example, PUFAs are precursors of different biologically active
compounds, including eicosanoids. These compounds are considered to be
secondary messengers (Graber et al., 1994) as well as take part in the direct
regulation of gene expression by altering the transcription of specific genes (Clarke
and Jump, 1994).
PUFA may be subdivided into families depending on the position of the double
bond in the molecules. Two of the important groups of PUFA in human nutrition
are the, n-6 or omega-6 fatty acids and the n-3 or omega-3 fatty acids. The
precursors of the n-6 and n-3 family of fatty acids are linoleic acid (LA, 18:2n-6)
and alpha-linolenic acid (ALA, 18:3n-3) respectively. These PUFA have to be
supplied by the diet (Nair et al., 1997). The n-3 and n-6 PUFAs are not interconvertible in the human body and affect eicosanoid metabolism, gene expression
and intercellular cell-to-cell communication (Simopoulos, 2000).
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LA and ALA can be elongated and desaturated into their longer-chain active
metabolite arachidonic acid (20:4n-6) and eicosapentaenoic acid (EPA; 20:5n-3)
and docosahexaenoic acid (DHA; 22:6n-3) (Roche, 1999). These two classes of
PUFA are metabolically and functionally distinct and in many cases have opposing
physiological effects (Simopoulos, 2000). Therefore, the absolute level and the
balance between n-6 and n-3 PUFA in the diet is considered to be an important
determinant of many metabolic functions in the human body (Table 11.4; Surai
and Sparks, 2001).
Table 11.4 Beneficial effects of n-3 fatty acids for human in prevention and management of human
diseases (Adapted from Surai and Sparks, 2001).
Disease
Disease
Embolism
Allergic problems
Chronic obstructive pulmonary disease
Prostate and breast cancer
Immunological functions
Autoimmune disease
Fetal brain and visual development
Improvement of learning ability
Positive effects on longevity
Industrial production of grain-based feeds (rich in n-6 PUFAs) for farm animals
results in production of meat which is rich in n-6 fatty acids and poor in n-3
PUFAs. While wild and free range animals tend to contain higher proportions of
n-3 PUFAs in their muscles or eggs (green grass and leaves are rich sources of
ALA) they are not the major sources of the fat and protein in human diet. As a
result of this shift to the n-6 PUFAs the ratio n-6 PUFAs/n-3 PUFAs in modern diet
comprises about 1:10-50 while evolutionary human diet was rich in n-3 fatty
acids and the n-6/n-3 ratio was at the level 1:2 (Simompoulos, 2000). Evidence is
accumulating that the metabolism of omega-3 and omega-6 fatty acids requires
the same desaturation enzymes, resulting in competition between these two families
(Sargent, 1997). Therefore, high proportions of n-6 fatty acids in the diet inhibit
conversion of ALA to EPA and DHA exacerbating the n-3 fatty acid deficiency
(Connor, 1999).
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Realisation that the long chain n-3 PUFAs are also important came to light
when the diets in the developed countries were already rich in n-6 PUFA (Sargent,
1997). The main PUFA in the Western diet is LA, found mostly in vegetable
oils such as safflower oil, sunflower oil, corn oil, soybean oil, etc. (Nair et al.,
1997). Because the typical Western diet, provides high levels of n-6 PUFAs
and low levels of n-3 PUFAs it is considered to be imbalanced and to be associated
with the increased incidences of certain diseases (Shahidi and Vanasundara, 1998).
This balance could be improved by reversing the trend for fish consumption
decrease (oily fish being an important source of DHA; Sargent, 1997). A survey
conducted in Nebraska (USA) indicated that fish was consumed only once per
two weeks (Lewis et al., 1995). Fish consumption in European countries averages
34 g/day and varies from 19 g/day (Netherlands) up to 92 g/day (Portugal)
providing n-3 fatty acids in a range 0.2-1.84 g/day (Gibney, 1997). Therefore
there is a recommendation to people who do not eat fish to obtain 200 mg of very
long chain n-3 PUFA daily from other sources (De Deckere et al, 1998).
It has been shown that inadequate intakes of n-3 polyunsaturated fatty acids
per se, particularly DHA, adversely affects cardiovascular function (Kinsella et
al, 1990) and, in neonates a deficient supply of this polyunsaturate may impair
the development and functioning of the retina and brain (Green and Yavin, 1998;
Uauy et al, 1996; Wainwright et al, 1994). The interest in this subject was
associated with observations in 1970s that the low incidence of heart attacks
among native fishing populations in Greenland, Japan and Alaska who ate very
large amounts of fish were associated with an increased level of omega-3 fatty
acids in their diet. More recently a significant inverse relationship was found
between fish consumption and the risk of a fatal myocardial infarction (Daviglus
et al., 1997). Similarly, an intake of 5.5 g of n-3 PUFA per month was associated
with a 50% reduction in the risk of primary cardiac arrest compared with no
dietary intake of n-3 PUFA (Siscovick et al, 1995).
There are a number of potential solutions to improve n-3/n-6 fatty acid balance
in our diet. For example:
1.
2.
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3.
Produce designer or so-called functional foods, enriched with n-3 fatty acids.
This option has been embraced during the last few years and there are now
many products in the market that are enriched with n-3 fatty acids, including
various oils, bakery products, infant formula milk, mayonnaise, margarine,
salad dressings and others (Simopoulos, 2000). Among them, eggs, as shown
in the chapter 12, could have a special place as an ideal delivery system for
n-3 fatty acids. In addition, chicken meat enriched with n-3 fatty acids could
also be a valuable option (Phetteplace and Watkins, 1989). Furthermore, it
is possible to alter fatty acid composition of pork, lamb, beef (Wood and
Enser, 1997; Wood et al., 1999) and milk (Mansbridge and Blake, 1997). It
is interesting, that recent survey in the UK revealed that poultry consumption
in 1999 was 236 g/person per week which is equal to total consumption of
beef, veal, lamb and pork (236 g; National Food Survey, 1999). This is
probably associated with lower chicken meat prices and healthier chicken
meat image from the point of view of fatty acid profile. However, chicken
meat enrichment with n-3 PUFA is associated with decreased quality in
terms of storage and flavour (Manilla and Husveth, 1999) and dietary
linolenic acid is not always effective in transferring to muscles (LopezFerrer et al., 2001). Indeed n-3 enriched meat is characterised by increased
susceptibility to lipid peroxidation (Li et al., 1996). Antioxidants are found
to be effective in decreasing lipid peroxidation in such meat (Li et al., 1996;
Wood and Esner, 1997). It seems realistic that a combination of increased
dietary antioxidant supplementation with new sources of high quality fish
oil could help produce meat with increased DHA concentration and without
off-flavour.
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day); Turkey (32 g/day), Sweden (38 g/day), Slovak Republic (38.2 g/day),
France and Germany (47 g/day) and Italy (49 g/day) (Reilly, 1996; Kadrabova
et al., 1998; Table 11.5).
70
Se intake (mcg/day)
60
50
40
30
20
10
0
1974
1986
1991
1994
1995
RDA
Figure 11.1 Selenium intake in the UK (Adapted from MAFF Food Surveillance Information sheet,
October 1997).
Did you know that selenium enters the food chain through
incorporation into vegetable proteins as the amino acids
such as selenomethionine
The decline in selenium intake is reflected in decreased serum and whole blood
selenium concentrations (Alfthan and Neve, 1996; MacPherson et al., 1997).
Indeed, in 1991 a French study showed a large-scale deficit in micronutrients,
including Se affecting 3040% of the healthy population (Hercberg et al., 1991).
This was confirmed by many other studies showing low plasma concentrations of
selenium. Plasma selenium concentrations are decreasing progressively in the
healthy European population since the 1980s, reflecting lower nutritional selenium
intake due to decreased nutrient Se content (Rayman, 1997; 2002; 2003; 2004).
The mean plasma concentration in various European areas (4085 g/l) is
substantially lower than the selenium concentration associated with a cancer
prevention activity according to the American Nutritional Cancer Prevention Study
or Se levels that required for maintenance of an optimal plasma GSH-Px activity
(Rayman, 2004).
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Country
g/day
Reference
2-36
7-11
11
15
11- 40
12-43
New Guinea
Czech republic
Nepal
Finland before selenium
fertilization
India,vegan low income
Egypt
Serbia
Slovenia
China
Croatia
Slovakia
Belgium
20
15- 50
23
26
27
29
30
30
26.0 - 37.2
27.3-33.9
27-38.2
28.4-61.1
Brazil
New Zealand
28.4-37.0
29-38
Sweden
29-44
France
Turkey
29-48
30-36.5
UK, 1994
UK, 1995
England
Spain
Germany
Portugal
Denmark
Italy
UK, 1985
India, conventional diet
Austria
Ireland
UK, 1974
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32
33
35
35
35-48
37
38-47
43
43
48
48
50
60
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Absorption
Urine Se
Retained Se
Broccoli
Selenate
Selenite
Broccoli
Selenate
Selenite
66.4
7.9
58.5
68.5
28.6
39.9
15.3
5.1
12.7
74.4
14.6
59.8
76.0
37.5
37.8
38.0
14.2
26.3
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Selenium deficiency
Selenium essentiality for human is proven and based on the following (Burk and
Levander, 1999):
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The signs and symptoms of selenium deficiency closely simulate each other for
animals and man. Deficiency states have been demonstrated for inhabitants of
regions where selenium supply is limited, in protein-energy malnutrition, and in
patients maintained on total parenteral nutrition without selenium supplementation.
Severe deficiency is characterized by cardiomyopathy while moderate deficiency
results in less severe, myodegenerative syndromes such as muscular weakness
and pain as well as a variety of other selenium-associated diseases (Koller and
Exon, 1986). For example, a suspected Se deficiency syndrome has been
demonstrated in a few patients treated with parenteral nutrition without added Se
(Rannem et al, 1996). Muscular pain and muscular and cardiac dysfunction have
been demonstrated in some patients, but no uniform symptomatology has been
described. A case of fatal cardiomyopathy caused by selenium deficiency during
parenteral nutrition for 6 consecutive years was also described (Fleming et al.,
1982). Furthermore, a case of fulminant heart failure in a middle aged woman
with a complex medical and surgical history including documented malabsorption
and selenium deficiency was presented where pathological examination of the
heart showed features consistent with Keshan disease (Burke and Opeskin, 2002).
Clinical manifestations of many of these disorders require contributory factors,
such as stress, to precipitate symptoms which are documented for animals and
implicated for humans.
Se deficiency in human population is associated with two diseases (Keshan
disease and Kaschin-Beck disease) reported in areas of China and some other
countries characterised by extremely low Se content in the soil and food. In
particular, the diseases occur in a geographic belt stretching from Heilonjiang
Province in north-east China to Yunnan Province in the south-west (Fordyce et
al., 2000).
Keshans disease is a cardiomyopathy, which mainly affects young children and
women of child-bearing age, has occurred in some areas of China where the soil
is low in Se (Chen et al., 1980). In fact, Keshan disease was first described in
Chinese medical literature more than 100 years ago, but not until 40 years after its
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multifocal necrosis
fibrous replacement of the myocardium
myocytolysis
It is important to note that there are other factors that affect this disease development
including (Burk and Levander, 1999):
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general population in those high risk areas was improved, there was also
improvement in general diet and as a result Keshan disease practically disappeared
from endemic areas (Yang et al., 1984). In a large, prospective, placebo-controlled
study conducted in China (Keshan Disease Research Group, 1979a), the incidence
of Keshan disease in selenium-supplemented children fell from 2.2% in 1974 to
1% in 1975, to 0.32% in 1976 and in 1977 no cases of the disease were reported.
Did you know that Keshan disease and Kashin-Beck diseases
are related to Se deficiency?
KaschinBeck disease, is an osteoarthropathy, a generative articular disease caused
by oxidative damage to cartilage that leads to deformation of bone structure (Tan,
1989; Ge and Yang, 1993). The disease is endemic in Tibet and other areas of
China, Siberia, and North Korea areas where Se deficiency is also endemic
(Sokoloff, 1989). This Se-responsive bone and joint disease has been detected in
children aged 5-13 years in China and less extensively in south-east Siberia. Indeed,
the disease occurs during preadolescence or adolescence (Allander, 1994). The
disease is characterised by joint necrosis - epiphyseal degeneration of the arm
and leg joints resulting in structural shortening of the fingers and long bones with
consequent growth retardation and stunting (FAO/WHO, 1996). Therefore,
affected subjects have varying degrees of joint deformation and limited joint
mobility. In the most severe cases, there is necrosis of growth plates and joint
cartilage (Moreno-Reyes et al., 1998). Therefore, necrotic degeneration of the
chondrocytes is the most striking pathologic feature of this disease. Dwarfism
and joint deformation result from these cartilage abnormalities (Tan, 1989; Burk
and Levander, 1999). As mentioned above, Kaschin-Beck disease occurs in areas
where the availability of soil Se for crop growth is low. The selenium contents of
hair and of whole blood are abnormally low and the blood content of GSH-Px is
reduced. The disease has been reported in white migrants to the areas of endemic
disease, and clinical improvement was observed in children who move to areas
where the disease is not endemic (Sokoloff, 1990). In addition to Se deficiency,
a number of other etiologic factors have been suggested for this condition,
including mycotoxins in grain, mineral imbalance, organic contaminants in
drinking water and some others. It has also been hypothesized that iodine deficiency
and KaschinBeck disease might be associated (Moreno-Reyes et al., 1998). A
spontaneous decrease in incidence from 1970 (44 percent) to 1980 (14 percent)
to 1986 (1 percent) has been attributed to general improvements in the nutritional
status of Chinese rural communities (FAO/WHO, 1996). However, the efficacy of
Se supplements in the prevention of KaschinBeck disease is still controversial.
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Selenium requirement
When establishing Se requirement for human there are the same problems as
mentioned above in relation to animals. The main problem is to decide what kind
of measurements/tests to use for such an assessment. On the one hand, Se dietary
doses providing maximal activity of GSH-Px have been actively used to establish
human Se requirement. On the other hand, GSH-Px is only one from at least 25
selenoproteins identified in human body. Furthermore, maximal expression of
GSH-Px does not mean that all other selenoproteins will have maximum expression
at the same Se dose. For example, recently it has been shown that expression of
SeP is not maximum at the Se levels providing maximum activity of GSH-Px
(Burk and Hill, 2005; Xia et al., 2005). Furthermore, TR activity is increasing
with Se doses much higher than those needed for maximal expression of GSH-Px
(Gromer et al., 2004). Since there are analytical difficulties to assess expression
and activities of all selenoproteins at certain Se doses there is always uncertainty
if a used parameter would be adequate. However, the bigger problem is related to
functional changes in the body depending on Se status. For example,
immunomodulating properties of Se are shown (see chapter 4) at doses much
higher than those considered to be adequate to maintain growth and development.
Similarly, anticancer properties of Se are also shown at doses much higher than
RDI.
There are also other factors affecting Se requirement:
diet composition
antioxidant concentration in the diet
level of stress
health status
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Recently a human study was carried out in New Zealand by Duffield et al. (1999).
In the study men and women were given Se supplements (placebo, 10,20, 30 or
40 g) for 20 weeks and they had an average dietary Se intake of 28 g/day. The
authors showed that GSH-Px activity reached a plateau only in the group with the
maximal Se supplementation and they proposed an intake of 90 g Se as
recommended daily allowance. The Institute of Medicine (USA) used two studies
(Chinese study of 1983 and aforementioned New Zealand study) to establish
RDA in selenium. As a result of this analysis the recommendations of the Panel
was for a daily RDA of 55 g for adults including men and women (Institute of
Medicine, 2000; Table 11.7). The British governments defined reference nutrient
intake is 75 g/day for men and 60 g/day for women (Rayman, 2000; Department
of Health, 1991).
Table 11.7 The 2000 Reference Dietary Intakes (RDI) for Selenium
Life stage
Age
Infants
0 6 months
7-12 months
1-3 years
4-8 years
9-13 years
14-70 years
>70 years
9-13 years
14-70 years
>70 years
Children
Males
Females
Pregnancy
Lactation
The Reference Daily Intake suggested by other countries are as follows (Levander,
1999):
The Nordic countries: 30 to 60 g/d for adult males and females (Standing
Nordic Committee on Food, 1989).
The German Estimated Value for Adequate Supply: 20 to 100 g/d for adults
(Committee on Nutrient Requirements, 1991).
The Australians: 85 and 70 g/d for men and women, respectively (Truswell
et al., 1990; Table 11.8).
Se status assessment
The definition of the exact role of selenium in human homeostasis has been
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Table 11.8 Australian recommended dietary intakes for selenium (Adapted from Tinggi, 2003)
Age group
Infants
0-6 months
7-12 months
Children
8-11 years
12-18 years
g/day
Age group
g/day
10
15
Adults
Males
Females
85
70
50
85
Pregnancy
Lactation
+10
+15
Due to the limited data available for Se dietary survey in Australia, the estimation for the
Australian RDI is based on the data from other countries.
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Table 11.9 Major Causes of Death in United States in 1995 and 2002(Adapted from Knight, 1999 and
Cancer Statistics, 2005)
1995
Cause of death
Heart disease
Cancer
Cerebrovascular
Chronic pulmonary diseases
Accidents
Pneumonia/influenza
Diabetes mellitus
HIV infection
Suicide
Chronic liver disease and
cirrhosis
2002
Rank
Number
died
Percent of
total
1
2
3
4
5
6
7
8
9
10
738,781
537,969
158,061
104,756
89,703
83,528
59,085
42,506
30,893
24,848
32.0
23.3
6.8
4.5
3.9
3.6
2.6
1.8
1.3
1.1
Number
died
Percent of
total
696,947
557,271*
162,672
124,816
106,742
65,681
73,249
58,8661
40,9742
33.8653
28.5
22.8
6.7
5.1
4.4
2.7
3.0
2.41
1.72
1.43
1
/Alzheimer disease; 2Nephritis; 3Septicemia
*In 2005 estimated number of new cancer cases in the USA is 1,372,910 and 580,280
Americans are expected to die of cancer, more than 1500 people a day (Cancer Facts and
Figure, 2005)
Cancer
Cancer is a major public health problem in the developed countries. It has been
calculated by Parkin et al (2005) that globally in 2002, there were:
The most commonly diagnosed cancers were (Figures 11.2 and 11.3):
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1400000
1200000
1000000
800000
600000
400000
Bladder
Oesophagus
Cervix
Liver
Prostate
Stomach
Bowel
Breast
Lung
200000
Figure 11.2 The most commonly diagnosed cancers worldwide in 2002 (Adapted from http://
info.cancerresearchuk.org/cancerstats/world/commoncancers
1200000
1000000
800000
600000
400000
Prostate
Pancreas
Cervix
Oesophagus
Breast
Bowel
Liver
Stomach
Lung
200000
Figure 11.3 The most common causes of death from cancer worldwide, 2002 estimates (Adapted from
http://info.cancerresearchuk.org/cancerstats/world/commoncancers
Overall, cancers of the lung, breast, bowel, stomach and prostate accounted for
almost half of all cancer diagnosed worldwide. Since 1975 the number of people
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being diagnosed with lung cancer worldwide has doubled, it is now the most
common cancer. The incidence rate for male lung cancer varies by 12-fold between
the different regions of the world, while the female rate varies by more than 30fold.
Did you know that the most common causes of cancer death
are lung cancer, stomach cancer and liver cancer?
The most common causes of cancer death are:
lung cancer (1.18 million deaths; five-year survival rates for lung cancer
are consistently poor at between 7-14% (Parkin et al., 1999) so mortality
patterns follow incidence patterns closely),
stomach cancer (700,000 deaths),
liver cancer (598,000 deaths).
Therefore lung cancer is the most common cause of death from cancer accounting
for 18% of all deaths from cancer. The most prevalent cancer in the world is
breast cancer (4.4 million survivors up to 5 years following diagnosis; Parkin et
al., 2005). One in ten of all new cancers diagnosed and almost one in four cancers
diagnosed in women worldwide is a cancer of the female breast. More than 1.1
million women are diagnosed each year and the numbers of women being
diagnosed annually worldwide has almost doubled since 1975. Incidence rates of
breast cancer are increasing in most countries, and the changes are usually greatest
in areas where rates were previously low. Breast cancer is the main cause of death
from cancer in women globally.
Each year an estimated one million people are diagnosed with bowel cancer
and it accounts for 9% of all cases. There have been steady increases worldwide
in the numbers of people being diagnosed with bowel cancer over the last 25
years. Bowel cancer is the fourth most common cause of death from cancer
worldwide accounting for 8% of deaths from cancer. Prostate cancer is the fifth
most commonly diagnosed cancer worldwide, it accounts for 6% of all cases, and
there have been large increases in the incidence of prostate cancer over the last
25 years (Parkin et al., 2005).
Currently, one in four deaths in the United States is due to cancer (Tables 11.10
and 11.11). In accordance with cancer statistics in the USA in 2005 (Jemal et al.,
2005):
Did you know that one in four deaths in the United States
is due to cancer?
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Table 11.10 Cancer incidence rates in the US in 1997-2001 (Adapted from American Cancer Society, 2005)
Males
Cancer
African American
rate
Prostate
Breast
Lung and bronchus
Colon and rectum
Oral cavity and pharynx
Ovary
Urinary bladder
Stomach
Kidney and renal pelvis
Non-Hodkin lymphoma
Pancreas
Leukemia
Myeloma
Larynx
Liver and intrahepatic bile duct
Esophagus
Brain and other nervous system
All malignant neoplasms
274.3
120.7
73.1
20.1
20.0
19.4
18.9
18.4
17.4
13.0
12.9
12.1
11.6
11.3
4.9
697.3
Females
White
rate
African American
rate
White
rate
171.2
82.3
64.4
15.9
41.2
10.2
16.5
24.5
12.6
17.0
6.7
6.8
6.7
8.1
8.8
568.3
118.6
55.3
56.1
5.9
10.3
7.8
9.8
10.0
11.2
14.2
8.0
10.2
2.7
3.9
3.9
3.2
400.2
143.2
53.5
46.8
6.6
15.0
10.2
4.5
8.2
16.9
9.5
10.1
4.2
1.5
2.6
2.0
6.1
435.1
Rates are per 100,000 and age-adjusted to the 2000 US population standard
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A total of 1,372,910 new cancer cases and 570,280 deaths are expected
corresponding to more than 1,500 deaths per day. Cancer accounted for
approximately 23% of all deaths, ranking second only to heart disease. Similarly,
in 2004 the American Cancer Society estimates 1,368,050 new cancer cases
and 536,700 deaths from the disease. Although cancer rates for different sites
vary widely, the 5-year relative survival rate for all cancers combined is estimated
at 63% (Borek, 2004).
The lifetime probability of developing cancer is 46% for men and 38% for
women. This means that during the lifetime almost one in two men and more
than one in three women will develop cancer.
Among men, cancers of the prostate, lung and bronchus, and colon and rectum
account for more than 56% of all newly diagnosed cancers
Prostate cancer alone accounts for approximately 33% (232,090) of incident
cases in men
The three most commonly diagnosed cancers among women in 2005 will be
cancers of the breast, lung and bronchus, and colon and rectum, accounting
for approximately 55% of estimated cancer cases in women
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Table 11.11 Cancer mortality rates in the US in 1997-2001 (Adapted from American Cancer Society, 2005)
Males
Cancer
African American
rate
Females
White
rate
104.1
70.4
34.3
16.0
5.6
13.3
11.7
9.3
76.6
28.8
24.8
12.0
7.9
5.8
7.4
6.1
9.0
9.0
7.5
7.4
6.3
5.4
3.3
352.2
10.5
4.4
3.9
10.8
6.2
2.3
6.0
250.5
African American
rate
White
rate
39.9
41.6
35.4
24.5
12.8
2.9
6.3
3.2
3.8
9.2
5.4
6.6
2.0
4.6
2.7
0.9
2.3
200.4
26.4
17.1
8.9
2.3
2.8
1.7
2.7
7.5
6.0
2.9
1.6
7.2
2.8
0.5
4.0
169.1
Rates are per 100,000 and age-adjusted to the 2000 US population standard
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Breast cancer alone is expected to account for 32% (211,240) of all new
cancer cases among women
Cancers of the lung and bronchus, prostate, and colon and rectum in men
and cancers of the lung and bronchus, breast, and colon and rectum in women
are the most common fatal cancers. These four cancers account for 50% of
the total cancer deaths among men and women. Indeed, colorectal cancer
is the second leading cause of mortality in the United States. In the United
States, the cumulative lifetime risk of developing colorectal cancer for both
men and women is 6%. Despite advances in the management of this disease,
the 5-year survival rate in the United States in only 62%. Four classes of
chemopreventive compounds have demonstrated efficacy in reducing
recurrent colorectal adenomas and/or cancer in randomized, controlled trials.
They are selenium, calcium carbonate, hormone replacement therapy, and
nonsteroidal anti-inflammatory drugs (Hawk and Levin, 2005).
When age-adjusted death rates are considered cancer is the leading cause of
death among men and women under age 85. Indeed, cancer is the leading
cause of death among women aged 40 to 79 and among men aged 60 to 79.
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For example, a total of 476,009 people under age 85 died from cancer in
the United States in 2002 compared with 450,637 deaths from heart disease
Among men, cancer of the lung and bronchus predominates in men aged
40 years and older. Colorectal cancer is the second most common cause of
cancer death among men 40 to 79 years old, and prostate cancer is the
second most common among men aged 80 and older.
Among women, breast cancer ranks first at ages 20 to 59 years, and lung
cancer ranks first at age 60 years and older. Indeed, lung cancer surpassed
breast cancer as the leading cause of cancer death in women in 1987. Lung
cancer is expected to account for 27% of all female cancer deaths in 2005.
Did you know that genetic damage, leading to the accumulation
of specific mutations, is a prerequisite for the cells
transformation from a normal into a malignant phenotype?
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Table 11.12 Age-specific (55-64 year group) cancer death rates in 1968 for white males in states different
in Se status (Adapted from Shamberger et al., 1976)
No of states
6
19
11
20
Se level, ppm
Probability
Very high,
0.26+
High,
0.1+
Medium,
0.06-0.09
Low,
0.01-0.05
392.0
P<0.001
429.7
P<0.001
450.0
P<0.001
516.0
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Table 11.13 Ecological studies showing an inverse relationship between dietary intake of Se and cancer
risk (Adapted from Whanger, 2004; Meuillet et al., 2004; Combs and Gray, 1998)
Effects
References
Yu et al., 1985
Cancer mortality rates were significantly lower in intermediateselenium and high-selenium counties in comparison to low-selenium
counties for total cancer and cancers of the lung, colon, rectum,
bladder, esophagus, pancreas, breast, ovary and cervix
Table 11.14 Observational studies examining the relationship between Se status and cancer risk (Adapted
from Whanger, 2004; Meuillet et al., 2004; Combs and Gray, 1998)
Effects
References
In a U.S. study of 11,000 hypertensives followed for 5 years showed a Willett et al., 1983
two-fold increase in risk for all cancers in those in the lowest
(<115 ng/ml) quitile compared to those in the highest quintile
(<154 ng/ml) of plasma selenium. The association between low
selenium level and cancer was strongest for gastrointestinal and
prostatic cancers. Serum levels of vitamins A and E compounded
the effect of low selenium; relative risks for the lowest tertile of
selenium were 2.4 and 3.9 in the lowest tertiles of vitamins E and A,
respectively
A matched-pair analysis was conducted with data based on a
prospective six-year follow-up of a random population sample with
8,113 persons in eastern Finland. Cases were 31- to 59-year-old men
and women initially free of cancer. One control was matched to each
case according to age, gender, daily tobacco consumption, and serum
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Effects
References
Patients who died of cancer during the follow up period had a 12%
Salonen et al., 1985
lower mean serum selenium concentration than the controls. The
adjusted risk of fatal cancer was 5.8-fold among subjects in the lowest
tertile of selenium concentrations compared with those with higher
values. Subjects with both low selenium and low alpha-tocopherol
concentrations in serum had an 11.4-fold adjusted risk.
Sera from 43 persons who developed thyroid cancer on an average
Glattre et al., 1989
4.8 years after blood sampling were compared with sera from controls.
Cases were significantly lower in serum Se than controls, and the
estimated odds ratio of thyroid cancer increased from 1 for levels
greater than or equal to 1.65 mol/l, to 6.1 for levels 1.26-1.64 mol/l,
to 7.7 for levels less than or equal to 1.25 mol/l
Low serum and plasma Se concentrations were associated with
increased risk of malignant oral cavity lesions. Mean serum Se levels
were 105, 101, and 77 ng/ml in the precancerous, controls, and
malignancy groups, respectively
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Effects
References
A 10-year prospective study of 1,738 Americans found initial plasma Clark et al., 1993
Se levels inversely correlated to both non-melanoma skin cancer and
colonic adenomatous polyps. Those with plasma levels below the U.S.
population median of 128 ng/ml plasma were four times more likely to
have one or more polyps than those with levels above 128 ng/ml.
The association between selenium and prostate cancer risk was in the
protective direction with individuals in the top four fifths of the
distribution having a reduced risk of prostate cancer compared with
individuals in the bottom fifth
Serum selenium was associated with a decreased risk of ovarian cancer Helzlsouer et al.,
among case participants diagnosed 4 or more years after blood
1995, 1996
collections (P for trend =0.02). Women in the highest tertile compared
with the lowest tertile were 4 times less likely to develop ovarian cancer
In Dutch patients the mean Se levels were significantly lower than
Kok et al., 1987
that of control values in men. The adjusted risk of death from cancer for
men in the lowest quintile of serum selenium (below 100.8 ng/ml) was
more than twice that of subjects with higher levels (relative risk = 2.7)
The association between toenail selenium and lung cancer was
van den Brandt et al.,
investigated in a cohort study of diet among 120,852 Dutch men and 1993a
women aged 55-69 years. The rate ratio of lung cancer for subjects in
the highest compared to the lowest quintile of toenail selenium, after
controlling for age, gender, smoking, and education, was 0.50 with a
significant inverse trend across quintiles. The protective effect of Se was
concentrated in subjects with a relatively low dietary intake of betacarotene or vitamin C.
In a multivariate analysis, the relative rates (RRs) of stomach cancer
van den Brandt et al.,
for subjects in increasing quintiles of toenail selenium level were 1.00, 1993b
0.44, 0.59, 0.84, and 0.64
Higher toenail Se levels were associated with a reduced risk of
Yoshizawa et al.,
advanced prostate cancer, OR for comparison of highest to lowest
1998
quintile was 0.49. After additionally controlling for family history of
prostate cancer, body mass index, calcium intake, lycopene intake,
saturated fat intake, vasectomy, and geographical region, the OR was 0.35
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Effects
References
Low selenium status was associated with increased risk for lung cancer Hartman et al., 2002
The OR for men with adjusted toenail selenium concentrations at the
75th percentile compared to those with the lowest selenium
concentrations ranged between 0.20 and 0.61
The association between cancer of the pancreas and serum Se was
significant when the data were analyzed as a whole but its effect was
seen principally in men
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Effects
References
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Effects
References
Serum samples from 9,101 cancer-free individuals were collected and Knekt et al., 1998
stored at -20 degrees C by the Finnish Mobile Clinic in 1968-1971
and 1973-1976. During follow-up until the end of 1991, 95 cases of
lung cancer were diagnosed. Selenium concentrations were determined
from the serum samples of the cases and 190 controls. Mean levels of
serum selenium in cases and controls were 53.2 ng/ml and 57.8 ng/ml,
respectively. The relative risk of lung cancer between the highest and
lowest tertiles of serum selenium, was 0.41. The association was
stronger at lower levels of alpha-tocopherol. The association was also
pronounced among current smokers.
In a prospective study of 39,268 men and women in Finland, risk for
several cancers (stomach, pancreas and lung) was significantly
elevated in men who had the lowest level of serum selenium
(53-63 g/l) , RR=2.5
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Effects
References
Toenail selenium level was not associated with cancer at any major
site, including uterine cancer, colorectal cancer, melanoma, ovarian
cancer, or lung cancer
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Effects
References
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Table 11.15 Prospective studies examining the role of selenium in cancer prevention (Adapted from
Donaldson, 2004)
Study
No.
No.
Outcomes
cases controls
Comments
Reference
Se = risk of
Result only in men
advance prostate
with PSA 4 ng/ml;
cancer (OR = 0.52,
13 years of follow up
95% CI = 0.280.98)
Physicians
586
Health Study
577
Li et al., 2004
Netherlands 540
Cohort Study
1,211
Baltimore
52
Longitudinal
Study of
Aging
96
Se = risk prostate
cancer (OR for
quartiles of Se = 1.0,
0.15, 0.21, 0.24
Washington
County,
Maryland
117
233
Health
181
Professional
Follow-up
Study
181
Se = risk of
advanced prostate
cancer
Adjusted OR = 0.35
Yoshizawa et al.,
(95% CI = 0.160.78) 1998
Prospective
study
136
238
A case
control
study
212
233
Se = risk of
prostate cancer
Criqui et al.,
1991
OR=0.71 (95%CI
Vogt et al.,
0.39-1.26) comprising 2003
highest to lowest
quartiles
Recent data (Woodson et al., 2003) suggest an effect of the MnSOD ala/ala
genotype on the development of prostate cancer. In fact, men homozygous for
the MnSOD ala allele had a 70% increase in risk of prostate cancer over men
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0.76
0.82
0.88
1.14
homozygous for the val allele. Furthermore men homozygous for MnSOD ala
(compared to MnSOD val/val or val/ala) showed a three-fold risk increase for
high-grade tumors. It is interesting to mention that among men with the AA
genotype, high selenium level (4th versus 1st quartile) was associated with a
relative risk (RR) of 0.3 for total prostate cancer; for clinically aggressive prostate
cancer, the RR was 0.2. In contrast, among men with the VV/VA genotype, the
RRs were 0.6 and 0.7 for total and clinically aggressive prostate cancer (Li et al.,
2005). The inverse association between baseline plasma selenium levels and risk
of advanced prostate cancer, even among men diagnosed during the post-PSA
era, suggests that higher levels of selenium may slow prostate cancer tumor
progression (Li et al., 2004).
However, there is a number of studies which failed to show an association between
Se in the diet and cancer incidence (Coates et al. 1988; Knekt et al. 1988; Menkes et
al. 1986; Nomura et al. 1987; Ringstad et al. 1988). In general, of the 36 observational
studies that were reviewed, approximately half supported an association with total
cancers and half did not (Trumbo, 2005). It was noted, that the greatest consistency
was observed for breast and prostate cancer.
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(Salonen et al., 1984; 1985; Wilett et al., 1983; Kim et al., 2003) and toenails
(Sakoda et al., 2005) were significantly lower in cancer patients than in control
subjects. However, there are quite a few cases when Se concentrations in body
fluids of cancer patients are not different from those in control groups. It is necessary
to mention that the reported reduction of Se levels in body fluids of cancer patients
in comparison with control subjects ranged between 5% and 35% (Alaejos et al.,
2000).
Table 11.16 Selenium concentrations in cancer patients (Adapted from Whanger, 2004; Combs and Gray,
1998)
Effects
References
Selenium was lowered in patients with carcinoma of the uterine cervix Bhuvarahamurthy et
al., 1996
Se concentration in whole blood and plasma in prostate cancer
patients was lower in comparison to healthy controls
Li et al., 1991
For the half of the New Zealand population with Se level below
Karunasinghe et al.,
97.8 ng/ml, lower serum selenium levels showed a statistically
2004
significant inverse relationship with overall accumulated DNA damage.
Plasma concentrations of selenium was significantly lower in colorectal Musil et al., 2005
cancer patients than in healthy controls over the monitored period
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Effects
References
Navarro-Alarcon et
al., 1998
Lopez-Saez et al.,
2003
McConnell et al.,
1980
Among subjects aged < or = 60 yr, mean serum selenium levels were
significantly lower in both patient groups (adenoma, 57.9 g /l;
cancer, 43.7 g /l) than in healthy controls (88.9 g /l) (p = 0.0001).
Subjects with higher selenium status had a lower probability to be in
the adenoma group than subjects with lower selenium status.
Fernandez-Banares et
al., 2002
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Effects
References
Serum selenium levels were significantly lower in lung cancer patients Miyamoto et al., 1987
than in the controls. Among families of adenocarcinoma patients, the
mean level was significantly lower (111 g /l) than that (122 g /l )
in age-ratio matched controls who did not have cancer patients among
their second-degree relatives.
In the nested case-control study of a stratified sample from the Belgian Kornitzer et al., 2004
population, serum selenium was an independent predictor of cancer
mortality in male subjects only
In Poland, Se concentrations in gastrointestinal cancer patients
(37.0 g /l or 38.4 g /l in stomach or colon cancer patients,
respectively) were significantly lower as compared to the healthy agematched control group (51.4 g /l)
In India, patients with head and neck cancer had serum selenium levels Yadav et al., 2002
significantly lower as compared with controls, and these levels
decreased further as tumour burden increased
In India, plasma selenium level of 75.4 ng/ml in cancer patients was
significantly less than control values (117 ng/ml) in normal healthy
individuals. The strongest association of plasma selenium level and
cancer was found in breast cancer (70.5 ng/ml) and gastrointestinal
tract cancer (73.1 ng/ml). Selenium level decreased with the progress
of disease and recurrence of disease.
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Effects
References
Mean serum Se levels were 101, 105 and 77 ng/ml in the controls,
precancerous and in patients with malignant oral cavity lesions,
respectively.
Table 11.17 Mean pre-diagnostic serum selenium concentrations in cancer cases and matched controls
from prospective cohort studies (Adapted from Waters et al., 2004)
Cases
Serum Se concentration,
g/l
P-value
Cohort
Case
Control
49.3
49.9
59.5
59.1
63.6
63.5
58.4
60.5
62.5
63.9
<0.05
<0.05
>0.05
<0.001
>0.05
Japan
35 males
38 females
105.2
97.4
112.8
102.7
0.18
0.25
Netherlands
40 males
29 female
116.7
130.6
126.4
129.3
0.04
0.83
Norway
26 male
34 female
124.8
123.2
130.3
127.9
0.08
0.36
USA
60 male
51 female
127.0
132.0
137.0
134.0
0.008
0.57
Finland
16 male smokers
14 male non-smokers
21 female non-smokers
597 males
499 females
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the cancer (Robinson et al., 1979; Lopez-Saez et al., 2003). The confirmation of
this idea came from the study showing that decreased Se levels came within normal
range after 1 year of radiotherapy in 10 patients with head and neck cancer who
were cured but in the remaining patients who had residual disease, levels remained
persistently low (Yadav et al., 2002). Similarly, low serum Se levels in patients
with acute leukemia are mostly dependent on tumor activity (Beguin et al., 1989).
On the other hand, data reported by Ujiie et al (1998) suggest that the low serum
Se level in cancerous patients may not be induced by the tumor but it was more
likely already present before the tumor. Results of Fernandez-Banares et al. (2002)
suggest that high selenium status may decrease the risk of large size adenomas in
a low selenium region, and that this preventive effect seems to be exclusive to
subjects < or = 60 yr
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Effect of selenium
References
Ip et al., 1992
El-Bayoumy et al.,
1992
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Effect of selenium
References
When fed at supplemental levels (1ppm) in the diet, Se in dairy protein McIntosh et al., 2004
was very effective at reducing tumours of the colon in azoxymethaneinduced rats
Significant inhibition in the incidence of DMBA-induced papilloma
formation (53-80%) and the cumulative numbers of papillomas per
papilloma bearing mouse were observed in the diphenylmethyl
selenocyanate-treated (2-3 mg/kg b.w.) groups as compared to the
carcinogen control group
Topical L-SeMet application was shown to protect against UV-induced Burke et al., 2003
skin cancer in a mice model
Pregnant CBA mice were administered selenium during the last week Popova, 2002
of pregnancy and for ten days following parturition. Selenium
significantly reduced the incidence of spontaneous hepatomas in adult
male progeny
Sodium selenite, selenocysteine and SeMet have been compared at
Mukhopadhyay-Sardar
4, 6, 8 and 10 ppm, in terms of their bioavailability and prolongation et al., 2000
of survival of Daltons lymphoma (DL) bearing mice. SeMet, at a dose of
8 ppm, was found to be the most bioavailable and least-cytotoxic form
that was capable of increasing the life span of the tumour bearing hosts
maximally (almost two-fold)
Rats receiving esophagoduodenal anastomosis surgery plus iron
Chen et al., 2000
supplementation were supplemented with sodium selenate at 1.7 mg/kg
Histopathological analysis showed that Se supplementation increased
the incidence of esophageal adenocarcinoma and the tumor volume
Supranutritional amounts of Se (1-2 ppm) supplied as high Se broccoli Finley et al., 2000;
to carcinogen-injected rats significantly decreased the incidence of
2001
aberrant crypts and aberrant crypt foci (preneoplastic lesions indicative
of colon cancer) compared with control animals
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Effect of selenium
References
Five-week-old heterozygotic male multiple intestinal neoplasia mice Davis et al.., 2002
were fed a basal diet containing either low-selenium broccoli or highselenium broccoli for 10 wk. Mice fed the selenium-enriched broccoli
had fewer small intestinal and large intestinal tumors than those fed an
equivalent amount of unenriched broccoli
In N-methylnitronitrosoguanidine-induced colorectal carcinogenesis Mukherjee et al., 2001
model treatment with SeMet either on initiation or on selection/
promotion, or during the entire experiment showed that SeMet was most
effective in regulating the cellular antioxidant defence systems, DNA
chain break control and reducing aberrant crypt foci in the colorectal
tissues of rats
Sodium selenite (2.5 ppm) given in drinking water to 8 months old
OF1 mice, for 4 consecutive months, reduced significantly the
mortality of mice with 6% and 50% mortality rate for Se and control
groups, respectively. In addition 80% of control deaths resulted from
a lymphoid cell neoplasma, while no one of Se supplemented mice
produced tumor
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Table 11.19 Protective effects of selenium on carcinogen-induced covalent DNA adduct formation in
rodents (Adapted from El-Bayoumy, 2001).
Form of Se
Carcinogen
References
Selenite
DMBA
F, SD rat, mammae
Selenite, selenateDMAB
Selenite
2-AAF
Selenite
AFB1
p-XSC
DMBA
p-XSC
o-,m-, p-XSC
RSeCN
BSC
F, A/J mouse
F, SD rat, mammae
F, SD rat, mammae
M, F344 rat, colon
NNK
DMBA
DMBA
AOM
1,4-phenylenebis(methylene)selenocyanate (p-XSC)
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
azoxymethane (AOM)
3,2'-dimethyl-4-aminobiphenyl (DMAB)
7,12-dimethylbenz(a)anthracene (DMBA)
2-acetylaminofluorene 2AAF
benzylselenocyanate- BSC
Cancer is a disease that develops slowly. Indeed, for most solid human tumors,
there is a 20-year interval from carcinogen exposure to clinical detection. During
this time, cancer cells acquire the capacity to divide, invade, and metastasise (Loeb
et al., 2003). Since, it is difficult to predict timing of carcinogen action and its
nature in real life, it is recommended Se to be supplemented continuously over
the entire lifespan. Indeed, decreased carcinogen-induced damage to DNA supports
a role for selenium inhibition of cancer at the stage of initiation, and increased
latency to tumor development suggests inhibition of tumor promotion (Birt et al.,
1986; 1988). The results of Bjrkhem-Bergman et al. (2005) suggest that selenium
is able to reduce the risk for liver cancer even when it is used only during a short
period of time covering the promotion phase of the carcinogenic process. Therefore
the carcinogenetic process may be prevented by selenium supplementation both
during the promotion and the progression phase. Similarly, most significant
beneficial effect of selenium during hepatocarcinogenesis was exerted potentially
in long-term continuous and/or before the initiation phase of carcinogenicity, rather
than in the promotion phase (Thirunavukkarasu and Sakthisekaran, 2003).
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Rat, mouse, and hamster models have been used to study liver, breast, colon,
skin, and pancreatic cancers. In general Combs and Combs (1986) estimated that
Se was used in more than 100 animal carcinogen studies and two-third showed
reduction in the incidence of cancer, with half showing a substantial (50% or
more) reduction. This was substantiated in later reviews (Combs and Gray, 1998;
El-Bayoumy, 1991; Whanger, 2004). It is necessary to mention that more than
90% of the selenium cancer chemoprevention experiments have used either sodium
selenite or selenomethionine as the test reagent because they are commercially
available (Ip, 1998). In animal models using chemically-induced cancerogenesis
selenite was more effective than SeMet irrespective of Se concentration in tissues.
Therefore it was suggested that the anticancer effect was due to various metabolic
forms of Se including methylated forms of selenium (Ip, 1998). In particular, it
was considered that anticancer effect of Se is independent on H 2 Se and
selenoprotein formation. Therefore, the effect of high-level Se supplementation
in cancer prevention studies is most likely due to direct chemical properties of Se
and its metabolites rather than being mediated through selenoproteins. The
methylated selenium products, particularly the monomethylated species, are
proposed to have the greatest anticarcinogenic potential (Szarka et al., 1994). In
particular, such Se analogues as selenobetaine and Se-methylselenocysteine, have
been successfully tested for anticarcinogenic potential (Reddy et al., 1994). In
particular, the authors showed that the partially methylated species have greater
efficacy as antitumorigenic agents in animal models than the fully methylated
species. Furthermore, doses of sodium selenite used in cancer prevention studies
using various animal models were quite high and in many cases almost toxic.
Therefore, to avoid Se toxicity various organo-selenocompounds were synthesized
and tested. For example, 1,4-phenylene-bis(methylene)selenocyante or xylene
selenocyanate (p-XSC), have been found to be very effective inhibitors of
mammary and colon carcinogenesis (Ip et al., 1994). At the same time this
compound was characterised by dramatic (almost 30-fold) decrease in toxicity
(el Bayoumy et al., 1992). Different forms of selenium supplementation, either
as inorganic Se (sodium selenite) or as organic forms such as selenomethionine,
methylseleninic acid, and other unspecified seleno-compounds in yeast and
broccoli, may have differing efficacies in cancer prevention (Chu et al., 2004).
Up to date there have been hundreds of chemicals that have been tested for their
anticancer activities in both in vivo and in vitro models. However, it is difficult to
translate all those data into specific recommendations for cancer prevention.
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Country Population
China
Hepatitis surface
antigen carriers
China
Hepatitis surface
antigen carriers
China
Double-blind,
Stomach
placebo controlled, cancer
general population
China
Double-blind,
Esophageal
placebo controlled, cancer
displasia
Li et al., 1993
Reverse smokers
Prasad et al.,
1995
200 g Se/day
Italy
Bonelli et al.,
1998
US
Clark et al.,
1996; 1998;
Duffield-Lilico
et al., 2003
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Oral lesions
Basal cell or
squamous cell
carcinoma
lung, colon,
prostate
The first trial in China. In the Qidong county of China, the incidence of
hepatocellular carcinoma (HCC) is particularly high. It is interesting to note
that in this region, about 15% of adults carry the hepatitis B surface antigen
and these individuals are 200 tines more likely to develop HCC (Rayman,
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The second trial in China. In the study of Yu et al. (1997) 2474 members
of families with high risk of primary liver cancer (PLC) were consuming
200 g of selenium per day in the form of Se-yeast or a placebo. During the
2-year period of study, 1.26% of the controls developed PLC vs 0.69% in
the placebo group. Furthermore, the authors studied 226 hepatitis B surface
antigen-positive subjects randomly assigned to either a placebo or Seenriched bakers yeast (200 g Se/d) for a 4-year period. They showed a
lower PLC incidence in the Se group compared with the placebo group (0
vs 7 cases, P<0.05).
The third trial in China. There were 3698 subjects randomly assigned to
an array of mixed treatments. The treatment consisting of Se-enriched bakers
yeast (providing 50 g Se/d) in combination with vitamin E and -carotene
was associated with modest reduction in stomach cancer mortality (by 21%)
and total mortality (by 9%; Taylor et al., 1994; Blot et al., 1995). Results
from the same study, published recently (Wei et al., 2004) showed a
significant inverse association between baseline serum Se and death from
oesophageal squamous cell carcinoma and gastric cancer.
The fourth trial in China. It was conducted from 1984 to 1991 in Linxian,
a rural county in Henan Province, characterised by exceptionally high
mortalities from esophageal cancer, a total of 29,584 adults were used to
assess the effect of vitamins and minerals on cancer (Blot et al., 1993).
During study 2127 subjects died from cancer. It was found that a
combination of Se (50 g Se/d as Se-yeast) with -carotene and vitamin A
significantly lowered total mortality and cancer mortality. There was a 13%
reduction in total and cancer mortality in the group treated with Se-enriched
yeast plus vitamin E and -carotene. The protective effects of this antioxidant
mixture was evident against cancers of the stomach, esophagus, and lung.
In particular, rate of lung cancer was substantially decreased (Blot, 1997).
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696
The fifth trial in China. Li et al. (1993) used randomly assigned 3318 subjects
with confirmed esophageal dysplasia who received a similar array of mixed
treatments of vitamins and minerals, including 50 g Se as selenate per day. In
general vitamin and mineral levels used were 2-3 times the recommended dietary
allowance levels. No significant treatment effects were observed on any cancerrelated end point. However, it is necessary to mention that justification of various
mixtures and doses of individual compounds in those mixtures are always a
problem. Therefore, it is difficult to answer the question if the trial was not
successful because of lack of effects of individual compounds, including
selenium, or because of possible synergistic-antagonistic interactions of those
compounds. Indeed, it seems likely that Se is more effective in cancer prevention
rather in reversing tumours as well as Se dose was not high enough to give the
best protection (Whanger, 2004).
The sixth trial in China. A double-blind intervention study was performed on
the participants aged 35 -74 years, who had matched at least one of the following
criteria: a medical history of stomach disorder, a family history of tumour, or
smoking and/or alcohol consumption. A total of 2,526 and 2,507 persons were
randomly enrolled into intervention group and control group respectively from
288 natural villages of seven communities in Qixia County, Shandong Province,
China. Each person of the intervention group orally took 200 mg synthetic
allitridum, an active principle of garlic, every day and 100 g selenium every
other day for one month of each year during November 1989 to December
1991 (Li et al., 2004). At the same time, people in control group were given 2
placebo capsules containing corn oil with the identical appearance to that in
the intervention group. In the first follow-up five years (1992 - 1997) after
stopping the intervention, after adjusting for age, gender, and other potential
confounders, relative risks (RRs) for all tumours and gastric cancer of the whole
population were 0.67 and 0.48, respectively, and for male group they were
0.51 and 0.36, respectively. No significant protective effect was found for the
female subgroup (Li et al., 2004).
The seventh trial in India. The trial was conducted with 298 rolled tobacco
smokers with pre-cancerous lesions in the oral cavity randomly assigned to a
placebo or a twice weekly supplement containing vitamin A, riboflavin, zinc,
and Se (Se-enriched yeast providing 100 g/d for 6 months, followed by 50
g/d for 6 months). After 1 year of treatment, subjects with precancerous oral
lesions in the treatment group had a significantly greater rate of complete
remission of lesions compared with those in the control group (57 vs 8%).
Subjects without lesions in the treated group also had significantly fewer new
lesions than those in the placebo group (12 vs 38%; Krishnaswamy et al.,
1995; Prasad et al., 1995).
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The eighth trial in Italy. A group of 304 patients with previous resected
adenomatous polyps who received a mixed treatment containing Se (200
g Se/d as L-SeMet) plus zinc, vitamin A, and riboflavin for 5 years had a
lower incidence of recurrent polyps than those receiving a placebo (5.6 vs
11%; Bonelli et al., 1998). However, this report was not published in peerreviewed journals.
The ninth trial in France. The SUpplementation en VItamines et
MinerauxAntioXydants (SU.VI.MAX) study was a randomized doubleblind, placebo-controlled, primary prevention trial designed to test the
efficacy of daily supplementation with antioxidant vitamins (vitamin C, 120
mg; vitamin E, 30 mg; and beta-carotene, 6 mg) and minerals (selenium,
100 g; and zinc, 20 mg), at nutritional doses (one to three times the daily
recommended dietary allowances), in reducing the frequency of major health
problems in industrialized countries, and especially the main causes of
premature death (cancers and cardiovascular diseases; Hercberg et al., 1998).
The study involves 12,735 eligible subjects (women aged 35 to 60 years;
men aged 45 to 60 years) included in 1994 in France. Sex-stratified analysis
showed a protective effect of antioxidants in men (relative risk, 0.69, but
not in women (relative risk, 1.04). A similar trend was observed for allcause mortality (relative risk, 0.63) in men vs 1.03 in women. Therefore,
after 7.5 years, low-dose antioxidant supplementation lowered all-cause
mortality in men but not in women (Hercberg et al., 2004). In the
SU.VI.MAX trial 5,141 men took either a placebo or a supplement daily for
8 years. During the follow-up, 103 cases of prostate cancer were diagnosed.
Overall, there was a moderate nonsignificant reduction in prostate cancer
rate associated with the supplementation (hazard ratio = 0.88). However,
the effect differed significantly between men with normal baseline PSA (<
3 g/l) and those with elevated PSA (p = 0.009). Among men with normal
PSA, there was a marked statistically significant reduction in the rate of
prostate cancer for men receiving the supplements (hazard ratio = 0.52).
However, in men with elevated PSA at baseline, the supplementation was
associated with an increased incidence of prostate cancer of borderline
statistical significance (hazard ratio = 1.54). The supplementation had no
effect on PSA or IGF levels (Meyer et al., 2005).
The tenth trial in the USA. The Nutrition Prevention of Cancer Trial (NPC
Trial) was designed in 1983 to test the hypothesis that an increase in Se
status would reduce the incidence of non-melanoma skin cancers (primary
endpoints) in a high-risk population. This was the first double-blind placebocontrolled intervention trial in the western population of this kind (Rayman,
2002). The participants with a history of non-melanoma skin cancer (1312
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Table 11.21 Cancer incidence (numbers of cancers) in selenium- and placebo-treated subjects (Adapted
from Clark et al., 1996)
Site
Skin carcinomas
Basal cell
Squamous cell
Lung
Prostate
Colorectal
Total carcinomas other than skin
Total non-carcinomas
Total cancer
Selenium
Placebo
Relative risk
P value
377
218
17
13
8
59
19
77
350
190
31
35
19
104
16
119
1.10
1.14
0.54
0.37
0.36
0.55
1.17
0.63
0.2
0.15
0.08
0.002
0.025
<0.001
0.65
0.001
Placebo
25
20
15
10
5
0
Lung
Prostate
Colorectal
Figure 11.5 Cancer incidence (numbers of cancers) in selenium- and placebo-treated subjects (Adapted
from Clark et al., 1996)
Furthermore, participants in the middle tertile of plasma Se, 107123 g/l, were
characterised by a more modest, but still protective effect of Se-treatment (RR =
0.39, P = 0.03). In contrast to participants with low Se level, those subjects in the
top tertile at entry to the trial, whose plasma Se concentration was >121 g/l had
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there are new human trials under way to further substantiate protective effects of
Se against cancer:
SELECT trial
Pre-Randimization period
Calendar Year
2001-2006
Randomized
Vit.E+
Se
20012013
Vit.E+
Placebo
Selenium +
Placebo
Placebo+
Placebo
Follow-up
Prostate cancer, other cancers, death
Figure 11.6 SELECT trial design (Adapted from Klein et al., 2003)
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Table 11.22 Expected incidence of prostate cancer in the SELECT trial (Adapted from Klein et al., 2003)
Group
Number
at risk
Proportion with
prostate cancer
Number with
prostate cancer
8100
8100
8100
8100
0.066
0.05
0.05
0.038
533
403
403
304
Placebo
Selenium
Vitamin E
Se + Vitamin E
Further prostate cancer studies at the Arizona Cancer Center and in Europe
and Australia using Se doses of 200-800 g/d are in progress (Rayman,
2004):
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Negative Biopsy trial: The hypothesis is that Se may prevent the progression
of cancer in 25% of men in this population who have early cancer missed
biopsy (Patrick, 2004). In the trial 700 men with persistently-elevated
prostate-specific antigen (PSA), but negative biopsies are randomly allocated
to placebo or 200 or 400 g Se/day with follow up period of 57 months
(Stratton et al., 2003a). Primary study endpoints include development of
prostate cancer and PSA velocity. Secondary biochemical endpoints include
change in chromagranin A and alkaline phosphatase. The trial will evaluate
the ability of selenium to halt or slow the preclinical progression of prostate
cancer and to decrease the incidence of clinical disease. The trial will be
directly generalizable to those patients at high risk of prostate cancer, but
who have not yet been diagnosed (Clark and Marshall, 2001). Randomization
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scheme was effective for selected parameters including age, body mass
index, smoking status, baseline PSA and baseline plasma selenium level.
Various data, including medical history, family history, and urological
symptoms and specimens (including blood and subsequent prostate biopsy
samples) had been collected at baseline, and throughout both the intervention
and follow-up stages of the protocol (Stratton et al., 2003a).
High-grade Prostatic Intraepithelial Neoplasia Trial: The trial will evaluate
the ability of Se to prevent the transformation of neoplasia to invasive cancer
(Patrick, 2004). For this purpose 470 men with high-grade prostatic
intraepithelial neoplasia, who are at risk for subsequent prostate cancer and
have had 2 or more biopsies that indicate no invasive prostate cancer, but
have not been treated with surgery or irradiation will be assigned to placebo
or to 200 g Se as selenomethionine. They will be followed for at least 3
years. The primary endpoint in the trial is the diagnosis of biopsy-proven
prostate cancer. Secondary endpoints include apoptosis and proliferation.
This study will assess the anticancer activity of Se immediately before
neoplastic growth becomes transformed into invasive growth.
Preprostatectomy trial: This trial will evaluate biomarkers of inflammation
and tissue selenium level. The study will examine prostate tissue from initial
biopsies and from prostate surgery. This will enable to examine tissue before
and after the administration of selenium. Eligible patients will be randomly
placed into one of the three groups. One hundred and ten men with localised
prostate cancer will be supplemented with 200 or 400 g Se/day between
biopsy and prostatectomy (Marshall, 2001). The third group will be given a
placebo pill that looks and smells exactly like a selenium pill, but contains
no selenium. This placebo group will serve as the control group so that the
effect of selenium in the other groups can be clearly determined. Participants
in this study will have a 66% chance of taking selenium.
Watchfull Waiting trial, a phase II, multi-center, randomized, double-blind,
placebo-controlled clinical intervention study testing the effects of two doses
of selenized yeast on progression of prostate cancer. Participants are men with
biopsy-proven prostate cancer who have elected to forgo therapy and be closely
followed by watchful waiting that includes quarterly prostate-specific antigen
(PSA) screening. The trial will evaluate 264 men with localised prostate cancer
and life expectancy <10 years and they will receive 200 or 800 g Se/day as
Se-yeast or matched placebo daily. Endpoints include time to disease progression
and PSA velocity. Secondary endpoints include time to initiation of therapy as
well as biochemical markers of disease progression including chromagranin A
and alkaline phosphatase. Immunohistochemical analyses for indicators of
apoptosis, proliferation and differentiation will be performed on baseline and
subsequent prostate biopsy specimens. (Stratton et al., 2003b).
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Table 11.23 Effect of Se (50 g/day as Se-yeast) on side effects in patients with ovarian cancer
undergoing chemotherapy (Adapted from Sieja and Talerczyk, 2004)
Vomiting
Stomatitis
Hair loss
Flatulence
Abdominal pain
Weakness
Malaise
Loss of appetite
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
Experimental Se-supplemented
group, n=31
Control group,
n=31
2.19
1.19
0.97
1.74
1.07
0.97
0.35
0.10
0.32
1.83
1.87
2.12
2.10
1.16
0.61
0.93
0.39
0.45
2.13
1.26
0.97
1.93
0.84
0.87
2.39
1.00
0.84
1.81
1.93
2.03
1.68
1.87
2.16
0.45
0.45
0.58
1.55
2.22
2.55
1.58
1.71
1.97
1.32
1.22
1.45
2.19
2.26
2.35
2.19
2.45
2.54
2.13
2.35
2.26
Scale of evaluation: 0- without signs, 1-mild signs, 2-moderate signs, 3-severe signs;
I-study after 1 month; II-study after 2 months, III- study after 3 months.
Furthermore, in vitro studies showed that the use of yeast Se in combination with
chemotherapeutic drugs can reduce the effective dosage of either Taxol and/or
Adriamycin in several different cancer cells, including ovarian cancer cells in
culture (Vadgama et al., 2000). Supplementation with Se improved the apoptotic
response. A combine selenium (200 g/day) and zinc (21 mg/day) treatment was
used with 30 patients during the course of chemotherapy treatment of colorectal
carcinoma (Federico et al., 2001). At the end of the chemotherapy course, 70% of
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The use of selenium, in the form of 5-methylselenocysteine (MSC), or selenoL-methionine (SeMet) as selective modulators of anticancer drugs is considered
as a novel way of improvement of cancer therapy. Indeed, results from Roswell
Park Cancer Institute (USA) have demonstrated that MSC and SeMet are highly
effective modulators of irinotecan cure rates in de novo sensitive and resistant
human tumor xenografts (Fakih et al., 2005). It is interesting to note that the
observed effects were not drug-specific or species-specific and are currently being
confirmed in ongoing clinical trials.
ANTIOXIDANT EFFECTS
Among the many possible causes of cancer ROS have been implicated in the
development of the disease. Indeed oxidative stress may induce gene mutation
and promote carcinogenesis. It has been shown that free radical tissue damage is
increased in proportion to prostate cancer (PCa) severity (Malins et al., 2001;
1997). In fact, the age-related increase in the proportion of hydroxyl radicalinduced DNA damage was a significant factor in the development of PCa.
Aforementioned studies have demonstrated that free radical-induced DNA damage
is greater in cancerous than in non-cancerous prostate tissues and lends support
to the hypothesis that the mechanism by which antioxidants may reduce the risk
of PCa is by their effect on reducing the production of free radicals. Furthermore,
ROS can modulate the apoptotic program, disregulation of which has a role
particularly in gastrointestinal cancer (Bjelakovic et al., 2004).
Experimental studies support this idea in part by showing that antioxidants that
prevent ROS damage can act as cancer preventive agents. However, once cancer
has occurred, radiation therapy and some forms of chemotherapy rely on ROS
formation and toxicity to eradicate tumor cells. It seems likely that at least some of
the 25 mammalian selenoproteins are involved in the mechanism of protection
against cancer by selenium. There is evidence that several of these proteins may
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be involved with the mechanism by which selenium provides its anticancer effects
(Diwadkar-Navsariwala and Diamond, 2004). First of all, GSH-Px is responsible
for removal of H2O2 and lipid hydroperoxides and in this way prevents formation
of hydroxyl radical responsible for DNA damages and carcinogenesis. Both TR
and Trx are critical for redox control at the cellular level; together, they are involved
in several biologic processes including antioxidant defense, cell proliferation, and
inhibition of apoptosis (Mustacich and Powis, 2000). Therefore in some cases
changes in activities of selenoproteins and redox status of the cell may encourage
cancerous cells to undergo apoptosis. Components of the TR-TRx system may
play an important regulatory role in the function of p53 (Pearson and Merrill,
1997; Ueno et al., 1999; Moos et al., 2003), a tumor suppressor gene that is either
deleted or suppressed in several human cancers. It seems likely that methionine
sulfoxide reductase B, responsible for prevention of SH-group oxidation in proteins
could also be involved in prevention of carcinogenesis.
Did you know that molecular mechanisms of anticancer effects of
selenium include: antioxidant effects, stimulation of DNA-repair
enzymes and activation of the tumor suppressor protein p53, effect
on gene expression, enhancement of immune function, induction
of apoptosis, inhibition of cell proliferation, alteration of
carcinogen metabolism, influence on estrogen and androgen
receptor expression, inhibition of angiogenesis and interactions
with heavy metals involving in cancer promotion?
Genetic data indicate functional polymorphisms in the genes for several
selenoproteins and the association of allelic variants with cancer risk. Furthermore,
allelic loss of selenoprotein genes during tumor evolution supports the possibility
that the deletion of these genes promotes cancer development. Indeed, allelic loss
at the GSH-Px-1 locus is an early event in the development of cancer of the head
and neck (Hu et al., 2004). Therefore, reduced levels of one or more selenoproteins
increase the likelihood of cancer development (Diwadkar-Navsariwala and
Diamond, 2004). The authors suggested that individuals with lower selenium
intake, attenuating polymorphisms, or haploinsufficiency may be those who will
benefit most from additional dietary selenium intake. Therefore, an optimal Se
status is an important factor responsible for selenoprotein synthesis and an effective
antioxidant defence. In this way Se can prevent damages to DNA leading to
cancer.
STIMULATION OF DNA-REPAIR ENZYMES AND ACTIVATION OF THE
TUMOR SUPPRESSOR PROTEIN P53
The presence of thousands of mutations in cancer cells suggests that among the
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108 cells in a human tumor at the time of diagnosis there are billions of different
mutations, and that gene mutations are present in one or more cells within a tumor
(Loeb et al., 2003). The hypotheses was proposed and the evidence was provided
showing that selenium supplementation can inhibit carcinogen-induced covalent
DNA adduct formation and can retard oxidative damage as a result of chemical
and physical insult to DNA, lipids, and proteins (El-Bayoumy, 2001). It is known
that carriers of BRCA1 mutations are characterised by significantly greater mean
frequencies of induced chromosome breaks per cell than did healthy non-carrier
relatives. It is known that such mutations are responsible for greatly increased
risks of breast and ovarian cancer. Indeed, the product of the BRCA1 gene is
involved in the repair of double-stranded DNA breaks and therefore increased
susceptibility to DNA breakage contributes to the cancer phenotype. In women
who are born constitutional heterozygous mutations of the BRCA1 gene the
frequency of chromosome breaks was greatly reduced following 1 to 3 months of
oral selenium supplementation (Kowalska et al., 2005). In the New Zealand
population characterised by comparatively low serum selenium levels (97.8 ng/
ml) a statistically significant inverse relationship (P = 0.02) with overall accumulated
DNA damage in blood leukocytes was shown. The authors suggested that the
selenium intake in half of this population is marginal for adequate repair of DNA
damage, increasing susceptibility to cancer and other degenerative diseases
(Karunasinghe et al., 2004). To evaluate the effects of dietary selenium
supplementation on DNA damage in prostate tissue and on apoptosis in prostate
epithelial cells an in vivo canine model was used (Waters et al., 2003). Sexually
intact elderly male beagle dogs were randomly assigned to receive an
unsupplemented diet (control group) or diets that were supplemented with selenium
(treatment group), either as SeMet or as high-selenium yeast at 3 g/kg or 6 g/kg
body weight per day for 7 months. The extent of DNA damage in prostate cells
and in peripheral blood lymphocytes was lower among the selenium-supplemented
dogs than among the control dogs but was not associated with the activity of the
antioxidant enzyme GSH-Px in plasma. Similarly, in human formation of DNA
adducts, an indicator of carcinogenicity, was significantly lower in the Sesupplemented subjects than in those receiving placebo (Liu et al., 1991).
It is important to mention that different forms and levels of selenium compounds
are critical factors with regard to cellular responses. SeMet-mediated redox
regulation of p53 and activation of the DNA-repair branch of the p53 pathway
has been reported (Seo et al., 2002). Similarly sodium selenite and methyl-seleninic
acid (MSA) also can affect p53 activity defined as trans-activation of a p53dependent reporter gene. It seems likely that specific mechanisms of p53 activation
differ among selenium chemical forms that may differentially modify p53 for
DNA repair or apoptosis in conjunction with a given level of endogenous or
exogenous DNA damage. In particular, it was shown that SeMet did not affect
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assigned to a 6-month trial with either 100 micrograms Se/d or a placebo (Peretz
et al., 1991). During selenium supplementation the proliferative response to
pokeweed mitogen increased significantly (+79% of baseline concentrations after
4 months, P<0.01) and reached the upper limit of the usual range for adults after
6 mo (+138%, P <0.001). Furthermore, Se positively affected the immune status
of advanced cancer patients. For example, the ability of selenium dioxide (SeO2)
to enhance the lymphocyte progression through the cell cycle in patients with
advanced (stage IV) cancer was studied (Mantovani et al., 2004). The addition
into culture of 1.5 microM SeO2 enhanced significantly the progression into S
phase of lymphocytes (an essential prerequisite for the physiological functioning
of the immune system) isolated from cancer patients, whilst no significant effect
was observed on lymphocytes isolated from controls.
It is important to mention that Se has a protective effect against age-related
immunosuppression and Se supplementation provides significant improvement
in elderly patients by increasing the humoral response after vaccination and could
have considerable public health importance by reducing morbidity from respiratory
tract infections (Girodon et al., 1999). Free radicals cause a cascade of intracellular
events resulting in liberation in cytoplasm of nuclear transcription factor kappa B
(NF B) from its inhibitory protein IB (Flohe et al., 1997) which permits its
translocation into the nucleus, where it binds to DNA, enabling the initiation of
the transcription process. NF B controls the production of the acute phase
mediators such as TNF-, lIL-2, and IL-2 receptors, which in turn activate NFB,
amplifying the inflammatory cascade (Berger, 2005). Selenium has been shown
to down regulate NFB and thereby to limit the extension of the inflammatory
response (Flohe et al., 1997; Kim and Stadtman, 1997). Indeed effect of Se on
human immunity is difficult to overestimate.
INDUCTION OF APOPTOSIS
Specific induction of apoptosis in pre-neoplastic/neoplastic lesions thereby
preventing the development of manifest tumors is considered as an important
mechanism of anticancer Se activity (Combs and Gray, 1998; Whanger, 2004).
The ability of cells to program their own death provides a mechanism to control
mutations. In fact, inhibition of cell growth can be accomplished by either a
decrease in cell proliferation or an increase in apoptosis or both. Apoptosis is
therefore an important cellular mechanism for growth regulation. Se was shown
to inhibit growth and stimulate programmed cell death in a variety of cell culture
systems. For example, it has been shown that selenium compounds can cause
apoptosis in cultured cells (Krishnaswamy et al., 1995). Both caspase-dependent
and -independent selenite-mediated apoptosis have been reported (Jnsson-
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Videster et al., 2004; Jiang et al., 2001) and it is associated with the inhibition of
several important intracellular signalling pathways (Gopee et al., 2004). In
particular, it was shown that p38 mitogen-activated protein kinases is a key
upstream mediator for the methylselenol-specific induction of vascular endothelial
caspase-dependent apoptosis, which is principally executed by caspase-3-like
activities (Jiang et al., 2004). Selenium can cause irreversible apoptosis with DNA
strand breaks (Spallholz, 1994; Davis et al., 1998; Spallholz et al., 2001) as well
as cell arrest and/or apoptosis independent of DNA strand breaks (Wilson et al.,
1992; Lu et al., 1995; Finley, 2005).
The effects of inorganic Se compounds on induction of apoptosis by which
Se compounds exert cancer chemopreventive activity have been studies recently
(Takahashi et al., 2005). With the use of HSC-3 human oral squamous cell
carcinoma cells, it has been shown that treatment with Se for 72 h, in the form of
SeO 2 and Na 2SeO 3, but not Na 2SeO 4, markedly induced apoptosis in a dosedependent manner. In particular, the authors suggested that modulation of the
mitochondrial redox equilibrium by Se contributes to the mitochondrial pathway,
regulating caspase-9-mediated apoptosis without a concurrent increase in ROS.
On the other hand, Se-methylselenocysteine (MSC), one of the most effective
selenium compounds at chemoprevention, induced apoptosis in HL-60 and ROS
played a crucial role in MSC-induced apoptosis (Jung et al., 2001). Recent data
of Jiang et al. (2004) support p38 MAPK as a key upstream mediator for the
methylselenol-specific induction of vascular endothelial caspase-dependent
apoptosis, which is principally executed by caspase-3-like activities. The signal
transduction pathways affected by Se compounds in biopsies of normal human
oral mucosa cells and human oral squamous carcinoma cells (SCCs) were
investigated (Ghose et al., 2001). Two Se compounds were tested. Firstly,
selenodiglutathione (SDG), the primary metabolite of selenite and the most
commonly used cancer-protective Se compound in animal models. Secondly, the
synthetic Se compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC), one
of the most potent chemopreventive pharmacological Se compounds. The authors
showed that:
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mice with PC3 tumors in the prostates were fed baseline selenium replete diet
(0.07 ppm), supplemented with different forms of selenium (sodium selenate,
selenomethionine, methylselenocysteine and selenized yeast) at 2 different
concentrations (0.3 and 3 ppm) in drinking water. It was shown that sodium
selenate significantly retarded the growth of primary prostatic tumors and the
development of retroperitoneal lymph node metastases, which was associated
with a decrease in angiogenesis (Corcoran et al., 2004). Furthermore, selenite
inhibited invasion of HT1080 human fibrosarcoma cells and adhesion of HT1080
cells to the collagen matrix (Yoon et al., 2001).
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Se action
References
Cox, 1985
Spallholz, 1994
Vogl et al., 1987
Yu et al., 1988
Pung et al., 1987
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Table 11.25 Antitumorigenic effect of different selenium compounds (Adapted from Whanger, 2002)
Compound
Se-methylselenocysteine
Selenobetaine
Selenobetaine methyl ester
Selenite
SeMet
Selenocysteine
2
2
2-3
3
4-5
4-5
Table 11.26 Growth inhibition of human tumor cell lines and normal diploid fibroblasts by
selenomethionine (adapted from Redman et al., 1998)*
Cell line
Concentration (IC50)
45 mM
50 mM
130 mM
40 mM
65 mM
1 mM
New clinical trials including SELECT and PRECISE are designed to further explore
the possibilities of cancer-prevention properties of selenium. However, it is necessary
to mention that a choice of selenium compounds for clinical trials is of great
importance. Indeed, from the point of view of general nutrition, Se-yeast providing a
natural mixture of selenocompounds in organic form would be the most beneficial.
Indeed, a profile of Se compounds of the yeast is similar to other natural Se sources,
such as grains and forages. Therefore, a choice of SeMet for the SELECT trial seems
to be risky. The problem is that SeMet is the main compound of the Se-yeast, but not
the only one. When results are obtained with Se-yeast it is not possible to be 100%
sure that they are just due to SeMet. As mentioned above, Se-yeast was used in
previous clinical trials and will be used in some other trials. Indeed, ideally increasing
Se consumption to have Se concentration in the blood >121 g/l would be a way to
address Se deficiency issues and simultaneously provide natural protection against
cancer. Therefore, there are two approaches to this:
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hypertension
hyperlipidaemia
smoking
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fact, the atherogenic index was significantly higher in the low selenium
intake group than in the middle selenium intake group and in the high
selenium intake group.
Table 11.27 Epidemiological follow-up studies of selenium status and CHD (Adapted from Alissa et al.,
2003)
Study
Region
No. of
cases
Age
Salonen et al.,
1982
Eastern
Finland
11,000
persons
35-59
<45
48
5-7
Salonen et al.,
1985
Eastern
Finland
30-64
<45
Virtamo et al.,
1985
Eastern
1110 men 55-74
and
Southwestern
Finland
<45
Ringstad and
Thelle, 1986
Norway
10,532
persons
37-87
<105
53- 74
<79
Salvini et al.,
1995
USA
>80
No significant risk
Kardinaal
et al., 1997
EURA1412
MIC study subjects
<70
92
persons
251
subjects
CHD- coronary heart disease; CVD- cardiovascular disease; IHD- ischemic heart disease;
MI- myocardial infract; CRVD- cerebrovascular disease
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Table 11.28 Protective functions of Se in prevention of CVD (Adapted from Alissa et al., 2003)
Reduction LDL levels, possibly by increasing peripheral catabolism, through its effect on
thyroid hormone metabolism
Inhibition of the oxidative modification of LDL in vitro
Increase resistance of LDL to oxidation in vitro and inhibition of foam-cell formation
Modification of cytokine-induced expression of ICAM-1 and VCAM-1 by human
endothelial cells
Se deficiency may induce a shift in prostaglandin production from prostacyclin to
thromboxanes
Serum Se levels are closely related to platelet aggregability, and possibly related to effects
on thromboxanes A2
Se deficiency is associated with increased lipid hydroperoxides that may cause endothelial
injury
Se has the potential to inhibit production of chemotactic products of LDL oxidation
Se deficiency is associated with impaired cell-mediated immune function, including
reduced circulating T-cells and reduced lymphocyte responsiveness
Selenoenzymes inhibit foam-cell formation and hence the release of growth factors that
stimulate vascular smooth muscle cell proliferation
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decreased SOD activity in rats arterial wall (Wu and Huang, 2004). In fact,
there is an inverse relationship between dietary Se and antioxidant capacity
of rat cardiovascular tissues (Wu et al., 2003; Wu and Huang, 2004).
increased injuries to arterial endothelium by cholesterol oxides in comparison
to the Se adequate animals (Huang et al., 2002). It seems likely that the
mechanism of selenium inhibition of cell apoptosis induced by oxysterols
in rat vascular smooth muscle cells was related with the antioxidant activities
of selenoproteins (Tang et al., 2005).
increased lipid peroxide levels in blood. In fact, dietary selenium is a potent
antioxidant against plasma and LDL lipid peroxidation and may thus be
considered antiatherogenic (Hussein et al., 1997).
decreased plasma nitric oxide content and vascular nitric oxide synthase
activity.
increased platelet aggregation, thromboxane B2 production and synthesis
of the lipoxygenase-derived compounds (Vitoux et al., 1996).
increased the collagen and ADP-stimulated platelet aggregation rate and
intensity in the Se-deficient rabbits (Turan et al., 1997). In human platelets,
the activity of GSH-Px is particularly high and is very sensitive to the
requirement of selenium. In these deficient subjects, Se administration
increases platelet GSH-Px activity and inhibits platelet hyperaggregation
and leukotriene synthesis.
increased serum total plasma cholesterol, low-density lipoprotein cholesterol
levels and very low density cholesterol (Stone et al., 1994 and decreased
serum high-density lipoprotein cholesterol level. Raised concentration of
LDL cholesterol is regarded as one of the risk factors associated with
atherosclerosis and ischaemic heart disease, whereas HDL cholesterol is
thought to protect against these diseases. Hypercholesterolaemia associated
with Se deficiency was related to increased 3-hydroxy-3-methylglutarylcoA (HMG-CoA) reductase (EC 1.1.1.34) activity in liver microsomes as
compared with control animals (Nassir et al., 1997). On the other hand,
supplemental intake of Se was associated with lower plasma concentrations
of total cholesterol and low-density lipoprotein cholesterol plus very low
density lipoprotein cholesterol (Poirier et al., 2002). Furthermore, Se
supplementation could increase the level of good cholesterol in HDL.
For example, serum Se levels in 26 healthy subjects were positively correlated
with high-density lipoprotein cholesterol concentrations (Luoma et al.,
1984). The high-density lipoprotein cholesterol/cholesterol ratio increased
during Se supplementation in a group excluding subjects with low
cholesterol. The results suggest a link running from low serum selenium to
reduced high-density lipoprotein cholesterol, and further to high coronary
risk. Therefore, in subjects with low selenium its supplementation may reduce
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From data presented above it is clear that Se could provide protective effect against
cardiovascular diseases. However, the therapeutic benefit of selenium
administration in the prevention and treatment of such diseases still remains
insufficiently documented and convincing proof of such an association can only
be obtained from large, controlled, prospective prevention trials.
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From data presented above it is clear that Se has a specific role in human
reproduction by maintaining high semen quality and noncomlicating pregnancies.
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only about half as likely to have asthma as those who consumed the least Se (2330 g/day). Clinical improvements were observed in non-allergic asthmatics after
Se supplementation (100 g/day) for 14 days (Hasselmark et al., 1993).
Significantly decreased consumption of corticoids by asthmatics was reported
after supplementation with 200 g/day of Se for 96 weeks (Gazdik et al., 2002).
It has been shown that children aged 4-16 who increased their intake of selenium
were 20% less likely to develop asthma (Ellwood et al., 2001). Similarly, for
children exposed to second-hand smoke, the decreased prevalence of asthma
with increased Se was 50% (Rubin et al., 2004). Furthermore, there is some
evidence indicating that Se-rich foods, such as whole-grain cereals and fish may
protect against asthma in children (Ellwood et al., 2001). However, not all trials
related to Se and asthma were successful. For example, a short-term Se
supplementation did not show any significant benefit in patients with intrinsic
asthma (Baker and Ayres, 2000).
It seems likely that optimal Se status is an important factor helping asthmatics.
Indeed, immunomodulating and antioxidant properties of selenoproteins are of
major importance in preventing/treatment of asthma.
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have been postulated (Braganza , 1998; Schulz et al., 1999). Indeed, free radicals
play a role in the etiopathogenesis of acute experimental pancreatitis (Lankisch et
al., 1989; Sanfey et al., 1985) and human pancreatitis of various origins, namely
ischemic, alcoholic and cholelithiasic (Sanfey et al., 1986). There is evidence
that patients with chronic pancreatitis have enhanced levels of free radical
production, cytochrome P450 induction and antioxidant deficiencies, in particular
selenium. The mean serum selenium levels were lower in chronic pancreatitis
patients than in control (Vaona et al., 2005). Indeed, in patients with acute
pancreatitis Se levels in toenails and in RBCs were significantly reduced (Musil et
al., 2005). Low GSH-Px activity and selenium concentration in severe acute
pancreatitis reflect the lower antioxidative ability in this form of disease
(Wereszczynska-Siemiatkowska et al., 2004). However, there are also publications
indicating no difference in Se and other antioxidant levels between healthy control
and pancreatitis patients. For example, there were no significant differences
between the antioxidant profiles of patients with chronic pancreatitis due to alcohol
excess and patients with idiopathic chronic pancreatitis, or between the antioxidant
profiles of patients with recurrent acute pancreatitis and control subjects (MorrisStiff et al, 1999). The pancreatic juice concentration of selenium is similar in
patients with chronic pancreatitis compared with age-matched controls (Scolapio
et al., 2004). The results therefore suggested that the effects of selenium on
pancreatic injury might be systemic rather than local tissue effect.
Did you know that free radicals are involved in the development
of pancreatitis and optimal Se supplementation could help to
control the disease?
The limited published literature in this field suggests that dietary antioxidant
supplementation may ameliorate the pain associated with chronic pancreatitis,
diminish the frequency of acute exacerbations and reduce the requirement for
pancreatic surgery (Bowrey et al., 1999). The selenium therapy caused a significant
increase in selenium, a moderate increase in activity of GSH-Px, a significant
decrease in MDA, whereas SOD remained unchanged (Wollschlager et al., 1997).
In a 20-week double-blind double-dummy crossover trial active treatment was
given as two types of tablets providing daily doses of 600 micrograms organic
selenium, 9000 IU -carotene, 0.54 g vitamin C, 270 IU vitamin E and 2 g
methionine. In the trial of 28 patients enrolled, 20 adhered to the full protocol. Six
patients had an attack whilst on placebo but none whilst on active treatment.
Analysis of visual analogue score sheets to compare background pain in the 10week period before entry and during each phase of the trial, endorsed the beneficial
effect of active treatment (Uden et al., 1990). The same trend emerged from
analysis of pain-score diaries by conventional and time series methods. An
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Selenium has been shown to protect the brain in models of focal cerebral ischemia
(Imai et al., 2001). Increasing evidence demonstrates that accumulation of
oxidation of DNA, proteins, and lipids by free radicals are responsible for the
functional decline in aged brains. A positive correlation between cognitive function
and Se serum levels in elderly was found (Smorgon et al., 2004). It was suggested
that Se may protect cognitive function and the oxidative stress in the brain could
be associated with some cases of depression. Five studies have reported that a
low selenium intake was associated with poorer mood (Benton, 2002). The
possibility that a sub-clinical deficiency of the trace element selenium might exist
in a sample of the British population was examined by giving a selenium
supplement for 5 weeks (Benton and Cook, 1990). Using a double-blind crossover
design 50 subjects received either a placebo or 100 g selenium on a daily basis.
On three occasions they filled in the Profile of Moods state. Mood did not change
when taking the placebo, whereas when taking the selenium the subjects reported
a substantial improvement after both 2.5 and 5 weeks. The lower the initial level
of selenium in the diet the more reports of anxiety, depression, and tiredness,
decreased following 5 weeks of selenium therapy (Benton and Cook, 1991). It is
interesting to note that in the trial subjects with baseline diets lower in selenium
evidenced a greater improvement. More recent results suggest that persons with
low selenium status might experience relatively depressed moods and support
the idea that selenium plays a particular role in the brain (Hawkes and Hornbostel,
1996). Indeed, metabolic feeding trials have shown that individuals fed marginal
selenium diets reported more symptoms of depression and hostility than individuals
fed higher selenium diets (Finley and Penland, 1998; Finley, 2005; Hawkes and
Hornbostel, 1996). In a randomized, double-blind, placebo-controlled selenium
therapy (200 g/day) trial with HIV+drug users the impact of selenium on anxiety,
depression, and mood state was determined (Shor-Posner et al., 2003). At the 12month evaluation, participants who received selenium reported increased vigor
(p = 0.004) and had less anxiety, compared to the placebo-treated individuals.
The risk for state anxiety was almost four times higher, and nearly nine times
greater for trait anxiety in the placebo-treated group.
Did you know that Se deficiency is associated with decreased
mood state and depression?
In a placebo controlled, double blind prospective study, 16 elderly subjects (3
males, 13 females) with a mean age of 63 years and Beck Depression Inventory II
(BDI) scores indicative of mild to moderate depression were randomly assigned
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to the liver tissue was minimal or absent. Furthermore, in the radiation-only group,
a reduction of the blood glutathione level and increases in serum values of
aforementioned enzymes activity were observed, whereas in the irradiation-treated
group, the reverse was found to occur. In an experiment conducted in Egypt,
animals were categorized into eight groups, receiving -tocopherol and/or Se
with or without whole-body gamma-irradiation (6.5 Gy). The results indicated
that antioxidant pre-treatments before irradiation had beneficial effects against
irradiation-induced injury (Noaman et al., 2002).
It seems likely that antioxidant pre-treatments prior to irradiation may also
have some beneficial effects against irradiation-induced intestinal injury. For
example, the effects of selenium and vitamin E (separately or in combination)
pre-treatments prior to whole abdominal irradiation on intestinal injury were
investigated in rats (Mutlu-Turkoglu et al., 2002). It was shown that irradiation
caused increased lipid peroxide and decreased GSH levels in the intestine. Selenium
and/or vitamin E pre-treatments ameliorated these disturbances in prooxidantantioxidant balance. Selenium pretreatment can also have protective effect against
damaging effects of radiation on the developing foetuses. A single dose of sodium
selenite (0.5 mg Se/kg b.w.) was injected intraperitoneally into mice on day 9 of
pregnancy, either 30 min or 2 h before 1.75 Gy whole body irradiation. The
results showed that administration of selenite 2 h (but not 30 min) before irradiation
resulted in a significant decrease in the number of malformed foetuses (Cekan et
al., 1985). The decrease in foetal malformations occurred proportionally for all
the major malformations observed, i.e. short or kinked tail, rib and vertebral
malformations, coloboma and deformation of retina and iris. Furthermore, selenium
pretreatment also protected against radiation-induced retardation of the sternum
of the foetus. In a different experiment the same authors used a dietary Se
supplementation of female mice to test the same hypothesis. The mice were fed a
standard pellet diet (containing 0.2 ppm Se) or the same diet supplemented with
0.8 ppm (low dose) or 3.4 ppm (high dose) of SeMet for 10 weeks. After mating
with males the mice were subjected, on the 9th day of pregnancy, to whole body
roentgen irradiation of 1.75 Gy. On day 18 of gestation the frequency of
resorptions, mortality and the incidence of fetal malformations were studied (Cekan
et al., 1985a). It was found that supplementation with Se-Met resulted in a
significant decrease of the number of malformed fetuses (from 62 per cent in the
irradiated controls to 47 per cent in the Se-supplemented groups). However, there
was no difference between two Se doses. Furthermore, the number of total
malformations as well as fetal resorptions were significantly decreased in a doseindependent manner in the supplemented groups. Similar to the previous
experiment described above, the decrease in fetal malformations occurred
proportionally for all the major malformations observed. GSH-Px activity in whole
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blood of SeMet-fed mice was significantly increased. These data indicated that Se
supplementation at 0.8 ppm was adequate to provide a protection against radiation
and further increase in Se dose was not required. Se-supplementation could also
improve immunity in radiation-exposed animals. For example, supplementation with
200 g/day of sodium selenite during therapy for squamous cell carcinoma of the
head and neck, e.g., surgery, radiation, or surgery and radiation, resulted in a
significantly enhanced cell-mediated immune responsiveness (KiremidjianSchumacher and Roy, 2001).
It seems likely that Se may provide an extended window of protection against
low-dose, low-dose-rate irradiation, including therapeutic potential when administered
before or after irradiation as well as can decrease toxicity of various radioprotective
compounds. The effect of Se pretreatment on the acute toxicity and radioprotective
effect of an effective radioprotector (WR-2721) was studied in male CD2F1 mice.
Injection of 1.6 mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased
the lethality of WR-2721 significantly (Weiss et al., 1987). At the same time, Se
injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival
significantly. Furthermore, a synergistic effect on post-irradiation survival was observed
when Se was injected 24 hr before WR-2721 (200-600 mg/kg IP 1/2 before irradiation).
Indeed, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice
were treated with both Se and 600 mg/kg WR-2721, and was 13% with WR-2721
alone (Weiss et al., 1987). Similarly, glucan, WR-2721, and selenium were evaluated
in C3H/HeN mice for survival-enhancing and hemopoietic-regenerating effects when
administered alone or in combinations before exposure to 60Co radiation. At LD50/
30 radiation doses (radiation doses lethal for 50% of mice within 30 days postexposure),
dose reduction factors varied from 1.02 to 1.66 and were highest when all three
compounds were combined in a single treatment (Patchen et al., 1990).
To better understand molecular mechanisms of radiation-protective effects of Se,
in vitro cell culture experiments were conducted using normal human skin fibroblasts
and squamous cell carcinoma cells. Combined treatment, i.e. radiation exposure +/sodium selenite in single-dose (0 to 7 Gy) and multiple fractionated-dose (5 x 2 Gy)
were used (Rodemann et al., 1999). The results indicate that sodium selenite under
both radiation exposure conditions provided the radioprotective effect. In contrast,
selenite treatment of human tumor cells did not affect their radiation sensitivity. In a
cell culture, protection against radiation-induced mutation was observed for both 30
nM sodium selenite or 4 mM aminothiol (WR-1065; Diamond et al., 1996). In
addition, the protection against mutation induction provided by the combination of
these agents appeared additive. However, in this experiment sodium selenite did not
provide protection against radiation toxicity when provided either alone or in
conjunction with WR-1065. Molecular mechanisms of protective effects of Se against
radiation injuries are not clarified. The authors of the aforementioned study suggested
that selenium and WR-1065 offer protection via independent mechanisms and that
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Table 11.29 Effect of organic selenium on the radiation-exposed rats (Adapted from Knizhnikov et al.,
1996)
Control
7
0
9
2.5
11
4.0
15
21
Average lifespan, days 578
Malignant tumour
8.5
number
Latent period of tumours, days
Breast cancer
562
Lung cancer
584
R+1.5Se*
R+5Se*
14
21
35
80
448
66.2
6
13
25
61
503
26.2
10
19
31
81
461
20.0
6
16
29
61
489
23.6
461
462
480
572
407
481
466
612
Selenium was added to the rat diets as Se-enriched yeast at 0.5, 1.5 and 5 ppm, a fortnight
after radiation exposure
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The treatment also enhanced rat resistance to radiation and reduced (1.5-3.5-fold) the
incidence of radiation-induced leukaemias and other malignancies, including breast,
thyroid and lung cancers. In the group with Se supplementation, latent periods were
longer than in exposed control without Se supplementation. According to these results
Se can reduce the risk of cancer when added to the diet long after the radiation.
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levels in the young-adult, middle-aged and elderly groups were 120.6 g/l, 97.2
g/l, and 90.8 g/l, respectively. Subjects with the lowest tertile of Se concentration
had significantly higher atherogenic index and lower HDL-cholesterol levels
compared to those with the highest tertile. However, only the serum HDLcholesterol level showed the dependency on the Se status as determined by stepwise analysis and this dependency was significant only in the subjects below the
age of 40. Similar relationship between Se and age was shown in animal models
as well. In an experiment conducted in Turkey male Wistar rats at ages of 1, 6 and
12 months were used. The activity of Se-GSH-Px and the level of GSH in rat
erythrocytes were decreased with age and the correlation between age and SeGSH-Px activity (r=-0.376, p<0.05) was negative (Ozturk and Gumuslu, 2004).
It is generally accepted that a well-preserved function of the immune system is
an excellent marker of health and longevity. Indeed, in ageing populations
immunity is often compromised. The elderly suffer a decline in immune function
that increases their vulnerability to infections. For example, detrimental changes
were observed in prematurely aging mice (PAM). The effect of ingestion during 5
weeks of a diet supplemented with antioxidants (vitamin C, vitamin E, -carotene,
zinc, and selenium) on several immune functions of peritoneal leukocytes from
PAM was studied (Alvarado et al., 2005). The results showed that, in macrophages,
chemotaxis and phagocytosis as well as the intracellular free radical levels (which
are depressed in PAM) increased after antioxidant supplementation. An increase
also occurred in lymphocyte chemotaxis, proliferative response to the mitogen
concanavalin A, and interleukin-2 release, as well as in natural killer cell activity.
However, the release of tumor necrosis factor-alpha, which increases with aging,
decreases after 5 weeks of supplementation. The effect of dietary (2.00 ppm for 8
weeks) supplementation of aged (24-month-old), mice with selenium (as sodium
selenite) on the lymphocytes functions was studied and the results suggested that
selenium restores the age-related defect in cell proliferation through an increase
in the number of high-affinity IL-2 receptors (Roy et al., 1995). Indeed, the
supplementation with selenium resulted in:
In another animal experiment it was shown that selenium and vitamin E facilitated
recovery from lipopolysaccharide-induced sickness in aged mice. Male mice were
fed diets that were low, adequate, or high in both -tocopherol (10, 75, or 500
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in the medium (P < 0.05). More importantly, the average life-span and the average
maximum life-span of flies in Se groups were increased significantly as compared
with control group (P < 0.05). It is interesting to note that the response of GSH-Px
activity and life-span of male flies to Se intake was better than that of females. It
seems likely that antioxidant supplementation could affect longevity of animals
and human. For example, the effect of daily dietary supplements of an antioxidant
mixture (AM) consisting of -carotene, -tocopherol, ascorbic acid, rutin,
selenium, and zinc on the survival of male mice starting at 2, 9, 16, and 23 months
of age was investigated. The results indicated that the survival of mice given AM
starting at 2 and 9 months of age increased significantly (from 86 to 108 days)
compared to the control (Bezlepkin et al., 1996). The times, of 50, 90, and 100%
mortality in mice given AM starting at 2 and 9 months of age increased by 169.5% compared to the control. It has been shown that beneficial effect of antioxidant
supplementation depended on the age when the supplementation started, since in
mice given AM, starting at 16 and 23 months of age, there was no effect of AM
supplementation.
Did you know that an optimal Se supplementation could improve
quality of life of elderly by improving their immunity and
preventing degenerative diseases?
The spatial distribution of the elderly, those aged 80 years or more, in 2408
counties, was compared with the prevalence of Kaschin-Beck and Keshan diseases,
both of which involve extreme Se deficiency. It was shown that far fewer people
of advanced age reside in those counties with Se deficiency than in unaffected
counties (Foster and Zhang, 1995). The authors suggested that possible reasons
for this include elevated mortality from endemic and chronic diseases in Se deficient
areas and accelerated ageing due to excessive cellular damage caused by free
radicals. To examine the hypothesis that inadequate plasma Se can adversely
affect the maintenance of optimal health the relationships between plasma Se and
mortality in an elderly population was studied. During the 2-year period from
1991 to 1993, 1389 men and women born between 1922 and 1932 were recruited.
In this 9-year study baseline plasma Se was higher (1.10 mol/l) in individuals
who were alive at the end of follow-up than in those who died during the followup (1.01 mol/l , P <0.0001). Furthermore, mortality rates were significantly higher
in individuals with low Se (relative risk (RR) = 1.56 ). When the underlying causes
of death were considered, an association with cancer-related mortality was found
(adjusted RR = 1.79; Akbaraly et al., 2005).
Dementia is one of the most pressing public health problems with social and
economic implication. The form called cognitive impairment non-dementia (CIND)
represents a subclinical phase of dementia. A range of various studies have shown
a possible effect of antioxidants and trace minerals on cognitive function. In a study
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status and Se is shown to be most important among others. Indeed cognitive impairment
is an important component of age-related dementing diseases. In particular,
observational studies of relationships between cognitive impairment and antioxidant
status are based on the evaluation of dietary intake or on the levels of various
antioxidants and trace elements in plasma. Despite some limitations, the comparison
between results obtained in various populations is becoming increasingly informative
and these studies argue for a protective effect of antioxidants on cognitive performance
(Berr, 2002).
Epidemiological data suggest that antioxidants may have a beneficial effect on
many age-related diseases including atherosclerosis, cancer, some neurodegenerative
and ocular diseases. The beneficial impact of antioxidants on various age-related
degenerative diseases may forecast an improvement in life span and enhance quality
of life. However, the widespread use of supplements is hampered by several factors
(Bonnefoy et al., 2002):
In China, selenosis was reported to occur in people who consumed selenium regularly
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at levels above 0.85 mg/day (Yang and Zhou, 1994) or 5 mg of selenium daily from
food or 1 mg/day as sodium selenite for a long period of time (Yang et al., 1983).
Epidemiologic studies and case reports have shown that chronic exposure to selenium
compounds is associated with several adverse health effects in humans (Vinceti et
al., 2001):
Reported symptoms
Garlic breath
Brittle nails
Brittle hair
Stomach upset
Dizziness
Serious Se-related toxicities
Blood chemistry and hematology
Patients characteristics
Plasma Se level, ng/ml*
Months on high Se treatment
Mean
Range
Age in years
0
1
0
1
1
0
Within normal limits
6
3
1
2
3
0
Within normal limits
492.2
639.7
14.2
5.5-22.7
73.6
11.5
1.0-23.6
70.5
*There was not relationships between Se toxicity symptoms and Se concentration in plasma
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Table 11.31 Selenium requirement and status (Adapted from Yang and Xia, 1995; Whanger, 2004).
Se requirement
g/day
Reference
Whanger et al., 2004
Yang and Xia, 1995
Levender, 1997
Yang and Xia, 1995;
Whanger et al., 2004
Food Nutrition Board, 2000
Department of Health, 1991
Tinggi, 2003
Department of Health, 1991
Tinggi, 2003
Poirier, 1994
Food Nutrition Board, 2000
Whanger et al., 2004
Whanger et al., 2004
Burk and Levander, 1999
Whanger et al., 2004
Yang and Xia, 1995
Food Nutrition Board, 2000
Yang and Xia, 1995
*an estimate of a daily exposure to the human population that is likely to be without an
appreciable risk of deleterious effects during a lifetime
It seems likely that there is a need for more research addressing upper range of
dietary Se intake. From the one hand, high Se exposure could be detrimental. On
the other hand, Se doses close to toxic are shown to be effective in cancer prevention
and treatment. The form of Se is also of great importance. As mentioned above
sodium selenite could be considered as a drug which can damage DNA, cause
pro-oxidant reactions and having other adverse health-related effects and therefore
should be used with extra caution. In contrast Se-enriched yeast is a food/feed
additive and can help meeting Se requirement of general population. It seems
likely that long-term consumption of organic selenium in the form of Se-yeast at
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doses 100-300 g/day for a long period of time is safe and could be beneficial for
those who live in areas with low Se status.
Conclusions
The main conclusion which can be drawn from this chapter is that an optimal Se
balance in the human body is an essential element of health maintenance. Indeed,
selenium is the most controversial trace element. Depending on the dose and
form it can be beneficial or detrimental for human health. Recent findings in the
field of molecular biology indicating a presence in human body of at least 25
selenoproteins can change our way of addressing Se requirement. Indeed, the
human Se requirement is developed based on the effect of Se on GSH-Px, an
important member of the selenoprotein family, but not the only one and probably
not the main one. In some instances, such as anticancer activity and
immunomodulation, Se requirement is much higher than needed to maintain
growth and development. It seems likely that we need to learn from nature a way
of Se supplementation. Indeed, food ingredients contain a range of
selenocompounds with Se-Met comprising 60-80% total selenium. Therefore, this
kind of organic selenocompound mixture could be an ideal for dietary
supplementation to meet Se requirement. There is also a scope for sodium selenite
and some synthetic selenocompounds as specific drugs to address specific
problems. Nevertheless, the global Se deficiency awaits a scientifically-based
solution. It seems likely that production of Se-enriched eggs, meat and milk could
be considered as a way forward in solving the Se deficiency. On the other hand,
anticancer activity of various Se compounds needs further investigation.
Diet is closely related to our life style, national traditions, preferences and
habits so the likelihood of the three ideal diets finding their way to our tables is
minimal. Since habit is a second nature it would be almost impossible to introduce
such diets in Western countries like the UK or USA. However, improvement of
the diet by balancing essential nutrients via designer/functional food without
changing peoples food preferences would bring health benefit. In this respect,
natural antioxidants have become a wonder remedy of the 21st century.
We are what we eat!
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Yang, G.Q. and Zhou, R.H. (1994). Further observations on the human maximum
safe dietary selenium intake in a seleniferous area of China. Journal of Trace
Elements and Electrolytes in Health and Disease 8: 159165
Yazar, M., Sarban, S., Kocyigit, A. and Isikan, U.E. (2005). Synovial fluid and
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12
SELENIUM-ENRICHED EGGS, MILK AND MEAT AS FUNCTIONAL
FOODS AND A SOLUTION TO SELENIUM DEFICIENCY
An egg a day keeps diseases at bay
Introduction
Relationships between diet and human health have received substantial attention
in the last few years with the realisation that unbalanced diets can cause serious
health-related problems. However not everyone eats the same foods and people
meet their nutritional needs in the various ways. In fact there is a range of groups
and factors affecting food choice (Tables 12.1 and 12.2; Blades, 2001). From
many food ingredients commonly present in our diet the natural antioxidants are
considered particularly important. It is well known that free radicals produced
under both normal and abnormal physiological conditions can have damaging
effects on polyunsaturated fatty acids, DNA and proteins. Antioxidant protection
is vital for either prevention or substantial reduction of the damage caused by free
radicals and products of their metabolism. Our food provides a major part of
natural antioxidants including vitamin E, carotenoids, flavonoids and selenium.
Particular interest in selenium was generated as a result of clinical studies showing
that dietary supplementation with organic selenium in the form of yeast grown on
a media enriched with this trace element decreased cancer mortality 2-fold.
Additionally, there are data indicating that inadequate selenium consumption is
associated with poor health and development of various viral and bacterial diseases.
Unfortunately, in many countries all over the world human food ingredients do
not provide sufficient selenium. As a result, finding solutions to this public health
problem are on the agenda of many government bodies. Results of various research
studies conducted over the last few years indicate that enrichment of animalderived foods with selenium via special supplements of food animal diets could
be an effective way of increasing human selenium status in countries where
selenium consumption falls below the Recommended Daily Allowances (RDA).
For example, selenium consumption in the UK is shown to be about 50% of
RDA.
809
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Table 12.1 Main groups affecting food choice (adapted from Blades, 2001).
Groups
Effect
Government
Food suppliers
Table 12.2 Factors influencing food choice (adapted from Blades, 2001).
Factor
Effect
Socio-economic factors
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(Table 12.3). When considering ways to improve human selenium intake, there
are several potential options. These include direct supplementation, soil fertilisation,
supplementation of food staples such as flour and production of functional foods.
Table 12.3 Selenium concentrations in various European food sources (adapted from Brown and Arthur,
2001).
Food
Eggs
Beef
Pork
Lamb
Poultry
Fish
Liver
Kidney
Milk
Dairy produce
Bread
Cereals
Fruit
Vegetables
Brazil nuts
a
/ BNF, 2001
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et al., 2002). However, since Se in high doses could be toxic and selenite is not
the optimal form of Se dietary supplementation, this approach is limited to the
specific areas of China and did not find a great support in other countries.
Soil selenium availability is dependent on both selenium content and soil pH.
Low crop selenium content owing to low soil pH is quite common situation in
various countries all over the world. For example, in Finland the availability of
soil Se for plants is poor owing to the relatively low Se concentration, low pH and
high iron content of the soil. In areas where soil selenium content is low (Finland
and New Zealand) sodium selenate was added to fertilizers used for both grain
and forage production (Alfthan, 1993; Oldfield, 1999; Arthur, 2003). Therefore,
since 1984 multimineral fertilizers have been supplemented with Se (16 mg/kg to
fertilizers for grain production and 6 mg/kg to those for fodder production) in the
form of sodium selenate. Within two years a three-fold increase of mean Se intake
was observed. The supplementation affected the Se content of all major food
groups with the exception of fish (Aro et al., 1995). As a result, there was a rapid
increase in the Se content of the crops and the foodstuffs derived from animals
consuming the crops, as well as the human Se status was improved (Arthur, 2003).
For example, the mean Se concentration in young adults and children in Finland
increased from 1.04 and 0.87 mol/l in 1985 to 1.59 and 1.31 mol/l in 1990
respectively (Wang et al., 1998). The Se level in human maternal milk was also
significantly increased (Kantol and Vartiainen, 2001). In 1990 the amount of Se
that was supplemented was reduced to 6 mg/kg for all fertilizers. This reduced the
mean Se intake by 30% and the serum Se concentration decreased by 25% from
the highest levels observed in 1989 (Aro et al., 1995). In fact, three different
methods of Se application were tested: seed pre-treatment, fertiliser enrichment,
and foliar application (Gissel-Nielsen, 1986). Seed pre-treatment had some
disadvantages while the two other methods proved to be efficient in a series of
experiments and in tests on a large number of farms all over Denmark. In general,
foliar application of selenium provided a higher efficiency for increasing the
selenium content of soybean than soil application (Yang et al., 2003) and
successfully used for production of Se-enriched rice in China (Hu et al., 2002).
This approach has been successful in Finland and New Zealand, but has had
limited interest in other countries because of environmental issues. For example,
in the USA, the use of Se fertilizers caused run-off of the element, resulting in its
accumulation in the aquatic biota (Maier et al., 1998). Furthermore, even in
Finland, simultaneous increase of total nitrogen, phosphorus, and selenium levels
in consecutive samples from some ground water pools indicated leaching of
selenium from the fertilizers into the ground water in certain areas (Makela et al.,
1995).
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Table 12.4 Common foods fortified with selenium (Combs, 2001; Surai and Sparks, 2001; Surai, 2002).
Selenium-enriched products
Several important factors must be considered when choosing the best food
supplementation strategy for a given population. Such factors are shown in Table
12.5. In general, main sources of dietary selenium could differ between different
countries. For example, in the UK meat and meat products provide 32% daily Se
consumption and dairy products and eggs are responsible for 22% Se consumption
(BNF, 2001; Figure 12.1). In contrast, in Russia about 50% Se in the diet originates
from bread and cereals and meat, milk and eggs provide about 20%, 10% and 5%
daily Se consumption respectively (Golubkina et al., 2002; Figure 12.2). In the
USA beef, white bread, pork, chicken and eggs account for half of the Se in the
diet (Schubert et al., 1987). In Ireland, meat and meat products (30%), bread and
rolls (24%), fish/fish products (11%), and milk and yoghurt (9%) were the main
contributors to mean daily Se intake (Murphy et al., 2002).
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Table 12.5 Some characteristics of food choice for Se-enrichment (adapted from Yaroshenko et al.,
2003a).
Comments
A part of traditional
meals for the population
Consumed regularly in
a moderate amount
Consumed by the
This is particularly important given that immune function is
majority of the population more likely to be compromised in groups such as children and
the elderly.
Affordable
Supplying a meaningful
amount of the nutrient
(e.g. at least 50% RDA)
Did you know that in the UK meat and meat products provide
32% daily Se consumption and dairy products and eggs are
responsible for 22% Se consumption?
Among animal-derived products, the egg is ideally suited to meet the list of
requirements mentioned in Table 12.5. The egg is a traditional and affordable
food in most countries and is consumed by people of all ages more or less regularly
and in moderation. It is also a very safe vehicle for supplementation given that a
toxic dose of selenium from eggs would require consumption of 30 eggs per day
over time, an impossible situation to imagine. There is an option of simultaneous
enrichment of eggs with several important nutrients, including omega-3 fatty acids,
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Figure 12.1 Estimated intake of Se from different food in the UK in 1997 (adapted from BNF (2001).
Vegetables
Potato
Fruit
Eggs
Milk and milk products
Fish
Bread and cereals
Meat
Figure 12.2 Estimated intake of Se from different food in the Russia (Ural region) (adapted from
Golubkina et al., 2002).
vitamin E, carotenoids (Surai and Sparks, 2002; Surai, 2002) and with a single
egg it is possible to deliver at least 50% of the RDA for selenium. It seems likely
that pork, beef, chicken and milk can also be enriched with selenium.
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Lipid
Phospholipid
Cholesterol esters
Triglycerides
Free fatty acids
Free cholesterol
Phospholipids
1.3
63.1
0.9
4.9
29.7
Phosphatidyl ethanolamine
Phosphatidyl serine
Phosphatidyl choline
Sphingomielin
Others
23.9
2.7
69.1
3.2
3.2
Did you know that compared with the hens egg, no other single
food of animal origin is eaten by so many people all over the
world and none is served in such a variety of ways?
Its proteins are highly digestible and remarkably complete, containing the most
important essential amino acids. The egg amino acid profile is similar to the ideal
balance of amino acids needed by men and women. It also supplies various
minerals, some in significant amounts, and contains a range of vitamins. For
example, in recent study in the USA it has been shown that eggs contributed 10%
to 20% of daily intake of folate and total, saturated and polyunsaturated fat, and
20% to 30% of daily intake of vitamins A, E and B12 (Song and Kerver, 2000). In
this respect, an egg can deliver (as % the daily value for a nutrient) protein: 10,
riboflavin: 15, vitamin B12: 8, vitamin A: 6, vitamin D: 6, folate: 6, vitamin B6: 4,
vitamin E: 3, thiamine 2, zinc 4 and iron 4. Many of those nutrients in the egg can
be manipulated by dietary means, however, a real value for improvement of human
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diet could have only those nutrients which are usually in short supply with other
products or have a positive effect on human health when consumed in excess.
Among them n-3 fatty acids, vitamin E, carotenoids and selenium have attracted
a lot of attention in nutritional sciences (Figure 12.3).
Omega-3 +
other nutrients
Omega-3
Linolenic
acid
DHA
Columbus
EggsPlus
Iodine
Egglands
best
Vitamin E
Others
Se
Lutein
Super Eggs
Figure 12.3 Designer egg production scheme (adapted from Yaroshenko and Surai, 2002).
The main problem with eggs consumption is that the image of the egg in the
mind of the consumer is not necessarily always good. Despite the nutritious
qualities of the egg, its comparatively high content of cholesterol has been the
driving factor causing declining egg consumption for the last 20 years. Indeed,
purchases of eggs for household consumption in the UK fell by 54% between
1975 and 1993 and by 9% between 1993 and 2000 (Buttriss, 2002). The problem
started when cholesterol was implicated as an important risk factor of coronary
heart disease (CHD), the leading cause of death in developed countries. The direct
relationship between the levels of plasma cholesterol and atherosclerosis was
experimentally demonstrated in an animal model (rabbits) 92 years ago
(Anischenkow and Charlatov, 1913). Since that time numerous studies have been
devoted to that subject. In particular, a consistent correlation was found between
levels of total cholesterol and CHD in populations living in different countries
(Keys, 1980). Therefore, dietary cholesterol became the centre of attention because
of its association with heart vascular disease development. That information
provided the stimulus for mass media blaming cholesterol for many pathological
conditions in the human body. As a result, many dieticians and doctors started to
recommend lower egg consumption to avoid excess of cholesterol in the diet.
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Similarly, it was initially believed that egg consumption was associated with a
rise in blood cholesterol (Yaffee et al., 1991) and as a consequence was deleterious
to health and life expectancy. The situation deteriorated when problems with egg
contamination with Salmonella were reported publicly. In the wake of health fears,
whole egg consumption in Britain has fallen by almost half in the past five years,
from more than three eggs a week to 1.70 eggs. Similar decreases in egg
consumption have been reported in other countries, including the US (Stadelman,
1999; Lewis et al., 2000). The proliferation of magazine articles and public
statements by organisations such as the American Heart Association relating eggs
to blood cholesterol and heart disease caused a downward trend in egg
consumption (McIntosh, 2000).
Did you know that misconception about relationship between
egg consumption and heart diseases was responsible for
sharp declining in egg consumption in the USA and many
European countries?
However, our understanding of cholesterol metabolism has substantially improved
since the initial furore over cholesterol, food animal products and heart disease.
Knowledge of reverse cholesterol transport by HDL from the vessel wall to the
liver for the catabolism (Lacko and Pritchard, 1990) was an important milestone
changing the current views of cholesterol intake. Dietary cholesterol is not regarded
anymore as the major determinant of the cholesterol level in the blood. There are
other more important determinants of this parameter such as saturated fat intake.
More is also known about the development of coronary heart disease (Shaper,
1987). Furthermore, detailed analyses of many cholesterol-lowering trials showed
that a decreased coronary morbidity did not reflect changes in total mortality
(Libbi et al., 2000), which was not changed. Furthermore, after analysing results
from 27 studies involved 30,902 person-years of observation, Hooper et al. (2001)
concluded that alterations of dietary fat intake had a positive effect on
cardiovascular events but practically no effect on total mortality.
Correlations drawn between nutrient intakes and longevity in the Japanese
since WWII illustrate this point. It was found that sufficient intakes of animal
protein and fat are crucial for attaining longevity (Shibata, 2001). It is interesting
that low serum cholesterol was deleterious for higher levels of functional capacity
and accelerated depressive status in the elderly in the community. High intakes of
milk and fats and oils had favourable effects on 10-year (1976-1986) survivorship
in 422 urban residents aged 69-71 (Shibata et al., 1992). The survivors revealed
a longitudinal increase in intakes of animal foods such as eggs, milk, fish and
meat over the 10 years. Furthermore, an increase in total cancer mortality in lung,
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prostate and colon was observed in men >60 years of age with low plasma
cholesterol in Switzerland (Eichholzer et al., 2000).
Results of many recent studies have shown that large numbers of eggs can be
consumed over lengthy periods without adverse changes to plasma cholesterol or
other lipid components. Furthermore, it is a general misconception that elevated
plasma cholesterol represents the main atherogenic stimulus. It seems likely that
the time has come to reconsider the eggs image and to distinguish between
scientific fact and conjecture.
Ginsberg et al. (1994) showed that consuming two eggs per day did not change
serum cholesterol; and consumption of four eggs per day had only a slight effect
on this parameter. Similarly, consumption of up to 14 eggs per week by students
did not alter serum cholesterol level (Vorster et al., 1992). Analyses of the results
from over 30 years in more than 2750 patients indicated that for the majority of
individuals modest changes in dietary cholesterol have little if any effect on plasma
lipoprotein cholesterol level (McNamara, 1995). McNamara also calculated that
a reduction in dietary cholesterol intake by 33% (from 450 to 300 mg/day) would
lower plasma cholesterol level by 3.7 mg/dl (approximately by 1.85%) and a 1%
decrease in energy intake from saturated fat decreases plasma cholesterol by 3
mg/dL (approximately 1.5%). Since dietary cholesterol has little effect on plasma
cholesterol in most individuals, the population-wide restriction on egg
consumption is not justified (McNamara, 1997). The same conclusion was drawn
by Dawber et al. (1982), who showed that differences in egg consumption were
unrelated to blood cholesterol level or to the incidence of coronary heart disease.
After adjusting for demographics (age, gender and ethnicity) and lifestyle variables
(smoking and physical activity), dietary cholesterol was found to be unrelated to
serum cholesterol concentration (Song and Kerver, 2000). When 116 male
volunteers between the ages 32 and 62 years consumed two whole fresh eggs
daily for 3 months no significant changes in mean serum cholesterol were recorded
and there was no significant association between dietary cholesterol intake and
either serum cholesterol or triglyceride (Flynn et al., 1979). Similarly a recent
study involving 37,850 men and 80,082 women concluded that the consumption
of up to one egg per day was unlikely to have a substantial overall impact on the
risk of cardiovascular disease or stroke among healthy men and women (Hu et
al., 1999).
Interestingly, recently it has been shown that people who reported eating four
or more eggs per week had significantly lower mean serum cholesterol than those
eating one egg or less per week (193 mg/dL vs. 197 mg/dl, p < 0.01) (Song and
Kerver, 2000). Furthermore, when dietary confounding factors were considered,
no association was found between egg consumption at levels up to one egg per
day and the risk of coronary heart disease in non-diabetic men and women
(Kritchevsky and Kritchevsky, 2000). Recent conclusions from a review of
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epidemiological and clinical data published by McNamara (2000) was that for
the general population, dietary cholesterol makes no significant contribution to
atherosclerosis and risk of cardiovascular disease. These suggestions are in line
with a recent finding that consumption of three eggs per day for 30 days by premenopausal women did not increase the risk of developing an atherogenic
lipoprotein profile (Herron et al., 2002). From another analysis of data
summarising 166 cholesterol-feeding studies conducted over the past 40 years in
3500 subjects, it was concluded that there was little justification for restriction in
egg consumption for general healthy individuals (McNamara, 2000a).
Did you know that recently it has been proven that there was
little justification for restriction in egg consumption for
general healthy individuals?
Once the public image of the egg is changed for the better, there are many
opportunities to produce designer eggs enriched with various nutrients with
health-promoting properties (Figure 12.3).
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Organic Se
added to the
feed, ppm
0
0.2
0.4
0.8
Se in egg
yolk, ng/g
298.3
605.3
854.0
1087.3
Se in egg
white, ng/g
50.7
193.7
403.7
621.7
Se per egg,
g
RDA from
one egg
7.10
18.04
30.67
43.35
11.4
28.9
49.1
69.4
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30
Fresh
1 week, 20 C
25
20
15
10
5
0
Control
0.2 Se
0.4 Se
0.8 Se
Figure 12.4 Effect of Se on lipid peroxidation in egg yolk, MDA ng/g (adapted from Yaroshenko et al.,
2003).
3.0
Yolk
White
2.5
2.0
1.5
1.0
0.5
0.0
Control
0.2 Se
0.4 Se
0.8 Se
Figure 12.5 Effect of Se on GSH-Px activity in egg yolk and albumin, U/g (adapted from Yaroshenko et
al., 2003).
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80
3 h, 37 C, in vitro
70
60
50
40
30
20
10
0
Control
0.2 Se
0.4 Se
0.8 Se
Figure 12.6 Effect of Se on lipid peroxidation in egg yolk during egg storage, MDA ng/g (adapted from
Yaroshenko et al., 2003)
It seems that Se in eggs is highly available. For example, a recent clinical trial
conducted in the Ukraine showed that consumption of two Se-enriched eggs per
day for eight weeks significantly increased the Se level of the plasma of volunteers
(Surai et al., 2004). In fact sixty volunteers (30 in control and 30 in experimental
group) successfully finished the trial. Eggs consumed in the control group contained
7-9 g Se/egg and experimental eggs were enriched with selenium (28-32 g Se/
egg). Blood was collected before the beginning and at the end of experimental
period and Se was determined in plasma by hydride generation atomic absorption
spectrometry with fluorometric detection. The level of selenium in plasma of
volunteers living in the Kiev area of Ukraine (0.055-0.081 g /ml) was on the low
side of the physiological range and was somehow lower than we reported earlier
in volunteers in Scotland (Surai et al., 2000). Consumption of commercially
available eggs for eight weeks only slightly increased Se in plasma, which reached
physiological level (0.075-0.085 g/ml). In contrast, consumption of two eggs
daily, which together delivered the daily requirement of 55-65 g Se, for eight
weeks was associated with a significant increase in Se concentration in plasma.
Plasma Se reached 0.09- 0.14 g/ml, indicating improving Se status of volunteers
(Surai et al., 2004). This is the first clinical trial to prove that selenium-enriched
eggs could be used as an important vector to improve selenium status in countries
with low Se consumption like Scotland or Ukraine. Se availability from eggs for
human needs further elucidation and effect of different dietary sources of Se on
its concentration in plasma probably depends on the Se status of the human. For
example, in our previous clinical study in Scotland a consumption of one Se-
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enriched egg per day for 8 weeks did not change Se concentration in blood which
was in a physiological range (Surai et al., 2000). In contrast, in the case of low
selenium status, the consumption of eggs enriched with Se during 3 months
significantly increased serum and hair Se level (Yu et al, 1996). Similarly, in
Chinese children from the area endemic for Keshan disease, Se content in hair
increased due to consumption of Se-enriched eggs during 3 years (Yu et al, 1998).
Did you know that recently in a clinical trial it has been
proven that consumption of Se-enriched eggs can improve
Se status of people?
After the successful clinical trial with Se-enriched eggs in Ukraine, the production
of such eggs, enriched with Se and vitamin E, started commercially and now the
eggs called Basket of life can be found in the supermarkets in Ukraine. This
development is very important for this region. From the one hand Se deficiency
was documented in people working in Chernobil area (Tutelian et al., 2002;
Golubkina et al., 2002). On the other hand selenium and other antioxidants can
be especially beneficial for people living in radionuclide-contaminated areas of
the Ukraine. Currently various companies all over the world market Se-enriched
eggs including Mega-Eggs in Ireland, NutriPlus eggs in Malaysia, as well as
selenium enriched eggs in Russia, Thailand, Australia and the US (Table 12.8).
Prices for those eggs varied from country to country and are similar to those for
free-range eggs.
In the UK the only designer egg available through the major supermarkets is
the Columbus egg produced by the Belgium company Belovo. These eggs,
enriched in n-3 fatty acids and vitamin E, first appeared in Belgium in 1997, and
since then they have been sold in the UK (1998), Netherlands (1999) and India,
Japan and South Africa (2000). Currently production of the Columbus egg exceeds
50 million eggs per year in Europe. These eggs are characterised by a balanced
nutritional lipid composition (C18, n-6:n-3=1:1) and a favourable structural lipid
ratio (long-chain PUFA, n-6:n-3 = 1:3). When fed to selected groups of people,
Columbus eggs have been shown to improve the circulating cell membrane fatty
acid composition by favourably altering the n-6:n-3 ratio (De Meester et al., 1998).
The level of alpha-linolenic acid in Columbus eggs is about 12.6% while DHA
comprises about 2% of total fatty acids in the egg yolk. Therefore, these eggs
could adjust a diet toward recent nutritional recommendations regarding dietary
fatty acid profiles. In particular it is recommended linoleic acid:alpha-linolenic
acid ratio in the diet to be 5:1-10:1 with n-3 PUFA to provide 0.4-2% of total
energy (FAO, 1998).
In this respect it is worth mentioning that recent n-3 PUFA consumption in the
UK provides only 0.23% of total energy and in the Netherlands, Belgium,
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Trade name
Countries
Columbus
Germany, Ireland and Italy this figure is even lower (Roche, 1999). Recently,
Columbus eggs were also enriched with selenium, delivering with a single egg
more than 50% RDA (35 g) of this trace element as well as 10 mg vitamin E
(66% RDA) and 75 g iodine.
Did you know that after long discussions about selenium
deficiency in the UK, a product (Columbus eggs) is available
on the supermarket shelves that can substantially improve
Se status of the major part of population?
Since these types of designer eggs find their way to supermarket shelves in other
countries the selenium status in those countries can also be improved. This could
be a result of successful knowledge transfer from developer and producer of the
feed additive (Alltech, Inc., USA) via scientific evaluation and development of
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the effective technology of designer egg production (Surai, 2002) to the egg
producer and food retailers to consumers. To satisfy consumer demand in the
UK, free range Columbus eggs enriched with n-3 PUFAs, vitamin E and selenium
are also on the supermarket shelves.
It is important to mention that in many countries there are several different
competitive companies producing Se-enriched eggs. For example in Russia
Seimovskaya poultry farm (Nizni Novgorod region) produces about 65,000 Seenriched eggs per day, while Magnitogorskaya (Magnitogorsk city), Aksaiskaya
(Rostov region), Voktinskaya (Izhevsk region) and Volzhaninskaya (Yaroslavl
region) poultry farms produce 40,000; 30,000; 15, 000 and 10,000 Se-enriched
eggs daily. In Belarus, Soligorskaya (Minsk region) poultry farm produces about
40,000 Se-enriched eggs daily. Se concentration in those eggs in Russian and
Belarus varies in a range of 20-30 g/egg. Similar range of Se in Se-enriched
eggs can be found in other countries with a target to achieve a 50% RDA in Se
with a single egg. There is also a possibility to produce eggs with much higher Se
content. For example, Combs (2000a) reported that using selenium in the form of
high-quality Se-enriched yeast in the chicken diet at the level 1.2 ppm it is possible
to achieve Se content in the egg up to 200 g.
If egg selenium content is enhanced along with increased levels of omega-3
PUFAs and vitamin E (Columbus) or vitamin E and specific carotenoids (Super
eggs, Table 12.9; Surai, 2001) in levels comparable with RDA, this could further
widen opportunities for producers and consumers to meet specific nutrient demands
that aid in maintaining a healthy life style. There is also an opportunity to produce
Se-enriched quail eggs (Figure 12.7) which can find their way into niche market.
Did you know that Se-enriched eggs are on the supermarket
shelves in more than 20 countries all over the world?
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Table 12.9 Major nutrients in a super egg (Adapted from Surai, 2001).
Nutrient in
the egg
Amount,
mg
% Recommended
dietary allowances
Vitamin E
19.3
129
Lutein
1.91
Selenium
0,032
50
209
100
49 g sardine
165 g Atlantic cod
170 g haddock
180 g carp
DHA
Selenium/egg, g
Control
Experimental
4
3
2
1
0
5 designer quail eggs = 50% RDA
Figure 12.7 Quail egg enrichment with selenium (adapted from Karadas et al. 2004).
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Table 12.10 Selenium content in meat from different countries, g/100g (adapted from Diaz-Alarcon et
al., 1996; McNaughton and Marks, 2002;Daun et al., 2001; Tutelyan et al., 2002; Golubkina et al., 2002;
Schubert et al., 1987).
Country
Pork
Year reported
Chicken
Year reported
USA (Ohio)
USA (Dakota)
USA
USA
Canada
France
Spain
Spain
Spain
Russia
Sweeden
Australia
UK
New Zealand
4,0
31.0
33
14.4-45.0
30.0
10.7
6.0
4.0
6.1-11.6
15.0-20.0
11.3
9.4-20.5
14.0
1.93-15.0
1980
1984
1987
1999
1982
1991
1983
1987
1996
2002
2001
2002
1991
2000
20
19.0-27.6
16.0
5.4
2.0
16.0
5.8-8.4
20.0
11.6-28.0
6.0-7.0
13.7-14.5
1987
1999
1982
1991
1983
1987
1996
2002
2002
1991
2000
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interesting that in addition to increased Se level in the meat there are other important
improvements in pork quality (Cole, 2000):
Table 12.11 Effect of dietary Se source and level on loin Se content of growing-finishing pigs, g/g
(adapted from Kim and Mahan, 2001)
Se doses
Selenite
Sel-Plex
0
0.154
0.154
10
15
20
0.333
3.375
0.277
5.927
0.323
10.311
0.322
7.648
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higher levels of 67 g/100 g (Finley et al., 1996; Hintze et al; 2001; 2002). The
opportunity of substantial enrichment of beef by Se is shown by analysis of beef
produced in a seleniferous area of South Dakota (Hintze et al., 2002). Indeed Se
levels in ribeye, sirloin, clod and round were 1.20; 1.19; 1.21 and 1.22 mg/g
respectively. After 14 weeks on high selenium diet (11.9 ppm from seleniferous
wheat and hay) the Se level in muscles further increased up to 2 g/g (Hintze et
al., 2002). Indeed, inclusion of such beef in any meat foods could substantially
enrich it with selenium. It is also interesting to mention that in the study high Se
intake for 14 week of the experimental period did not affect feed intake, average
daily gain and final weight of steers.
Table 12.12 Selenium content of common meats in a grocery store in North Dakota (adapted Finley,
1999).
Item
Concentration, Standard
g/g
serving, g
0.37
0.06
0.08
0.33
0.20
0.45
0.35
0.30
85
85
85
85
85
85
85
85
Se in
serving, g
%
RDA
31.5
5.1
7.1
28.3
16.9
38.1
30.1
25.3
57.3
9.3
12.9
51.5
30.7
69.3
54.7
46.0
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0.9
Se-supplemented
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Drip loss
Fluid loss
Figure 12.8 Drip loss (%) and fluid loss (%) in beef during storage (adapted from Simek et al., 2002).
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Control
Se-enriched
250
200
150
100
50
0
Breast Muscle
Leg muscle
Figure 12.9 Se-enriched chicken meat ng/g (adapted from Yaroshenko et al., 2004).
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the image of meat purchased at the supermarket (Janssens, 1998). There are many
meat quality characteristics that attract consumer attention. They include
appearance, texture and flavour (Liu et al., 1995) as well as tenderness, juiciness,
aroma (Janssens, 1998) and other subjective characteristics. Among these
appearance has a major impact on the initial decision of the customer to purchase
or reject the product (Sheehy et al., 1997). Consumers prefer fresh meat with a
minimum loss of water during handling and cooking. Therefore water-holding
capacity of the meat (Mahan and Kim, 1999) as well as colour (Froning, 1995)
and absence off-flavours (Sheehy et al., 1997) are considered among most
important meat quality characteristics.
It has been shown that sensory quality of meat is affected by muscle
biochemistry and modern processing technologies (Ouali, 1991). For example,
grinding increases oxygen incorporation into muscle and cooking releases proteinbound iron into the intracellular pool (Chan and Decker, 1994). In this process
free radical production and lipid peroxidation cause membrane structure and
quality disruption, which leads ultimately to significant losses in food quality,
including off-flavour, off colours, poor texture etc. (Stanley, 1991).
One approach to enhancing oxidative stability of meat is to add antioxidants
either into the animal diet or directly during processing (Decker, 1998). For
example, an increasing body of evidence indicates that increased vitamin E
supplementation is an effective means of meat quality improvement in chickens,
turkeys, cattle, pigs and lambs (Sheehy et al., 1997; Wulf et al., 1995; Buckley et
al., 1995; Liu et al., 1995). Based on the data presented above and taking into
account antioxidant interactions, it is possible to suggest that synergism between
Se and vitamin E could work to enhance meat quality. In fact, it has been shown
that GSH-Px activity in muscles did not change significantly over 8-day storage
of beef (Renerre et al., 1996). This means that once GSH-Px activity is elevated it
is maintained post-mortem. Therefore we might expect a stabilising effect of dietary
Se supplementation during meat storage. Indeed supplementing broiler diets with
0.25 ppm Se substantially increased GSH-Px activity in breast (2.1-fold) and leg
(4.1-fold) muscle; and as a result decreased lipid peroxidation was detected (2.5fold in breast muscle and 3.3-fold in leg muscles) after 4 days storage at 4C
compared to the control group (DeVore et al., 1983). These data clearly indicate
that GSH-Px significantly contributes to the overall antioxidant defence of muscle,
decreasing tissue susceptibility to lipid peroxidation and that increasing oxidative
stability of skeletal muscle can be accomplished by organic Se supplementation
of the diet. Additionally, Avanzo et al. (2001) have shown that deficiencies of tocopherol and Se caused multiple alterations in the antioxidant system and
adversely affected the redox state of chicken superficial pectoralis muscle.
Protective effects of Se may be not direct, but are mediated via improvement of
other chains of antioxidant defence. For example, Se in combination with vitamin
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Table 12.13 Selenium content in cows milk in various countries (adapted from Tinggi et al., 2001)
Country
Se, g/l
Year reported
Finland
7.9
1981
Germany
23.2
1978
Norway
10.1
1983
Netherlands
16.4 (winter)
1989
Belgium
10.1 (summer)
11.3 (summer)
17.3 (winter)
1989
1988
1988
Poland
10.5
1992
UK
15.0
1995
Israel
73.0
1990
Canada
28.0
1980
USA (Ohio)
8.0
1979
32-138
1984
Japan
23.1
1982
New Zealand
2.9-9.7
1973
Australia
15.8
1983
vehicle for dietary selenoprotein enrichment (McIntosh and Royle, 2002). The
authors showed that using organic selenium in the cows diet it is possible
substantially increase Se concentration in milk. In fact there was a significant and
rapid increase in milk Se concentration with the introduction of 2 and 6 mg Se
daily supplements as Sel-PlexTM. Therefore, Se concentration in the milk increased
from 6.9 up to 25.2 g/l. It seems likely that this concentration is still too low to
substantially affect Se consumption of the humans. However, in the same study it
was shown that when dietary Se content was increased to supply 25 mg Se/day,
milk concentrations plateaued at about 150 g/l without any clinically detectable
signs of Se excess (McIntosh and Royle, 2002). It is possible to suggest, that at
longer period of organic Se supplementation (in the reported study it was only 16
days), it is possible to achieve similar Se concentration with lower Se doses.
Therefore, about 200 ml of such Se-enriched milk could provide about 50% RDA
in Se. Since milk-processing products would also be enriched with Se, they
potentially could be a substantial source of Se for improvement of human diet
especially in regions low in natural Se level in the soil, feeds and foods. Therefore,
Se-enriched milk is under the consideration in Australia. In 2003 one of the
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biggest raw milk producers in Korea, Seoul Dairy Co-op. launched their Seenriched milk under the SELK name. SELK is enriched with Se, Ca, vitamins A,
D and E. In general, Se concentration SELK is about 50 g/l being 2.5fold
higher than commercial milk and they sell Se-enriched milk on premium price.
The co-op is running an educational program in a weekly magazine and in daily
newspapers on the importance of selenium for human health. Indeed, education
is a key for further development and successful marketing of animal-derived
products enriched with selenium. Unfortunately in many cases in public eyes Se
is a pollutant and toxicant and it will take some time and a lot of efforts from
scientist to change this gloom picture.
Did you know that recently Se-enriched milk has reached
supermarket shelves in Korea?
It seems likely that Se bioavailability from milk is quite high and comparable with
that for sodium selenite (Mutanen et al., 1986). It is interesting that Se availability
from milk depends on the dietary source of Se for cows. For example, the
experimental milks had their Se content increased by feeding cows either sodium
selenite (selenited milk) or Se-enriched barley (barley milk). The Se in the barley
milk was significantly more available than that in the selenited milk when the
plasma Se level was the response criterion (Mutanen et al., 1986). Absorption of
selenium from milk protein and isolated soy protein formulas in pre-school children
was evaluated using stable isotope tracer 74Se (Solomons et al., 1986). In the
experiment, mean fractional absorption of selenium from the formulas based on
milk, isolated soy protein, and milk-soy were 64.2, 73.4 and 45.0%, respectively,
with the combined formula having a significantly lower intestinal uptake for added
selenite than the casein formula. It is also interesting that the apparent digestibility
coefficient and retention of selenium in rats were higher for the goat milk diet
than for the cow milk-based diet (Barrionuevo et al., 2003).
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Recently, six major targets in relation to functional food science have been
identified (Roberfroid, 2000):
gastrointestinal functions;
redox and antioxidant systems;
metabolism of the macronutrients;
development in foetal and early life;
xenobiotic metabolism and its modulation;
mood and behaviour or cognition and physical performance.
In the same review the author has stated that the health benefit of a functional
food will be limited if the food is not part of the diet. Therefore functional foods
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must remain foods and they must achieve their effects in amounts normally
consumed in a diet (Contor, 2001).
Eggs have not traditionally been regarded as a functional food, primarily due
to concerns about their adverse effects on serum cholesterol levels (Hasler, 2000).
However recent finding described above indicating that there is little if any
connection between dietary cholesterol and blood cholesterol levels as well as
between moderate egg consumption and heart diseases could help changing a
bad image of eggs. In this respect, eggs enriched with selenium as well as with a
combination of Se, DHA, vitamin E and lutein, ideally fit in the category of
functional food enabling to substantially improve the diet. Eggs are consumed
regularly by the most of the population and are served as an integral part of
traditional English or Mexican breakfast. The development of super egg
enriched with these 4 nutrients received a lot of publicity through mass media
including radio, TV and newspapers. Some of newspaper titles are shown in Table
12.14. They clearly show great public interest in improvement of egg quality and
in creation healthy eggs.
Table 12.14 Some newspaper titles related to the super egg development (adapted from Surai, 2002)
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Title
Newspaper
Date
Daily Mail
The HERALD
Super egg
Cambridge Evening
News
The EXPRESS
The TIMES
The EXPRESS
August 2, 1998
The HERALD
April 5, 1999
The GUARDIAN
April 7, 1999
April 5, 1999
METRO
April 7, 1999
The EXPRESS
April 8, 1999
Daily Mail
April 7, 1999
Farmers Guardian
April 9, 1999
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Therefore, from information provided above, it is clear that vitamin E, lutein and
selenium are major antioxidants to consider for egg enrichment. An egg has also
a great potential in terms of improvement of human diet. These and other options
of egg use in the human diet need further investigation. Indeed, most of studies,
including our own 8 week study, with designer eggs have been conducted during
a limited length of time and it is not clear what kind of benefit we could expect
when, for example, super eggs are in the diet during 6-12 month of study.
The data indicate that a designer egg enriched in vitamin E, lutein, DHA and
Se can be not only a good nutritional product but also a good vector for the
delivery of four essential nutrients vital for human health. A crucial feature of
these designer eggs is the synergistic combination of n-3 fatty acids with major
antioxidants, vitamin E, lutein and Se, as an important approach to the improvement
of the human diet. These eggs will not be able to replace vegetable and fruits as a
major source of natural antioxidants and fish products as a source of DHA.
However, they can substantially improve the diet, especially in countries like
Scotland, significantly contributing to the recommended daily intake of vitamin
E, lutein, DHA and Se. Commercially, it is possible to produce designer eggs
enriched with 4 nutrients as mentioned above or with 3, 2 or 1 nutrient depending
on the consumer demand. It seems likely, that egg yolk can also be enriched with
vitamin D (Mattila et al., 2003). As a result, price for the production of such eggs
could substantially vary. Therefore the way of egg to the functional food category
started successfully and now it is consumer education which is needed to fulfil
the idea of using eggs as functional food. Indeed, many categories of people will
benefit from using designer eggs as part of their everyday diet. However, more
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DeVore, V.R., Colnago, G.L., Jensen, L.S. and Greene, B.E. (1983). Thiobarbituric
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Diaz-Alarcon, J.P., Navarro-Alarcon, M., dela-Serrana, H.L.G. and Lopez-Martinez,
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Dvorska, J.E., Yaroshenko, F.A., Surai, P.F. and Sparks, N.H.C. (2003). Seleniumenriched eggs: quality evaluation. Proceedings of the 14 th European
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13
ANTIOXIDANT-PROOXIDANT BALANCE IN THE INTESTINE: FROM
HEALTHY GUT TO GENERAL HEALTH
A good beginning makes a good ending
Introduction
A delicate balance between antioxidants and pro-oxidants in cells is an important
determinant of various physiological processes and maintenance of this balance
is the main aim of so called an integrated antioxidant system built in the human
body. This system was developed during evolution to provide an antioxidant
defence and give a chance for animals to survive in oxygenated atmosphere.
Recent data suggest that the antioxidant-pro-oxidant balance starts in the intestine.
Relationship between food and human health has attracted attention of scientists
for many years. It is now widely accepted that fruits and vegetables are important
dietary components responsible for maintenance of good health. However,
molecular mechanisms of protective effects of fruits and vegetables have not
been fully elucidated. One of the attractive ideas is that various antioxidant
compounds of fruits and vegetables are responsible for prevention of oxidative
damage in digestive tract. In fact, antioxidant-prooxidant balance in digestive
tract is considered to be a major determinant of human and animal health (Surai et
al., 2003; 2004).
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Table 13.1 Natural food sources of some antioxidants (Adapted from Shahidi, 1997).
Compounds
Source
Vitamin C
Carotenoids
Flavonoids/isoflavonoids
Phenolic acids/derivatives
Catechins
Extracts/essential oils
When food is consumed and appears in the stomach and then in the small intestine,
it contains a range of antioxidants but may also contain a range of potentially
dangerous substances (Figure 13.1). To keep a balance between antioxidants and
prooxidants in the intestine is a very important task in which diet plays a crucial
role. In general, prooxidants which can be found in the digestive tract could be
summarised as follows (Surai et al., 2003).
tocopherols and tocotrienols
coenzyme Q
carotenoids
flavonoids
ascorbic acid
spices, essential oils
vitamin A
selenomethionine
BOA, BOT
Antioxidant system
maintained,
good health
oxidized PUFAs
oxysterols
persistent organic pollutants
Fe2+ and Cu+
nitrites
heavy metals
mycotoxins
alcohol
Antioxidant system
compromised, poor
health
Figure 13.1 Antioxidant-prooxidant balance in the intestine (adapted from Surai et al., 2003).
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OXYSTEROLS
Cholesterol oxidation products (oxysterols) are commonly found in foods of animal
origin and they are formed during food processing, storage and cooking. In fact,
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more than 60 cholesterol oxides have been identified (Schroepfer, 2000; Savage
et al., 2002). Recent findings suggest that oxysterols may modulate cytotoxicity
by inducing apoptosis. Furthermore, the generation of an oxidative stress may be
a significant event occurring during 7-beta-oxysterol-induced apoptosis
(OCallaghan et al., 2002) simultaneously with a significant decrease in GSH
levels and an increase in the activity of SOD and caspase-3. When cytotoxicity of
various oxysterols was examined in a human monocytic blood cell line
(OCallaghan et al., 2001), it was shown that they are cytotoxic and the mode of
cell death was by apoptosis. However, mode of cytotoxic action of various
oxysterols varied substantially, since some oxysterols induced apoptosis without
changes in the GSH concentration or the SOD activity in the U937 cells. It is
interesting that 7-ketocholesterol is considered to induce apoptosis and the
substantial overproduction of superoxide radical was obtained with 7-betahydroxycholesterol and 7-ketocholesterol (Miguet-Alfonsi et al., 2002).
Furthermore, oxidation of triglyceride-rich lipoproteins was significantly greater
in rabbits fed oxidized cholesterol compared to the pure cholesterol-fed animals
(Vine et al., 1998). Oxysterols can also replace cholesterol in membranes,
perturbing permeability, stability and other membrane properties (Guardiola et
al., 1996), which are extremely important in relation to enterocyte membranes in
the intestine.
Sources of cholesterol oxides in the diet are represented mainly by processed
animal-derived products including egg products, dairy products and meat products.
For example, fresh egg yolk contains negligible levels of cholesterol oxide
(Paniangvait et al., 1995), however, spray-dried egg yolk powder is a rich source
of cholesterol oxides. Similarly, fresh meat is almost free of cholesterol oxides
but cooked meat products are considered to be a substantial source of cholesterol
oxides in the diet (Savage et al., 2002). Cholesterol was oxidised in meat samples
during household cooking and the rate of oxidation differed according to the
cooking conditions (Pie et al., 1981). In general, cholesterol oxide consumption
could be substantial in many populations. For example, the average diet in the
Netherlands provides about 1 mg of 7--hydroxycholesterol and 0.5 mg of
cholesterol--epoxide per day (Van de Bovenkamp et al., 1988). It is necessary
to underline that certain oxidation products of cholesterol are carcinogenic
(Pearson et al., 1983).
Therefore, cholesterol oxides can be found in the diet and they are cytotoxic
and potentially can be involved in lipid peroxidation in the intestine.
IRON IONS
Iron is recognised as an essential nutrient and its reference nutrient intakes (RNI)
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in the UK are 8.7 mg/day for men and postmenopausal women and 14.8 mg/day
for premenopausal women with USA RDA being 8 mg/day and 18 mg/day
respectively (Kelly, 2002). However, iron absorption from a diverse diet has been
shown to be about 15% (Department of Health, 1991). The main problem with
iron nutrition is its reactivity and possible involvement in free radical generation.
Iron ions are considered to catalyse the formation of the hydroxyl radical and
accelerate the decomposition of lipid hydroperoxides (Davies and Slater, 1987;
Donovan and Menzel, 1978) and to stimulate lipid peroxidation (Braughler et al.,
1986, Minotti and Aust, 1987). In fact the potential risks of an excess intake of
iron, associated with an increase in free radical generation were first proposed
more than 40 years ago (Richmond, 1959). Numerous compounds, including
heme and nonheme iron, have been reported to act as catalysts of lipid oxidation
in food systems (Pearson et al., 1983). In fact muscle tissue is rich source of iron
bound to proteins in the form of myoglobin, Furthermore, meat can also contain
some hemoglobin residues from residual blood and iron-containing cytochromes.
Post-slaughter events such as early post-mortem pH drop, carcass temperature
and tenderising techniques such as electrical stimulation are involved in disruption
of cellular compartmentalisation and release of catalytic metal ions (Morrissey et
al., 1998). The next step of lipid peroxidation in the meat is associated with meat
handling, processing, storage and cooking. It is widely believed that during heat
treatment of the meat products nonheme iron is released from high molecular
weight sources such as haemoglobin, myoglobin, ferritin and haemosiderin
becoming the major pro-oxidant in cooked meat (Pearson et al., 1983; Morrissey
et al., 1998).
Iron fortification of various foods could represent another source of potentially
dangerous free iron in the digestive tract. Fortification of cereal products with Fe
has been in practice for a number of years throughout the developing world. In
fact thirty countries throughout the developing world now have a range of products
fortified with iron including wheat flour, maize flour, milk, rice and weaning foods
(Darnton-Hill and Nalubola, 2002). Ferrous sulfate was considered to be the first
choice for food fortification and used to fortify infant formula, bread and pasta
and wheat flour (Hurrell, 2002). At the same time this form of iron is the most
active one in promoting lipid peroxidation. Therefore, iron supplements could
also be involved in lipid peroxidation in the intestine. In fact, iron supplements
are commonly prescribed for pregnant women at doses ranging from 30 mg/d in
the United States to as high as 240 mg/d where prevalence of anemia is high
(Knutson et al., 2000). However, undesirable gastrointestinal side effects of iron
supplementation are high, suggesting iron-related oxidative stress in the intestine
(Hollan and Johansen 1993). For example, iron supplementation in relatively high
doses resulted in abnormal iron accumulation and increased lipid peroxidation in
rats (Knutson et al., 2000). Furthermore faecal iron available to the Fenton reaction
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HEAVY METALS
Agricultural uses of phosphate fertilizers and sewage sludge and industrial uses
of cadmium have been identified as a major cause of widespread dispersion of
the metal at trace levels into human foodstuffs. Approximately, 1/3 of cadmium
dietary intake is attributed to the ingestion of animal products, while plant products
provide the remaining 2/3 (Nasreddine and Parent-Massin, 2002). Recently it has
been calculated that daily cadmium intake to be 30 g being within the safe intake
level of 70 g per day recommended by WHO/FAO (Satarug et al., 2003). The
dietary intakes of lead for European countries vary between 16 g/day (Spain)
and 280 g/day (Italy) and intakes of arsenic and mercury comprise 38-286 g/
day and 0.7-13.5 g/day respectively (Nasreddine and Parent-Massin, 2002).
Mercury is a common contaminant of fish. For example, fish with a total mercury
content between 0.5 and 1.5 ppm include fresh and frozen tuna, swordfish and
shark. Mercury levels in freshwater fish varies, but in general bass, pike,
muskellunge and walleye have high levels (Wooltorton, 2002).
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It is well known that heavy metals can cause oxidative stress and stimulate
lipid peroxidation. For example, cadmium increased lipid peroxidation in liver,
kidney, and testes of rats and reduced metallothionein and total sulphydryl in
liver and kidney (Khandelwal et al., 2002). In renal tubular epithelial cells of rats
significant decrease in activities of GSH, GSH-Px, SOD and increased MDA
formation were observed as a result of the treatment with lead and cadmium (Wang
et al., 2002). In fact prooxidant effect of cadmium is much more complex.
Cadmium depletes GSH and protein-bound sulfhydryl groups, resulting in
enhanced production of reactive oxygen species such as superoxide, hydroxyl
radicals, and hydrogen peroxide (Stohs et al., 2001). The ROS production is
associated with lipid peroxidation, enhanced excretion of urinary lipid metabolites,
modulation of intracellular oxidized states, DNA damage, membrane damage,
altered gene expression, and apoptosis (Stohs et al., 2001). On the other hand,
reduced glutathione (GSH) and -tocopherol offer significant protection against
cadmium toxicity in rats by diminishing oxidative stress via raising GSH
concentration and reducing lipid peroxidation (Singh and Rana, 2002).
It is believed that lead can alter certain membrane bound enzymes and may
cause oxidative stress. For example, exposure of HepG2 cells to lead ions decreased
cell viability and stimulated lipid peroxidation of cell membranes decreasing the
fluidity in the polar surface of cell membranes (Chen et al., 2002). Levels of lipid
peroxidation products such as MDA, conjugated diene and hydroperoxide were
increased in liver, lung and kidney of lead-treated rats. Consumption of lead in
drinking water by rats imposed oxidative stress increasing lipid peroxidation in
peripheral blood mononuclear cells and liver (Ercal et al., 2000). Administration
of exogenous antioxidants in the lead treated animals significantly reduced the
prooxidant effect of the toxicant (Upasani et al., 2001). Mercury is also a strong
prooxidant able to increase lipid peroxidation and decrease GSH content in liver
of Swiss albino mice. In particular, mercury treatment enhanced lipid peroxidation
in kidney, testis and epididymus of rats (Mahboob et al., 2001). Mercury can
interact with other potential prooxidants. For example, mercury or selenite when
injected alone, did not alter hepatic or renal lipid peroxidation in mouse, however,
simultaneous exposure to these compounds significantly increased lipid
peroxidation (Farina et al., 2003).
Did you know that our food could contain a range of prooxidants in various concentrations, including oxidised lipids,
oxysterols, iron, nitrites, nitrates, heavy metals, persistent
organic pollutants and mycotoxins? A combination of those
compounds could promote lipid peroxidation in the gut.
Heavy metal concentrations in major food sources are quite low, however, in
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MYCOTOXINS
At least 25% of worlds grain production is contaminated with mycotoxins, which
are a worldwide problem (Fink-Gremmels, 1999). For example, aflatoxins are
considered to be unavoidable contaminants of food, since they cannot be prevented
or eliminated by current agricultural practice (Peraica and Domijan, 2001). The
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intake of aflatoxin M1 from milk was calculated to vary from 0.1 ng/person/day
in Africa up to 12 ng/person/day in Far Eastern countries (Creppy, 2002). Milk
and dairy products purchased at Egyptian markets and breast milk from lactating
mothers were analysed. Three of 15 cows milk samples were found positive for
AFM1 with mean value 6.3 mg/kg. Twenty percent of hard cheese samples
contained detectable levels of AFM1 and one sample from ten contained AFB1
and AFG1. For breast milk, two of ten samples were positive for AFM1 with mean
value 2.75 g/kg, while 3 of 10 samples were positive for OA (Ei-Sayed et al.,
2000). In Turkey, the incidence of AFM1 in cheese was 89.5% with the highest
concentration to be 810 ng/kg (Huseyin and Sonal, 2001). From fifty four samples
of fresh full cream and skimmed milk, powdered milk, yoghurt and infant formula
collected in Kuwait, 28% were contaminated with AFM1 with 6% being above
the maximum permissible limit of 0.2 g/l (Srivastava et al., 2001).
Therefore the chance of getting traces of mycotoxins in our diet are very high.
For example, the results of survey of 313 UK retail foods and 153 UK cereal
samples showed that ochratoxin A was detected in 25% samples with 27 samples
containing OA at concentrations above 4 ng/g (Atkins and Norman, 1998). OA
was found in a number of samples of feed and food from various countries with
its detection in human blood (Peraica and Domijan, 2001). In fact, OA can be
detected at levels greater than 0.1 ppb in more than 90% of human and swine
blood samples in central European countries (Petzinger and Weidenbach, 2002).
In fact, OA has been found in human blood samples in number of countries in
cool temperate areas of the Northern Hemisphere (Creppy, 2002). When blood
analyses were performed in Scandinavian blood donors the mean plasma levels
of OA was 0.18 g/l in Oslo and 0.21 g/l in Visby (Thuvander et al, 2001). In
particular in Croatia OA consumption was estimated to be 0.4 ng/kg body weight
(Peraica and Domijan, 2001). In the UK composite duplicate diet samples from
50 individuals and corresponding plasma and urine samples were obtained over
30 days. Average intake of OA varied in a range of 0.24-3.54 ng/kg body weight/
day and OA was detected in all plasma samples and in 92% of urine samples
(Gilbert et al., 2001). In Germany the absolute OA intake was approximately
28.7-290.8 ng/day in 1996-1999 and the calculated total daily intake of OA varied
between 0.9 ng/kg body weight in Germany and 4.6 ng/kg body weight in Italy
(Petzinger and Weidenbach, 2002). It is necessary to mention that OA altered
both barrier and absorption function of the intestinal epithelium causing intestinal
injuries, including inflammation and diarrhea (Maresca et al., 2001). Ochratoxin
in combination with aflatoxin showed a synergistic toxicity (Campbell et al., 1983).
For ochratoxin the elimination half-life in human was calculated to be 840 hours
(Creppy, 2002).
Consumption of mouldy sorghum or maize containing high levels of fumonisin
B1 caused an outbreak a human disease in India involving gastrointestinal
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ALCOHOL
Exposure of the intestinal mucosa to ethanol leads to morphological injuries and
impairs the absorptive capacity creating an oxidative stress in the small intestine
(Kaur et al., 1998). The diet seems to affect this process since enrichment of the
rat diet with fish oil increased the detrimental effect of alcohol. The in vivo formation
of the -hydroxyethyl free radical metabolite of ethanol has been demonstrated
(Knecht et al., 1990). Free radical formation in ethanol-treated rats has been
detected (Knecht et al., 1995). In the study of Reinke et al. (1997) ascorbyl radicals
and spin adducts of dietary alcohol or endogenous compounds, such as lipids,
were detected with higher frequency in bile from alcohol-fed rats than in
corresponding samples from rats fed control diets. Furthermore, formation of lipid
radicals was enhanced after acute alcohol administration. Liver microsomes from
alcohol-fed rats also metabolized ethanol to the 1-hydroxyethyl radical at higher
rates than controls. During ethanol metabolism the antioxidant system is
compromised, with reduced levels of vitamin E (Kawase et all, 1989), selenium
(Aaseth et al., 1980; Dworkin et al., 1985) and GSH (Trimble et al., 1993).
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IMMUNE SYSTEM
The immune system is considered to be an important source of ROS in the human
body (Surai, 2002) and intestinal immunity is not an exception. Indeed, the
intestinal tract is considered to represent the largest immune organ of the human
body responding to the challenge of bacteria or food antigens by production of
ROS (Halliwell et al., 2000). Mucosal surfaces covered by a layer of epithelial
cells represent the most critical interface between the organism and its environment
since the mucosal interstitia of the intestine is continuously exposed to large
amounts of dietary and microbial antigens. Therefore epithelial cells engage in
cross talk with luminal bacteria and their products and produce mediators and
signals that are key components of host innate and acquired mucosal immunity
(Maaser and Kagnoff, 2002). The mucosal immune system is a first line of defence
against foreign antigens, including microbial and dietary antigens and under
normal circumstances it employs tightly regulated dynamic mucosal intra- and
internets consisting of inductive (e.g. Peyers patch) and effector (e.g. intestinal
lamina propria) tissues and maintains an appropriate immunological homeostasis
between the host and mucosal environments (Kiyono et al., 2001). Therefore the
mucosal immune system has evolved efficient mechanisms to distinguish potentially
pathogenic from nonpathological antigens. For example, the mucosal immune
compartment must be able to choose the appropriate effector function (e.g.,
tolerance vs. clearance) necessary to deal with each encountered antigen whether
it be innocuous or pathogenic in nature (Laroux et al., 2001).
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VITAMIN E
Vitamin E is main biological chain-breaking antioxidant, found in food in the
form of 4 tocopherols and 4 tocotrienols. Biological activity of vitamin E in tissues
is mainly due to -tocopherol, but in food the main form of vitamin E is tocopherol. It is possible that the gut is a special place for -tocopherol and
tocotrienols to play their antioxidant role. Vitamin E is not stable and is easily
oxidized during food processing. Synthetic antioxidants added to the food can
inhibit vitamin E oxidation. Vitamin E (-tocopherol) in the tablet or capsular
form is mainly produced in the stable esterified form or as a mixture of tocopherols.
The major vitamin E sources in the diet are vegetable oils (wheat, soybean,
sunflower and corn) and some other plant-derived foods. For example, in middleaged Japanese, vitamin E was mainly of vegetable origin with main contributions
coming from spinach, safflower oil and pumpkin (Imaeda et al., 1999). In the
UK, the average daily vitamin E intake is 11.7 mg in men and 8.6 mg in women.
Similar consumption was reported in the USA and other countries (Weber et al,
1997) with margarines and mayonnaise supplying 23% of total vitamin E
consumed. These levels are in the line with the RDA. In fact the Food and Nutrition
Board of the Institute of Medicine recently published dietary reference intakes for
vitamin E, which is 15 mg for adults being 50% greater than the generous
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COENZYME Q
Coenzyme Q, known also as ubiquinone, was discovered in 1957. The name
ubiquinone is related to its ubiquitous presence in all cells and the name coenzyme
Q reflects the chemical structure of the compound containing one quinone group
and 10 isoprenyl units. These two names have been used interchangeably for
more than 40 years. Coenzyme Q10 (CoQ10) exists both in an oxidised and a reduced
form, ubiquinone and ubiquinol, respectively (Overvad et al., 1999). It was found
that the ratio of ubiquinol-10 to ubiquinone-10 was about 95/5 in human plasma
from healthy donors. A significant increase in the oxidized form (ubiquinone-10)
content was observed in plasmas of patients with hepatitis, cirrhosis, and hepatoma
when compared with normal subjects, reflecting increased oxidative stress in these
patients (Yamamoto and Yamashita, 1997). Therefore the ubiquinol/ubiquinone
ratio is considered to be a sensitive marker of oxidative stress and an altered
ubiquinol/ubiquinone ratio is the first sign of lipoprotein exposure to oxidative
stress (Lagendijk et al, 1997).
The highest concentrations of CoQ10 in human tissues are found in heart, liver
and kidney (70-110 g/g) and the lowest concentration (8 g/g) was detected in
the lung. In human plasma CoQ10 was found in the range of 0.75-1,00 g/ml with
the total content in the body to be about 1.0-1.5 g (for review see Overvad et al.,
1999). A statistically significant circadian rhythm in human plasma CoQ 10
concentration was demonstrated, but no differences were found in CoQ 10
concentration between male and female subjects. On the other hand, some racial
differences were demonstrated with lower plasma CoQ levels in Caucasian
compared to African subjects (Reis et al., 2002). Coenzyme Q is the only fatsoluble antioxidant synthesised in the body. Therefore, tissue CoQ originates from
endogenous synthesis and from food. In fact, there are two major forms of CoQ
in the human tissues namely CoQ10 comprising 95-97% in heart, kidney, liver
and spleen and 92% in brain and 87% in lung (Dallner and Sindelar, 2000), the
rest of this compound is represented by CoQ 9 . In accordance with CoQ 10
concentration human tissues can be placed in the following descending order:
muscle>>heart>kidney> liver>>spleen>>brain> lung (Dallner and Sindelar, 2000).
Contents of CoQ10 in foods varied from 157.9 g/g to below the detection limit
with meat, meat products (55%) and fish (9%) to be major sources of this
compound. Vegetable oils, dairy products, vegetables and fruits plus berries provide
18%, 8%, 6% and 4% of daily CoQ10 consumption respectively. In the same foods
CoQ9 is also found varying from 8.5 g/g (reindeer), 0.4 g/g (beef and chicken)
down to lower levels in other foods. Daily intake of CoQ10 was calculated to be
5.4 mg for men and 3.8 mg for women and CoQ9 intake was shown to be 0.6 mg/
day for men and 0.4 mg/day for women (Mattila and Kumpulainen, 2001). It is
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believed that CoQ is quite stable during food cooking loosing only 15-30% its
initial value (Weber et al., 1997).
CoQ10 is characterised by comparatively slow absorption in humans with blood
values reaching a maximum after 6 h following intake of 100 mg CoQ10 with a
secondary peak been shown in the blood 24 h after intake with half-life in the
plasma to be about 33 h (Overvad et al.,1999). Potentially, slow absorption of
CoQ could be an advantage for antioxidant protection in the GIT. Animal studies
showed that the total uptake of dietary CoQ supplement comprised only 2-3%
(Zhang et all, 1995). However, high consumption of CoQ10 is associated with a
substantial (2.8-fold) increased concentration in human plasma, but tissue response
was shown to be much less pronounced. In particular, only liver and spleen
responded to CoQ supplementation in rats (Dallner and Sindelar, 2000).
Administration of the labelled CoQ10 to rats intraperitoneally resulted in an efficient
uptake into the circulation, with high concentrations found in spleen, liver, and
white blood cells; lower concentrations in adrenals, ovaries, thymus, and heart;
and practically no uptake in kidney, muscle, and brain (Bentinger et al., 2003). In
liver homogenate most CoQ appeared in the organelles, but it was also present in
the cytosol and transport vesicles. In particular mitochondria had a very low
concentration of labelled CoQ, which was mainly present in the lysosomes. All
organs that took up the labelled lipid also contained water-soluble metabolites
(Bentinger et al., 2003).
In general, dietary supplementation of CoQ does not affect the endogenous
synthesis of CoQ in tissues. However, oxidative stress (physical exercise, thyroid
hormone treatment, cold adaptation, vitamin A deficiency, etc.) is associated with
increased CoQ synthesis reflecting a cellular adaptation (Ernster and Dallner, 1995).
Therefore, CoQ synthesis is considered to be an adaptive mechanism in response
to stress conditions when other antioxidants are depleted. For example, in vitamin
E and Se deficient rats CoQ concentration elevated and CoQ-dependent reductase
system is activated (Navarro et al., 1998).
It is believed that exogenous CoQ, protects cells from oxidative stress by
conversion into its reduced antioxidant form by cellular reductases. In particular
cytosolic NADPH-CoQ reductase is responsible for cellular CoQ redox cycle as
an endogenous antioxidant (Kishi et al., 1999). The plasma membrane
oxidoreductase and DT-diaphorase are two such systems, likewise, they are
overexposed under oxidative stress conditions (Genova et al., 2003). In addition,
the selenoenzyme thioredoxin reductase is an important ubiquinone reductase
and can explain how selenium and coenzyme Q, by a combined action, may
protect the cell from oxidative damage (Xia et al., 2003).
Possible mechanisms of action of dietary CoQ 10 administration on organ
function include (Dallner and Sindelar, 2000):
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CAROTENOIDS
Carotenoids comprise a family of more than 600 compounds responsible for a
variety of bright colours in fall leaves, flowers (narcissus, marigold), fruits
(pineapple, citrus fruits, paprika), vegetables (carrots, tomatoes), insects (ladybird),
bird plumage (flamingo, cock of the rock, ibis, canary) and marine animals
(crustaceans, salmon) (Pfander, 1992). These pigments provide different colours
from light yellow to dark red and when complexed with proteins they can produce
green and blue colorations (Ong and Tee, 1992). Yellow, orange and green fruits
and vegetables provide a range of carotenoids. -carotene, -carotene and cryptoxanthin are the major provitamin A carotenoids in human and lutein,
zeaxanthin and lycopene are major carotenoids in the diet which are not converted
to vitamin A. Biological functions of these natural pigments in relation to animals
or humans are not well defined but their antioxidant properties seem to be of
major importance. In mixture with other antioxidants they could be much more
effective than on their own, and the GIT could be a major place for these
compounds to exert their activity. In some conditions, carotenoids can be
prooxidants. However, it is well recognised that this possibility is not likely to be
the case in physiological conditions, including in the GIT when an array of other
antioxidants is present.
Approximately 40 carotenoids are commonly consumed in the U.S. diet and
approximately 20 can be detected in human serum and tissues (Cooper et al.,
1999). Most nutrition research was concentrated on the six carotenoids found in
the highest concentrations in human blood: -carotene, lycopene, -carotene,
lutein, zeaxanthin and -cryptoxanthin. The major dietary lutein sources in the
human diet are green vegetables and fruits, including spinach, squash, grapes
(Sommerburg et al, 1998), broccoli, parsley, peas etc. (Hart and Scott, 1995).
Carotenoid consumption and their serum profile vary substantially depending on
the origin of the population studied. For example France presents the highest
levels of serum lutein and -carotene and Spain shows the lowest level of carotene, along with the highest levels of -cryptoxanthin (Olmedilla et al, 1997).
American women consume approximately 6 mg of total carotenoids per day (Chug-
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Ahuja et al, 1993), the average daily intake of major carotenoids in Spanish
population is 3.5 mg/day (Olmedilla et al, 1997) and in Germany total carotenoid
intake amounts to 5.33 mg/day with average lutein intake being 1.91 mg/day
(Pelz et al, 1998). Daily consumption of lutein and zeaxanthin in American elderly
subjects was 2.7 mg for men and 3.09 mg for women (Tucker et al, 1999). In
general, the recommended daily intake of carotenoids can only be achieved by
consuming 100-200 g/day of vegetables and fruits with a particularly high
carotenoid content (Muller, 1996).
Low lutein consumption reflects low consumption of fresh vegetable and fruits,
changes in nutritional habits and use of highly processed food. According to
National Health Interview Surveys, the intake of lutein declined among different
categories of people in the USA between 1987 and 1992 (Nebeling et al, 1997).
There were also significant seasonal differences in plasma carotenoid
concentrations in the UK reflecting a higher intake of lutein during the spring
compared with summer and autumn (Scott et al, 1996). It is interesting to mention
that there is also a high positive correlation of lutein (r=0.889) between maternal
plasma concentrations and cord plasma (Yeum et al, 1998) indicating that the
nutritional status of mothers is the major determinant of the lutein status of their
babies. In addition it has been shown that breast milk is the major source of lutein
to the infants (Thurnham et al, 1997). An increased intake of another carotenoid,
-carotene, by lactating women increases the supply of milk -carotene available
to their breast-fed infants (Canfield et al, 1998).
In general carotenoids are not toxic for humans. There is no evidence that
conversion of -carotene to vitamin A contributes to vitamin A toxicity. Probably
this process is metabolically regulated. However, extremely high consumption of
carotenoids for a long time could cause hypercarotenemia. It can lead to yellowing
of the skin which eventually returns to the norm after carotenoid exclusion from
the diet. Diabetes mellitus, hypothyroidism, hypothalamic amenorrhoea, anorexia
nervosa, liver disease, and some other metabolic disorders can increase carotenoid
absorption, leading to hypercarotenemia (Dawson, 2000).
Carotenoid assimilation from the diet varies significantly depending on many
various conditions, however, it seems likely that a substantial proportion of ingested
carotenoids could be found in all segments of the digestive tract. Therefore, in
combination with other dietary antioxidants carotenoids could promote antioxidant
defence in the GIT. Furthermore carotenoid activities related to the promotion of
cell differentiation, regulation of cell proliferation and intracellular communication
via gap junctions, as well as regulation of the detoxifying enzymes and
enhancement of immune system (Surai, 2002) could also be of great importance
in the GIT.
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VITAMIN A
Vitamin A is a generic term combining all-trans-retinol or its esters (acetate,
palmitate, etc.), and all-trans-retinal. Most of food products are poor source of
vitamin A, however it can be efficiently synthesised in the GIT from carotenoids.
Vitamin A is present in foods mainly in the form of esters with long-chain fatty
acids. The richest food source of this vitamin is liver. Biological functions of
vitamin A are diverse (vision, testicular function, development, bone growth,
differentiation, hematopoiesis, pattern formation during embryogenesis, etc.), but
its antioxidant properties can be also of importance (Livrea et al., 1996) especially
in combination with other antioxidants in the GIT. Intake requirements were
calculated to amount to 1000 retinol equivalents (RE) for men, 800 RE for women
(Gerster, 1997). One RE is equal to 1 g retinol or 6 g -carotene. Unlike most
carotenoids, vitamin A and most retinoids are highly toxic when taken in excessive
amounts (Bates, 1995). The US NRC Committee on Dietary Allowances has
recommended that adults should not consume more than 7500 retinol equivalents
daily (Dawson, 2000).
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GLUTATHIONE
Glutathione (GSH) is the most abundant non-protein thiol in mammalian cells,
and is considered to be an active antioxidant in biological systems providing cells
with their reducing milieu. Cellular GSH plays a key role in many biological
processes and can be synthesised in the human body. GSH is abundantly distributed
in the mucosal cells of GIT in man and its highest concentration is found in the
duodenum (Loguercio and Pierro, 1999). Cellular GSH plays a key role in many
biological processes: the synthesis of DNA and proteins, including cell growth
and proliferation, regulation of programmed cell death, immune regulation, the
transport of amino acids, xenobiotic metabolism, redox-sensitive signal
transduction (Sen and Packer, 2000). Furthermore, GSH thiolic group can react
directly with H 2 O 2 , superoxide anion, hydroxyl radicals, alkoxyl radicals,
hydroperoxides (Lenzi et al., 2000; Meister and Anderson, 1983). Therefore, a
crucial role for GSH is as free radical scavenger, particularly effective against the
hydroxyl radical (Bains and Shaw, 1997), since there are no enzymatic defences
against this species of radical. Usually decreased GSH concentration in tissues is
associated with increased lipid peroxidation (Thompson et al., 1992). Furthermore
in stress conditions GSH prevents the loss of protein thiols and vitamin E
(Palamanda and Kehrer, 1993) and plays an important role as a key modulator of
cell signalling (Elliott and Koliwad, 1997). Animals and human are able to
synthesise glutathione. In addition to body synthetic activity food also provide
GSH.
FLAVONOIDS
Flavonoids are low molecular weight polyphenolic substances based on the flavan
nucleus. They are widespread in nature, occurring in all plant families, and are
found in considerable quantities in fruits, vegetables, grains, cola, tea, coffee,
cocoa, beer and red wine (Skibola and Smith, 2000). The list of flavonoids
substantially increased from more than 4000 in 1996 (Cook and Samman, 1996)
to over 8000 individual compounds known in 2000 (Pietta, 2000). The major
flavonoid classes include flavonols, flavones, flavanones, flavanols (catechins),
anthocyanidins, isoflavones, dihidroflavonols and chalcones (Cook and Samman,
1996). Representatives of major groups of flavonols were characterised as having
antioxidant properties in vitro and in vivo (Pietta, 2000; Bors et al., 1996; Cook
and Samman, 1996; Larson, 1988).
These compounds have received substantial attention in recent years. The major
driving forces of research in the field were positive effects of fruits and vegetables
on human health and their preventive role in the development of various diseases,
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especially cancers. The flavonoid content in fruits and vegetables can be as high
as 300 mg/kg fresh weight (Ishige et al., 2001). The major problem with
antioxidant properties of these compounds is their low availability from the dietary
sources. For example, in human blood or urine polyphenol concentrations was
shown to be in a range 1-2 M (Bell et al., 2000; Lapidot et al., 1998) in
comparison to the general concentration of antioxidants in human plasma to be
about 1000 M (Benzi and Strain, 1999). Therefore it has been suggested that the
digestive tract is the major site of antioxidant defence afforded by polyphenolic
compounds such as flavonoids (Kanner and Lapidort, 2001; Halliwell et al., 2000).
Daily intake of flavonoids varies substantially between different countries and
in Asian population and in vegetarians it is the highest. In particular, average
daily intake of flavonols in Asian countries comprised about 68 mg and isoflavones
20-240 mg (Skibola and Smith, 2000). In contrast the mean intake of flavonols of
the German population was about 11.5 mg, mainly derived from fruits and
vegetables, but also from black tea and red wine (Bohm et al., 1998). Indeed,
naturally occurring polyphenolic compounds may play a role in the protective
effects of fruits and vegetables against cancers in general, and they appear to
have considerable potential as chemopreventive agents against neoplastic changes
in the alimentary tract (Gee and Johnson, 2001). In general flavonoids can prevent
LDL oxidative modification by scavenging ROS, chelating transition metal ions
or inhibiting lipoxygenase and this leads to the prevention of atherosclerosis. For
example, a number of studies have shown that consumption of soy is
antiatherogenic and that the isoflavones genistein, diadzein and biochanin, which
inhibit lipoprotein oxidation in vitro and suppress formation of plasma lipid
oxidation products in vivo, are most likely responsible for this effect (Patel et al.,
2001).
Did you know that flavonoids and other phenolics could provide
efficient antioxidant protection in the lower gut due to their
comparatively low efficiency of absorption in the small intestine?
However, there are no data available on the long-term effects of flavonoid dietary
supplementation on humans. A serious problem with flavonoids is that, depending
on conditions, they could be antioxidants or prooxidants, antimutagens or
promutagens. Therefore unregulated use of flavonoid-containing supplements
can have a detrimental effect on human health. For example, the results obtained
by Silva et al. (2000) suggest that there is a range of flavonols whose genotoxicity
in eukaryotic cells depends on their autooxidation. These flavonols can autooxidize
when the pH value is slightly alkaline, such as in the intestine, and therefore can
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OTHER POLY(PHENOLICS)
Cereal brans contain significant quantities of the phenolic ferulic acid and diferulic
acid and their potential health benefits (protection of LDL from oxidative
modification and reduction in atherogenesis as well as inhibitory effects on tumor
promotion and chemopreventive properties) have been related mostly to their
antioxidant activity (Andreasen et al., 2001).
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SELENOMETHIONINE
In most diets, the main food sources of Se are cereals, meats and fish (Combs,
2001). Selenomethionine (SeMet) represents the major natural form of selenium
in feed and food ingredients. It seems likely that SeMet has specific functions on
its own beyond being an important source of Se for selenoprotein synthesis. In
fact, there is strong evidence that this compound itself can be considered as an
antioxidant (for review Surai, 2002). Therefore it is most likely that SeMet can
contribute to the total antioxidant potential of the GIT.
SYNTHETIC ANTIOXIDANTS
Antioxidants in foods may be endogenous origin or may be added externally to
preserve their lipid components from peroxidation. Synthetic antioxidants such
as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl
gallate (PG) and tert-butylhydroquinone (TBHQ) are commonly used in food
formulations. However, due to safety concerns, public interest shifted from
synthetic to natural antioxidants. As a result mixed tocopherols, herbal extracts
such as those of rosemary and sage, as well as tea extracts have been
commercialised for food and nutraceutical applications (Shahidi, 2000).
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protective effects of HSP are seen in heat stress, infection, and inflammation
(Malago et al., 2002). Similarly, glucocorticoid protection of rat intestinal cells
against oxidant-induced stress was mediated by HSP72 (Urayama et al., 1998).
The molecular mechanisms of heat shock response-induced cytoprotection
are beyond this review. However, they involve inhibition of proinflammatory
cytokine production and induction of cellular proliferation for restitution of the
damaged epithelium (Malago et al., 2002). HSPs play an important role in gastric
mucosal defence under conditions of stress. For example, exposure of rats to
restraint and water-immersion stress caused rapid HSP70 mRNA expression and
HSP70 accumulation in gastric mucosa and the extent of HSP70 induction inversely
correlated to the severity of mucosal damage (Rokutan, 1999). Therefore HSP70
is involved in repair of partially damaged proteins and substantially contributes to
protection of the gastrointestinal mucosa against various necrotising factors
(Tsukimi and Okabe, 2001).
Heme oxygenase (HO-1), known as HSP 32, can be induced by various stresses.
It has been shown that HO-1 induction and the maintenance of its appropriate
activity is critical in protecting the intestinal epithelial cells from oxidative injury
(Fujii et al., 2003). It is interesting that in the aforementioned experiment HO-1
was markedly induced following LPS treatment in the mucosal epithelial cells in
the upper intestine (duodenum and jejunum) but not in the lower intestine (ileum
and colon). It seems likely, that there is a delicate interaction between HSPs and
other antioxidant defence mechanisms to maintain mucosal integrity and repair
of acute mucosal damage.
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protecting the plasma vitamin E in high cholesterol-fed rabbits (Jeon et al., 2001).
In addition, synergistic effect of different antioxidants is also of great importance.
For example, lycopene acts synergistically, as an effective antioxidant against
LDL oxidation, with several natural antioxidants such as vitamin E, the flavonoid
glabridin, the phenolics rosmarinic acid and carnosic acid, and garlic (Fuhrman
et al., 2000). Similarly synergistic interactions between isoflavones and ascorbic
acid have been shown (Patel et al., 2001). It is believed that rat intestine and
mesenteric lymph possess efficient antioxidant defences against preformed lipid
hydroperoxides and (peroxyl) radical mediated lipid oxidation (Mohr et al., 1999).
It was also shown that supplementation of vitamin E alone or in combination
with ascorbic acid protects the GIT of Fe-deficient rats against Fe-mediated
oxidative damage during Fe repletion (Srigiridhar and Nair, 2000). Since flavonoids
are consumed in concentrations usually much higher than other antioxidant
compounds, their protective effect during digestion is of great importance. For
example, recently it has been shown that plant antioxidants such as flavonoids
not only prevented an accumulation of peroxidized lipids but also could switch
prooxidant properties of heme-proteins to antioxidant ones (Kanner and Lapidot,
2001). Dietary polyphenols can also modulate in vivo oxidative damage in the
gastrointestinal tract of rodents (Giovannelli et al., 2000) supporting the hypothesis
that dietary polyphenols might have both a protective and a therapeutic potential
in oxidative damage-related pathologies.
The dietary concentration of linoleic acid significantly affected oxidation of
pig jejunal mucosa and vitamin E has a protective effect. In particular, a histological
study of the jejunal mucosa of pigs showed lower cell desquamation in groups
supplemented with -tocopheryl acetate and a higher cell desquamation was found
in the groups fed diets containing the higher concentration of linoleic acid (Lopez
Bote et al., 2001). In the same study in vitro-induced oxidation of jejunal mucosa
homogenates was lower in pigs fed diets supplemented with -tocopheryl acetate.
Vitamin E also protects the rat colon from oxidative stress associated with
inflammation. In fact, vitamin E supplementation, which resulted in increased
colonic vitamin E levels, reduced colonic weight and damage score, prevented
lipid peroxidation and diarrhea, reduced interleukin-1 beta levels and preserved
glutathione reductase activity and total glutathione levels (Gonzalez et al., 2001)
There are other inducable antioxidant enzymes in the intestine. For example,
feeding mice with BHA induces phase II detoxifying enzymes and intestinal and
hepatic peroxiredoxin I, a stress-inducible antioxidant, in a manner similar to the
induction of glutathione S-transferases (GST, Ishii et al., 2000) and it was suggested
that the induction of this antioxidant may be important to protect the cells and
tissues against toxic electrophiles and reactive oxygen species. Similarly, in small
intestine GST activity changed in response to the different fatty acid supplemented
diets and increased as a result of the elevated oxidative stress (Giron et al., 1999).
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There are other antioxidant applications in relation to GIT health. For example,
irradiation caused increased lipid peroxide and decreased GSH levels in the
intestine. Intestinal SOD and GSH-Px activities were increased, but GST activity
decreased following irradiation of rats. Selenium and/or vitamin E pre-treatments
ameliorated these disturbances in prooxidant-antioxidant balance in the GIT
(Mutlu-Turkoglu et al., 2000). This amelioration has been confirmed with
histopathological findings. Common mucosal immune responses in orally
immunized aged mice were depressed and dietary supplementation with vitamin
E restored their mucosal and systemic humoral immune responses to mature adult
levels (Enioutina et al., 2000).
When the antioxidant system of the GIT is compromised, various diseases can
be observed. For example, in rats at weaning it was shown that early chronic
diarrhea and severe protein-energy malnutrition impair the antioxidant defence
system in both the small and large intestine (Nieto et al., 2000). Similarly, ulcerative
colitis in rats induced by trinitrobenzenesulfonic acid damages the intestinal mucosa
and is accompanied by a shift in the antioxidant enzyme activities, and low levels
of glutathione (Nieto et al., 2000a).
Indeed, the antioxidant-prooxidant balance in various parts of the intestine
would ultimately depend on the level of antioxidants and prooxidants provided
with the diet and released by cells themselves as well as on the level of absorption
of both antioxidants and prooxidants. Therefore in the stomach the conditions are
favourable for lipid peroxidation since major antioxidants and prooxidants are
there before absorption or major metabolism, and the pH is also favourable (Kanner
and Lapidot, 2001). In the small intestine this balance will be more variable since
many antioxidants, mainly vitamins E, A, C and carotenoids will be effectively
absorbed. However efficiency of absorption of iron is quite low (Department of
Health, 1991) therefore it will be available for stimulation of peroxidation. On the
other hand, after lipid absorption, concentrations of substrates of peroxidation
will be substantially decreased. Furthermore, various flavonoids will also be
available for antioxidant defence. Finally, in the colon/rectum, levels of vitamin
E, A, C are low, although various flavonoids and some carotenoids are present
(Halliwell et al., 2000) as well as iron, but the lipid concentration would be low.
Therefore in each part of the digestive tract there is a possibility of oxidative
stress and damage to various biological molecules. Indeed, it seems likely that
the protective effects of vegetables and fruits against development of various
cancers, especially cancers related to the digestive tract, are based on provision
of a variety of antioxidants which are not always well absorbed, thus providing
antioxidant protection in the large intestine and preventing oxidative damage and
possible mutagenic effect. It is possible to suggest that there is a reason for some
antioxidants not to be absorbed completely and in that way providing antioxidant
protection in lower parts of the intestine. For example, tocotrienols are common
compounds of major food and feed ingredients and possess high antioxidant
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activity (Surai, 2002). It is well appreciated that they are not well absorbed from
the food and feed and would be found in the digesta in colon providing antioxidant
protection in the lower intestine. However, this idea needs further investigation.
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Table 13.2 Healthy meals via antioxidant enrichment and decreased lipid peroxidation (adapted from
Surai et al., 2004).
Meat
Egg
Fish
+++
+++
+
+++
+++
+++
+++
+
+++
Food preparation
Cooking oil
Boiling vs frying
Spices and herbs
Olive
+
+++
Olive
+++
+++
Olive
+
+++
Food serving
With vegetables
+++
+++
+++
Meal composition
Fruit
Fruit juice
Red wine
Tea
+++
+++
+++
+++
+++
+++
+
+++
+++
+++
+
+++
Technological improvement
at the producer level
Vitamin E enrichment
Selenium enrichment
Carotenoid enrichment
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To choose olive oil for frying food. This will decrease accumulation of
oxidation products (Quiles et al., 2002) and will be beneficial in terms of
decreasing omega-6 PUFA consumption and increasing MUFA consumption
in accordance with recent health-related findings (Tuck and Hayball, 2002).
Rapeseed oil enriched with tocopherols is also an important choice.
To use more spices and herbs during cooking. This will decrease oxidation
and prevent accumulation of the lipid peroxides.
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Therefore, since we cannot avoid pro-oxidants in our food we need to make sure
that they are compensated by consumption of increased levels of natural
antioxidants. For this reason, it would be advantage if our meat and fish meals are
served with plenty of vegetables. Various sauces (e.g. tomato sauce) could provide
additional antioxidants. Red wine could also add additional flavonoids as a source
of antioxidants. Various juices are also good sources of natural antioxidants as
well as fruits. If a meal is finished with tea, this will also add to antioxidant potential
of the digesta.
Did you know that simple changes in our diet and ways of
the food preparation could help in preventing prooxidant
formation in the digestive tract?
All these suggestions are in a line of traditional meals serving in various countries
of the worlds. Antioxidants compensate prooxidants and a positive balance in the
digestive tract is the first step to healthy life. All the future exists in the past.
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Conclusions
Recent achievements in biochemistry and molecular biology, together with
epidemiological data have changed our thinking about food. It became
increasingly clear that our diet plays a pivotal role in maintenance of our health
and a disbalanced diet can cause serious health-related problems. It seems likely
that antioxidants are among the major regulators of many physiological processes
and therefore a balance between antioxidants and prooxidants in the diet, GIT,
plasma and tissues is an important determinant of the state of our health. Plants
consumed by humans contain thousands of phenolic compounds. Among them
the effects of dietary polyphenols are of great current interest due to their higher
consumption in comparison to other antioxidants such as vitamin E and their
antioxidant and possible anticarcinogenic activities. It could well be, that some
dietary constituents which are not well absorbed could have health-promoting
properties by maintaining antioxidant-prooxidant balance in the large intestine,
where concentration of other antioxidants (vitamin E, carotenoids, ascorbate) could
be low, but prooxidants (iron) and substrates of oxidation are still present. This
protective effect in the large intestine could be responsible for bowl cancer
prevention. Therefore, there could be a biological reason for some nutrients not
to be absorbed, but be involved in antioxidant protection in the lower gut.
Realising an importance of the food choice issue fundamental solutions for
food improvement should be found and production of functional food is one of
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14
CONCLUSIONS: LOOKING AHEAD
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also belongs to the second level of antioxidant defence, since it is responsible for
detoxification of lipid hydroperoxides formed as a result of reaction of lipid
peroxides with such antioxidants as vitamin E. Indeed, an optimal interaction
between vitamin E and GSH-Px provides an important mechanism of antioxidant
defence. Recently it has been shown that SeMet could affect activity of DNArepairing enzymes. This means that Se also belongs to the third level of antioxidant
defence. The understanding that all antioxidants in the body are working as a
team, called the antioxidant system, creates an additional interest in antioxidant
interactions. Indeed, vitamin E recycling is shown to be the most important
mechanism of the antioxidant defences. This means that if the recycling is effective,
even low levels of vitamin E could provide a substantial antioxidant protection.
For example, in the brain the vitamin E level is usually comparatively low and
levels of polyunsaturated fatty acids are high but in physiological conditions it is
very difficult to detect any products of peroxidation there. On the other side, if
the recycling is broken probably even high vitamin E supplementation would not
provide the adequate antioxidant protection. There are various additional
mechanisms of the antioxidant protection including redox signalling and changes
in gene expression, stress-protein synthesis as well as apoptosis. Recent
understanding of essentiality of free radicals in various physiological processes
shifted research to antioxidant-prooxidant balance rather than studying simply
the lipid peroxidation. For example, free radicals are synthesised in phagocyte
cells and used as a weapon to kill pathogens. This process is under strict metabolic
control, since an excess of the free radical production could escape from the
phagosome and damage various biological molecule including damages to
phagocytes themselves. It seems likely that some toxicants, such as mycotoxins
could affect this regulation causing an excessive free radical production and
creating an oxidative stress. In general, ingestion of excessive amounts of
antioxidants is presumed to shift the oxidant-antioxidant balance toward the
antioxidant side. This is supposed to be beneficial; however, this may also
adversely affect key physiological processes that are dependent on free radicals,
including prostaglandin production, cell division and differentiation. Free radicals
are important signalling molecules in spermatozoa capacitation, they are involved
in neurone communications, blood vessel tone maintenance and in some others
important physiological processes. Furthermore, recent evidence suggests that
cellular oxidation can induce changes in gene expression during normal
development. Conversely, antioxidants such as ascorbate, glutathione, tocopherol or carotenoids are inhibitory to differentiation in many types of cells.
New data coming from understanding important role of antioxidant-prooxidant
balance in gene expression and it seems likely, that antioxidants can affect gene
expression by mechanisms independent on their antioxidant activity. For example,
it has been demonstrated that GSH, in addition to its antioxidant and protective
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function against oxidative stress, has a specific signalling role in redox regulation.
Indeed, recently it has been shown that 2,016 genes regulated by H2O2 (Fratelli et
al., 2005). Of these, 215 genes showed GSH-dependent expression changes,
classifiable into four clusters displaying down- or up-regulation by H2O2, either
potentiated or inhibited by GSH depletion. In particular, the biological process
categories overrepresented in the largest cluster (genes whose up-regulation was
inhibited by GSH depletion) were NF-kappaB activation, transcription, and DNA
methylation. Furthermore, this cluster also included several cytokine and
chemokine ligands and receptors, the redox regulator thioredoxin interacting
protein, and the histone deacetylase sirtuin. The cluster of genes whose upregulation was potentiated by GSH depletion included two heat shock proteins
(HSP40 and HSP70) and the AP-1 transcription factor components Fos and FosB
(Fratelli et al., 2005).
In 1973 GSH-Px was shown to be a selenoprotein and antioxidant role of Se
was established. Since then a family of selenoproteins in human and animals was
shown to include at least 25 different selenoproteins. In fact, many of new
selenoproteins have been described only recently and therefore the knowledge of
their functions and regulation is far from being complete. Traditionally
selenoproteins have been considered to function as antioxidants. This is particularly
true for GSH-Px, but also for TR and MSR. In fact there are six different forms of
GSH-Px and they are located in different parts of the cell, in different tissues and
in some cases are differently regulated. It seems likely that this is an adaptive
mechanism to more effectively deal with free radical production. For example,
PH-GSH-Px is specifically located in membranes and is able to deal with lipid
peroxides directly without necessity to release them from membranes by
phospholipases. GI-GSH-Px is considered to be the most important defensive
mechanism against lipid hydroperoxide absorption. In fact, GI-GSH-Px destroys
lipid peroxides in GI tract preventing them from absorption. However, low GIGSH-Px activity could potentially fail to destroy all peroxides and some of them
can be absorbed and incorporated into lipoproteins. This incorporation could be
a triggering mechanism of the lipid peroxidation in LDL leading to changes related
to CVD development. The role of GI-GSH-Px in the maintenance of antioxidantprooxidant balance in the GI tract needs further investigation. Furthermore, sperm
nuclear GSH-Px is also of great importance for sperm quality maintenance. For
the question if GSH-Px is the main selenoprotein in the human and animals my
answer would be, probably not. A great body of information related to the role
and functions of GSH-Px accumulated for the last 30 years put this enzyme at the
top of the selenoprotein list. However, TR, described as a selenoprotein 23 years
later than GSH-Px and methionine sulfoxide reductase B (MSR-B), described as a
selenoprotein only in 2003 are going to change that view on the importance of
different selenoproteins. TR is involved in regulation of such processes as DNA
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The plant absorbs Se from the soil in the form of selenite or selenate and synthesises
selenoamino acids with SeMet representing more than 50% of the Se in cereal
grains and other feed ingredients. In many cases the variability in results of Se
specification investigations of plant material reflects analytical difficulties. Recently
strong cation-exchange chromatography (SCX-HPLC) was used in conjunction
with inductively coupled plasma mass spectrometry (ICP-MS) to investigate cationic
selenium species present in leaf extract of wild-type Brassica juncea supplemented
with selenite. Total amount of Se accumulated by the leaves was found to be 352
g/g (Yathavakilla et al., 2005). Major cationic selenium species identified in the
present study were methylselenomethionine and methylselenocysteine while SeMet
was found in minor quantities. By high performance liquid chromatography with
inductively coupled plasma mass spectrometry (HPLC-ICPMS) it was shown that
the main Se species in bulbs, leaves or flowers of the Se-enriched garlic, onions,
cabbage and ashitaba were selenate, Se-methylselenocysteine or gamma-glutamylSe-methylselenocysteine, while those in fruit bodies of the peppers and pumpkin
were selenomethionine bound to protein (Yoshida et al., 2005).
Recent advances in Se biochemistry have provided a deeper understanding of
the principal differences in metabolism of the two forms of Se namely inorganic
Se (sodium selenite or selenate) and organic Se (mainly SeMet). Selenite is taken
up by red blood cells within several minutes, reduced to selenide by glutathione,
and then transported to the plasma, bound selectively to albumin and transferred
to the liver (Suzuki and Ogra, 2002). Contrary to selenite, intact selenate is either
taken up directly by the liver or excreted into the urine. The chemical speciesspecific metabolic pathway for Se was explained by the metabolic regulation
through selenide as the assumed common intermediate for the inorganic and
organic Se sources and as the checkpoint metabolite between utilization for the
selenoprotein synthesis and methylation for the excretion of Se (Suzuki and Ogra,
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2002). In particular, organic Se, which can be found in grains, forages and other
feed ingredients, is primarily in the form of SeMet and is metabolised in the same
way as methionine. It is actively transported through intestinal membranes during
absorption and actively accumulated in such tissues as liver and muscle. It is well
known that methionine is not synthesised by animals or humans and therefore it
is an essential amino acid. The same is true for SeMet, which is not synthesised in
animals or humans and must be derived from feed sources. In fact SeMet is
considered to be a storage form of Se in the body. Indeed, when organic Se is
used in the diet, the Se reserve is built in muscles in the form of SeMet. These
reserves can be used in stress conditions when the Se requirement increases but
feed consumption decreased. The skeletal muscles are the major Se-storage organ,
accounting for about 46.9% of the total Se in the human body, while kidney
contains only 4% of Se reserves. In humans, whole body Se depends on the
regional location and varies from 3-6 mg up to 13-20 mg. In stress conditions
protein catabolism by proteasomes can release SeMet, which could serve as a
source of Se for newly synthesied selenoproteins, such as GSH-Px, thioredoxin
reductase and methionine sulphoxide reductase. Those enzymes can deal with
overproduction of free radicals and prevent decrease in productive and
reproductive performance of farm animals. It was proven that Se from both selenite
and SeMet is readily available for synthesis of the selenoenzyme GSH peroxidase
in rat tissues. There are also several lines of evidence confirming the idea that Se
accumulated in tissues in the form of SeMet can be available for selenoprotein
synthesis (see relevant references).
Studies in our laboratory indicated that chicks hatched from eggs enriched
with Se by means of using Se yeast had higher liver and muscle selenium and
GSH-Px activity not only at hatching, but more importantly, even at 21-28 days
posthatch (Surai, 2000; Surai et al., 2005; Pappas et al., 2005). This could be
explained by usage of SeMet accumulated in tissues as a result of Se transfer from
the egg during embryogenesis. In experiments with rats supplemented with high
doses of selenium in the form of selenite or SeMet it was shown that half-life of
decay of GSH-Px to be 4.2 and 9.1 days, respectively indicating that SeMet was
used for the GSH-Px synthesis at time of Se deprivation. Furthermore, in SeMet
or Se-yeast supplemented mice, liver GSH-Px activities declined more slowly
during Se depletion than in mice given selenite. Similarly, in human supplemented
with organic selenium, during Se deprivation the decline in the level of
selenoprotein P was slower than in individuals who had been supplemented with
selenite. When wheat and selanate were used as Se sources in a supplementation
study in Finnish men it was shown that once the supplements were withdrawn,
platelet GSH-Px activity declined less in the group given wheat Se. After several
weeks supplementation with high-Se bread, plasma Se remained elevated when
supplementation ceased. Furthermore, in Finish children Se-yeast provided a longer
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assimilated from feed and better accumulated in tissues. Indeed with the same
dose of supplementation organic selenium can deliver more element to the target
tissues and because of toxicity of high selenium levels this could be a solution to
avoid adverse effect of selenium overdose. If the body has selenium reserves in
the form of SeMet in muscles, during acute phase response it would be liberated
as a result of skeletal muscle protein catabolism by proteosome action and would
be used for re-synthesis of new selenoproteins which could decrease oxidative
stress. In chickens, first two weeks posthatch represent most important period of
immune system development and maternal diet is shown to have a profound
effect on this process. In particular, the first week of chick life is a period of rapid
expansion of leukocyte population, seeding of lymphoid organs and other events
ultimately leading to the production of unique clones of lymphocytes that will
mediate immunity in postnatal development. In this respect effects of various
combinations of natural antioxidants and n-3 PUFA await investigation. As a result
of antioxidant (selenium) deficiency increased oxidative stress of a host can lead
to increased virus mutation rate and change in a viral pathogen resulting in
emerging viral pathogens with new pathogenic properties. Therefore, Se deficiency
was associated with a change to the viral genotype, converting the virus from a
benign to a virulent strain. This possibility was not exploited in animal/poultry
production, but seems important to study more extensively. There is a need to
study antioxidant composition and fatty acid profile of immunocompetent tissues
depending on animal age and nutritional supplementation of antioxidants and n3 PUFAs.
The second important application of Se nutrition of animals and human is
related to its effect on reproduction. Animal and human spermatozoa are unique
in structure and chemical composition and are characterized by high proportions
of PUFAs in the phospholipid fraction of their membranes. This feature of these
highly specialized cells is a reflection of the specific needs of their membranes
for high levels of fluidity and flexibility, which are necessary for sperm motility
and fusion with the egg. This functional advantage conferred by PUFAs is, however,
associated with disadvantages in terms of the susceptibility of sperm to free radical
attack and lipid peroxidation. Therefore antioxidant protection is a vital element
in maintaining sperm membrane integrity, motility and fertilizing ability. It has
been suggested that natural antioxidants (vitamin E, ascorbic acid and glutathione)
together with antioxidant enzymes (superoxide dismutase and glutathione
peroxidase) build an integrated antioxidant system in semen capable of protecting
it against free radicals and toxic products of their metabolism. The delicate balance
between free radical production and antioxidant defense is considered to be an
important determinant of semen quality and in particular its fertilizing ability. The
essentiality of selenium for mail fertility was shown in the early 1980s. For many
years sperm capsular selenoprotein (SCS) was considered as the major protein
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expressed in spermatozoa. However, recently it has been establishes that, PHGSH-Px can be converted into structural selenoprotein and it is the protein which
was previously named SCS. The role of PH-GSH-Px as a structural protein may
explain the mechanical instability of the mitochondrial midpiece that is observed
in selenium deficiency. Recently a fifth member of Se-dependent glutathione
peroxidases: a specific sperm nuclei GSH-Px (sn-GSH-Px), has been characterized.
In particular, in Se-depleted rats the concentration of sn-GSH-Px decreased to a
third of the normal level and chromatin condensation was severely disturbed and
it seems likely that the main function of sn-GSH-Px is protamine thiol crosslinking during sperm maturation.
There are species-specific differences in the Se level in semen. For example,
the level of Se in seminal plasma was lowest in the human and the stallion, higher
in ram and boar, with the highest levels in the bull. Selenocysteine is shown to be
the main form of Se in rat sperm and selenocysteine and selenomethionine were
found in ovine sperm. It has been shown that Se concentration in chicken semen
depends on the Se in the diet and by inclusion of organic selenium in the diet it is
possible to significantly increase Se level in the spermatozoa (Pappas et al., 2005a).
GSH-Px in the sperm is considered to be the main enzyme, which removes
peroxides and thereby protects cells against damage caused by free radicals and
the products of lipid peroxidation in vivo.
There is species and tissue-specificity in GSH-Px expression. For example,
GSH-Px activity has been found to be expressed in the semen of several mammalian
species including ram, dog, human, goat, bull. It is interesting to note that in
bulls, for example, GSH-Px is exclusively associated with the seminal plasma
and not found in spermatozoa, while GSH-Px activity in seminal plasma was low
in man and ram and not detectable in boar and stallion. Furthermore, bull and ram
seminal plasma GSH-Px activities per mg protein were comparable, but when
expressed per ml seminal plasma, activity of the bull was more than 7 times the
activity of ram seminal plasma. It has been shown that approximately two thirds
of GSH-Px activity in bull semen was non-Se-GSH-Px. Among avian species, in
seminal plasma total GSH-Px activity was the highest in turkey and lowest in
duck and goose. In spermatozoa, on the other hand, the highest GSH-Px activities
were found for goose and duck and much lower GSH-Px activity was recorded
for guinea fowl, turkey or chicken. Recently, it has been shown that despite a
high proportion of PUFAs and a low level of vitamin E, duck spermatozoa have
the same susceptibility to lipid peroxidation as chicken spermatozoa. It has been
suggested that an increased activity of Se-GSH-Px in duck semen compensates
for the relatively low concentrations of other antioxidants. It was proven that
inclusion of Se in the chicken diet increased GSH-Px activity in semen and
improved the antioxidant defences. In boars it was shown that low Se diet had a
greater detrimental effect on semen quality that diets inadequate in vitamin E.
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Table 14.1 Effect of selenium on goose reproduction (Adapted from Tverdochlebov et al., 2005)
Items
Fertility, %
Hatchability of fertile eggs, %
Hatch of eggs set, %
Weight of day-old gosling, g
94.4
67.2
63.4
93.9
96.7
71.3
68.9
96.4
97.1
75.0
72.8
98.8
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importance for piglet growth and development. The data presented clearly showed
that only organic Se could solve this problem. Indeed, Se transfer via placenta, to
the colostrum and milk are shown to be much higher when organic selenium was
in the diet. Furthermore, usage of sodium selenite in the sows diet could potentially
have a detrimental effect on the level of splay leg and stillborn piglets. In contrast,
organic Se can improve the situation. Recently published data of the experiment
conducted in the commercial conditions in Iowa (USA) confirmed a positive effect
of organic selenium in the form of Sel-Plex on the sows and piglets (Tables 14.2
and 14.3). Substitution of inorganic selenium in diets fed commercial sows with
Sel-Plex (0.3 ppm added Se) resulted in more piglets weaned with lower preweaning mortality (9.76 vs 11.3%; Gourley et al., 2005). Furthermore, culls were
reduced in nursery pigs weaned from sows given organic selenium. Se
accumulation in the pig muscles as a result of organic Se inclusion into the diet
was a background information for the development of the technology for Sepork production.
Table 14.2 Effect of sow selenium source on litter parameters (Adapted from Gourley et al., 2005)
Item
Number of sows
Litter size, number
Total
Live
Stillbirths
Weaned
Pre-wean mortality, %
383
377
11.32
10.44
0.95
9.26
11.3
11.49
10.65
0.76
9.61
9.76
Table 14.3 Effect of sow and nursery diet selenium source on growth performance (Adapted from
Gourley et al., 2005)
Sow diet:
Selenite
Wean diet:
Number of pigs
Daily gain, g
Death, number
Culled, number
Total removal, %
Pig sold, number
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Sel-Plex
Selenite
Sel-Plex
Selenite
Sel-Plex
250
390
3
12
6.0
235
250
390
3
9
4.8
238
250
390
1
8
3.6
241
250
376
0
7
2.8
243
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systematic review and meta-analysis of the literature. Sixteen studies (eleven cohort
studies and five case-control studies) that examined the association between intake
of selenium and the risk of prostate cancer were included in the final analysis.
The results of the systematic review suggest that selenium intake may reduce the
risk of prostate cancer (Etminan et al., 2005). In fact, the pooled relative risk of
prostate cancer for any intake of selenium was 0.72 for cohort studies and 0.74
for case-control studies. The pooled relative risk of moderate intake was 0.74 for
cohort studies and 0.74 for case-control studies. Furthermore, prediagnostic
selenium concentrations measured in archived toenails were inversely associated
with bladder cancer risk in women (P for trend=0.02), but not in men, in a nested
case-control study of 338 cases and 341 matched controls (Michaud et al., 2005).
Similarly, a strong association of selenium with breast cancer in the Indian
population was shown. In particular a hospital based case-control study to examine
the associations of vitamin C, vitamin E and selenium with breast cancer was
conducted in India (Singh et al., 2005). One hundred and sixty breast cancer
patients and an equal number of normal healthy individuals constituted the study
population. The mean vitamin C, vitamin E and selenium levels were lower in
patients as compared to the controls. There was a 7% lower risk of breast cancer
if the level of selenium was increased by 1 unit.
Up to date there were 10 human trials to test protective effect of Se against
cancer and six of them were conducted in China, a country characterised by a
number of Se-deficient regions. The main outcome of the mentioned trials is a
protective effect of Se (in most cases Se-Yeast) against cancer. Recently, an
investigation evaluated the association between selenium supplementation and
prevalent and incident colorectal adenomas and colorectal cancers detected during
the Nutritional Prevention of Cancer trial follow-up. In addition to being associated
with a reduced risk of incident colorectal cancers, selenium supplementation was
associated with a significantly reduced risk of prevalent adenomas, but only among
subjects with either a low baseline selenium level or among current smokers (Reid
et al., 2005).
Aforementioned data provided a strong incentive to design a definite trial for
selenium and vitamin E with prostate cancer as a primary end point. Therefore
there are new human trials under way to further substantiate protective effects of
Se against cancer, including SELECT trial employing 32,000 participants without
evidence of prostate cancer from more that 400 participating study sites in the
USA, Puerto Rico and Canada. Further prostate cancer studies at the Arizona
Cancer Center and in Europe and Australia using Se doses of 200-800 g/d are in
progress.
There is a strong evidence to suggest a protective effect of Se supplementation
against side effects of cancer chemotherapy. Recently twenty patients have been
enrolled in the study. Each of the participants received 1 mg sodium selenite
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intravenously or orally daily for 3 wk during the pre-, intra-, and postoperative
period (Zimmermann et al., 2005). There was an inverse correlation between the
severity of the lymphedema and the whole-blood/plasma selenium concentration
and GSH-Px activity. In addition, a positive correlation between the ROS
concentration and the extent of lymphedema was observed. A significant reduction
of lymphedema occurred in the sodium selenite-treated group.
Molecular mechanisms of anticancer effect of Se are diverse and include
antioxidant effects of selenoproteins, stimulation of DNA-repair enzymes and
activation of the tumor suppressor protein p53, effect on gene expression,
enhancement of immune function, induction of apoptosis, inhibition of cell
proliferation, alteration of carcinogen metabolism, influence on estrogen and
androgen receptor expression, inhibition of angiogenesis, interactions with heavy
metals involving in cancer promotion and some others. Indeed, redox modification
of thiol/disulfide interchange in proteins by selenium could lead to protein
unfolding. Recent data supported the idea that unfolded protein response could
be an important mechanism in mediating the anticancer activity of selenium (Zu
et al., 2005) and endoplasmic reticulum stress response is shown to be important
in mediating the anticancer effect of selenium (Wu et al., 2005).
It has been shown that, ideally, increasing Se consumption to have Se
concentration in the blood >121 g/l would be a way to address Se deficiency
issues and simultaneously provide natural protection against cancer. It is not clear
at present how protective effect of increased Se concentrations in plasma happened.
It seems likely that in those people with the highest Se concentration in plasma
activity of GSH-Px is not much different from those who are in the lower Se
groups. The only difference would be the level of SeMet in the plasma. This
could also indicate higher Se reserves in the body of those patients. Therefore, it
could well be that the Se reserves in the body which could be protective in stress
conditions may play a crucial role in anti-cancer effects. Indeed, various stresses
are important in mutagenesis and further cancer initiation. Therefore, if SeMet
accumulated in tissues could be used for additional synthesis of selenoproteins,
this could provide an essential defence dealing with those stress conditions and
overproduction of free radicals leading to mutagenesis. Furthermore, SeMet per
se is shown to up-regulate DNA repairing enzymes increasing defences against
possible mutagenesis. Therefore, this could explain why those patients with
comparatively high Se in the blood and respectively high proportion of SeMet
(this could be indicative of Se reserves in the body) are more protected from
cancer than those with lower Se level in tissues and plasma.
In general, as mentioned above there are two approaches to achieve Se
protective effect against cancer. Firstly, nutritional approach including consumption
of Se-enriched food, such as eggs, meat, milk as well as various vegetables. This
could also include dietary supplements in the form of Se-yeast. There is a great
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Data are actively accumulating to indicate that from the one hand, Se deficiency
is related to reproductive disorders in human, including poor semen quality and
pregnancy complications and from the other hand, the Se dietary supplementation
could potentially have prevent those changes. Human and animal studies suggest
that the developing fetus is susceptible to metabolic programming throughout
gestation (Maloney and Rees, 2005). Although there are still controversial areas,
there is at present sufficient scientific evidence for foetal programming to be
regarded as an additional risk factor for chronic disease, in interaction with genetic
and lifestyle risk factors (Delisle, 2002). Indeed, evidence from both human and
animal studies suggests that diseases of adult life are induced by the fetal
environment (Haimov-Kochman, 2005). Evidence is also emerging that suggests
programming of hormonal systems in response to an adverse fetal environment
may be one of the mechanisms underlying these long-term consequences of early
life events. In particular, alterations in the neuroendocrine response to stress may
play an important part (Phillips, 2004). The available evidence suggest that nutrient
sensing regulatory systems are present in many tissues during early development
(Maloney and Rees, 2005). Programming agents seem to include growth factors,
cytokines and hormones, all of which can be altered by stress. As a consequence,
such stress-modified systems of the offspring are more susceptible to
environmental influences during later life, e.g. the development of atopic diseases
upon exposure to antigens (Knackstedt et al., 2005). It seems likely that oxidative
stress is also involved in programming. Indeed, oxidative stress may be a common
link underlying the superficial programming associations between adverse fetal
growth or preterm birth and elevated risks of certain chronic diseases. The
mechanisms of oxidative stress programming may be through directly modulating
gene expression or indirectly through the effects of certain oxidized molecules
(Luo et al., 2005). Experimental investigations have well demonstrated the role
of redox balance in modulating gene expression, and recent studies indicate that
both the insulin functional axis and blood pressure could be sensitive targets to
oxidative stress programming. For example, reduced lifespan in rats subject to
prenatal protein restriction is a consequence of enhanced oxidative processes
promoting apoptosis and loss of tissue function (Langley-Evans and Sculley, 2005).
Furthermore, maternal treatment with cholesterol-lowering agents or antioxidants
greatly reduces fetal and postnatal atherogenesis, indicating a pathogenic role of
lipid peroxidation and a potential involvement of oxidation-sensitive signaling
pathways (Palinski and Napoli, 2002). Taking into account that Se can affect
concentration of the above-mentioned programming agents, it could well be that
Se plays an important role in the foetal programming.
An optimal Se status is shown to be beneficial in asthma, rheumatoid arthritis,
diabetes, HIV, pancreatitis, brain and neurodegenerative disorders. It is also
protective against radiation and can be considered as an anti-ageing agent. For
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example, a recent study with rats has shown that a period of 5-week diabetes was
enough to cause some important and degenerative changes in the structure of the
bone tissues, and it has demonstrated that selenium treatment of the diabetic rats
could normalize these alterations (Ozdemir et al., 2005). Furthermore, it has been
shown that oxidative stress is involved in the etiology of diabetes-induced downregulation of heart function via depressed endogenous antioxidant defense
mechanisms and Se supplementation could prevent such detrimental changes
(Ayaz and Turan, 2005). Further clarification of the Se role in diabetes came from
the data indicating that diabetic ischemia increased neuronal death by augmenting
the production of reactive oxygen species, such as superoxide radical and by
suppressing the antioxidant selenoprotein P (Tagavilla and Li, 2005). Further
studies of effect of selenium on arthritis are related to gene expression. For
example, the effects of SeMet on gene expression, activation of mitogen-activating
kinases, and DNA binding of nuclear factor-kB (NF-kB) and apolipoprotein-1
(AP-1) in articular chondrocytes have been recently determined
(Andriamanalijaona et al., 2005). Pretreatment with 0.5 M SeMet prevented IL1beta-induced matrix metalloproteinases (MMP-1) and aggrecanase-1 expression,
and reduced the cytokine inhibitory effect on type II collagen, aggrecan core
protein, and transforming growth factor-beta receptor II mRNA levels. Therefore,
SeMet stimulated the signalling pathway. Effects of Se on pancreatitis are also of
great interest. For example, intravenous selenium given 24 hours after induction
of experimental acute pancreatitis was associated with a reduction in the histological
stigmata of pancreatic injury and a dramatic reduction in broncho-alveolar lavage
protein content (Hardman et al., 2005). To further examine the hypothesis on
relationship between Se status and optimal health the relationships between plasma
selenium and mortality in an elderly population was studied in the EVA (Etude du
Vieillissement Arteriel) study (Akbaraly et al., 2005). The EVA study was a 9year longitudinal study with 6 periods of follow-up. Baseline plasma selenium
was higher in individuals who were alive at the end of 9 year follow-up (1.10
mol/L) than in those who died during the follow-up (1.01 mol/L). Furthermore,
mortality rates were significantly higher in individuals with low selenium (relative
risk (RR) = 1.56). After various potential confounding factors were taken into
account, this association remained significant (RR = 1.54). When the underlying
causes of death were considered, an association with cancer-related mortality
(adjusted RR = 1.79) was found (Akbaraly et al., 2005). Indeed, it is clear that
more research should be done in an area related to health-promoting properties of
various Se compounds.
The analysis of current literature indicates that an enrichment of eggs, meat
and milk with Se is a valuable option to improve Se status of the general population.
Such eggs are currently being produced in more than in 25 countries worldwide
delivering approximately 50% RDA in Se with a single egg. There are also various
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Table 14.4 Selenium concentration (ng/g wet yolk) in egg yolks of free-living avian species (Adapted
from Pappas et al., 2005)
Common name
Scientific name
Country
Yolk Se (ng/g)
Gull
Moorhen
Black coot
American coot
Canada goose
American crow
House sparrow
Barn swallow
Tree swallow
Yellow headed
blackbird
Brewers blackbird
Blackbird
Song thrush
Starling
Larus fuscus
Gallinula chloropus
Fulica atra
Fulica americana
Branta canadensis
Corvus brachyrhynchos
Passer domesticus
Hirundo rustica
Tachycineta bicolour
Xanthocephalus
xanthocephalus
Euphagus cyanocephalus
Turdus merula
Turdus philomelos
Sturnus vulgaris
UK
UK
UK
Canada
Canada
Canada
Canada
Canada
Canada
744
430
394
503
732
1190
939
Canada
Canada
New Zealand
New Zealand
New Zealand
1033
1026
1081
989
1373
34
34
39
36
124
153
75
1896
2238
65
11
95
54
85
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Index 955
INDEX
955
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956
p-GSH-Px 73-74
PH-GSH-Px 67- 72
snGSH-Px 74-76
Role 76- 79
SOD 7, 9
Cu,Zn-SOD 10
Extra-cellular Cu,Zn-SOD 10
Fe-SOD 10
Mn-SOD 10
Antioxidant-prooxidant balance 487, 589, 599, 601, 857, 889, 891, 895, 897, 924, 948, 954
Antioxidant protection
Brain 325-329
Kidney 331
Liver 322-325
Lung 330
Muscle 331-332
Seminal plasma 415-422
Spermatozoa 404-415
Yolk 311-316
YSM 329-330
Antioxidant recycling 25, 87, 422
Antioxidant system 1, 7, 9, 14, 16, 23, 25, 27, 30, 101, 102, 106, 248, 258, 259, 279, 286,
303, 304, 324, 344, 347, 363, 368, 373, 377, 381, 383, 385, 392, 397, 400, 414, 422, 446,
451, 474, 540, 550, 595, 610, 615, 624, 734, 834, 838, 857, 865, 870, 878, 891, 923,
926, 930, 935
Apoptosis 18, 19, 25, 28, 33, 51, 61, 62, 64, 70, 71, 72, 77, 83, 86, 101, 108, 111, 112, 116,
250, 256, 258, 259, 318, 322, 342, 347, 373, 374, 492, 612, 613, 614, 700, 703, 707,
709, 710, 711, 713, 714, 715, 717, 718, 726, 732, 751, 862, 866, 870, 879, 888, 896, 924,
926, 942, 943, 944
HIV 735
Mycotoxin 326-330
Asbestosis 8
Ascaridia galli 244
Ascorbic acid
Antioxidant defence 13, 279, 286
Antioxidant properties 14-15
Cancer 676
Embryonic tissues 372-385
GIT 116, 857, 858, 882,890
Horses 596
Mycotoxins 325-326, 344
NE 373
Se absorption 183
Semen 279,286
Sodium selenite 112, 114, 192, 197
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Index 957
TR 87,107
Vitamin E recycling 23, 24, 25, 106, 372
Ascites 410, 423, 447
Aspergillus 317, 318, 319, 602
Asthenospermia 69
Ataxia 19, 96, 370, 617
Atherosclerosis 8, 644, 720, 721, 726, 727, 751, 818, 821, 884, 943
Autoimmune disease 8, 215, 259, 652
Barley 176, 177, 190, 296, 319, 320, 321, 322, 459, 554, 837
Beta-carotene 31, 601, 676, 697
Bile 384, 608, 617, 658, 669, 670, 678, 688, 864, 870
Bilirubin 25, 595
Bioavailability
Se 66, 154, 162, 163, 169, 171-181, 197, 478, 526, 527, 547, 548, 607, 611, 625, 627,
689, 813, 833, 837, 929
Brain
Antioxidant protection 25, 378
CoQ 876, 877
GSH-Px 57, 58, 59, 62, 69, 80, 183, 376, 380
ID 89, 90
Lipid peroxidation 372, 373, 377, 609
Mycotoxins 326
NE 106
Se 50, 166, 180, 390, 391, 419, 658, 739, 740
SeMet 164
SeP 95, 96, 97
SeW 92, 93, 94
TR 88
Broccoli 157, 158, 657, 689, 690, 692, 880
Brucella 233, 249, 332, 954
Bursa of Fabricius 88, 221, 234, 243, 334, 336, 341, 399, 410
Butylated hydroxytoluene (BHT) 886
Butylated hydroxyanisole (BHA) 373, 603, 886
Calcium 104, 154, 254, 365, 492, 528, 534, 537, 608, 614, 646, 670, 675, 680
Cancer 666-671
Se 671- 729
Carnosine 377
Carotenoids
Antioxidant system 7, 9, 13, 14, 26, 29, 102, 260
Chick embryonic development 29, 374, 375
Liver 376
Other tissues 377, 379, 380
Diet and food 650, 651, 809, 816, 827, 841, 857, 858, 880- 882, 894, 948
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958
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Index 959
Cystic fibrosis 665, 940
Cytokines 10, 106, 365, 539, 603, 944
Immunity 214-262, 712, 872, 930
Mycotoxins 339
Selenoproteins 65, 78
Cytotoxicity 11, 64, 65, 94, 111, 112, 113, 114, 712, 862
Immunity 216, 221-223, 237-241, 250
Mycotoxins 330-331, 343-344
Daidzein 650
Deficiency of
Selenium 250-252
Cattle 487-521
Diagnosis 534
Dogs 604
Fish 616
Horses 589, 604
Human 659-661, 665, 725-735, 740, 746, 749, 810, 825, 832, 842, 892, 923-932,
935-945
Pigs 446-455, 471, 476
Poultry 363-374, 394, 418
Vitamin E 23, 102, 104, 105, 106, 107, 229, 234, 237, 241, 256
Cat 604
Fish 617
Human 875
Pigs 446- 453
Poultry 364 - 372
Ruminants 488-506
Dehydroascorbic acid 14, 87
Delayed-type hypersensitivity (DTH) 222, 240, 254, 332, 334, 612, 613
Dendritic cells 214, 222
Deoxynivalenol, see Mycotoxins
Depression 652, 740, 741, 742
Diet
Hunter-gatherer 644-650
Ideal 644, 645, 650, 651, 754
Japanese 644-650
Mediterranean 644, 645, 646, 647, 648, 651
Paleolithic 645-650
Western 653, 654
Digestive system 151, 383, 419, 470, 646
Diketo-L-gulonic acid 24
Dioxine 30
DNA
Adduct formation 112, 324, 325,342, 343, 687, 688, 690, 691, 710,861, 870
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Damage 4, 5, 17, 18, 19, 61, 62, 111, 114, 115, 197, 320, 343, 649, 683, 708, 709, 710,
711, 861, 866, 868, 948
Repair system 17, 18, 19, 25, 115
Strand breaks 6, 17, 18, 31, 61, 111, 114, 326, 714, 715
Drip loss 115, 197, 408, 414, 422, 423, 473, 474, 478, 556, 560, 830, 831, 832, 936
Drosophila melangaster 46, 50, 68, 748
Duck 105, 106, 365, 366, 367, 369, 394, 400, 401, 411, 455, 604, 933
Embryo 384
Mycotoxins 319, 326
Sperm 279, 280, 284, 292, 293
Duodenum 163, 521, 883, 889
E coli 11, 83, 243, 338, 409
E tenella 243, 251
Egg, see Vitamin E, Carotenoids, Selenium
Eggs
Designer 817-827
Greek 843, 946
n-3 (Omega-3) designer 818-841
Super 818-841
Eicosanoids 214, 218, 248, 254, 257, 279, 371, 651, 873, 930
Embolism 652
Embryonic degeneration 105
Emphysema 8
Encephalomalacia 102, 105, 106, 321, 364, 367, 370-373, 385
Endoplasmic reticulum 84, 90, 97, 100, 101, 617, 942
Endotoxin 8, 62, 614
Environmental pollutants 858, 867, 868, 874, 896, 948
Enzymes
5I-apurinic endonuclease 17, 329
Aconitase 12
Cyclooxygenase 77
DNA glycosylase 17
DNA polymerase 17
Gastrointestinal GSH-Px 46, 50, 60, 74, 78, 107, 886, 887, 893, 925
Glucose-6 phosphate dehydrogenase 24, 286
Glutamic-oxalacetic transaminase (GOT) 449, 450
Glutathione reductase 9, 16, 23, 24, 53, 56, 57, 82, 85, 87, 165, 368, 595, 610, 887, 890
Glutathione-S-transferase 56, 79, 81, 82, 617, 715, 734, 861,890
Iodothyronine 5-deiodinase 46, 50, 60, 89-92, 177, 178, 470, 511, 541, 938
Lactate dehydrogenase (LDH) 71, 111, 286, 449, 490, 492, 742
5-lipoxigenase 254
Mieloperoxidase 228, 613, 872, 873
NADPH oxidase 28
Phospholipase A2 (PLA2) 82, 371
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Index 961
Phospholipid hydroperoxide GSH-Px (PH-GSH-Px) 46, 49, 50, 54, 59, 67-72, 75, 77, 78,
92, 96, 99, 107, 289, 304, 458, 531, 925, 933
Plasma GSH-Px 46, 73-74
Proline hydroxylase 12
Protein kinase 86, 714
Protein phosphatase 86
Ribonucleotide reductase 12, 51, 83, 86
Selenophosphate synthetase 47, 97
Specific sperm nuclei glutathione peroxidase 67, 74, 75, 289, 925, 933
Succinate dehydrogenase 12
Thioredoxin 9, 19, 21, 51, 82, 83, 84, 87, 96, 227, 290, 291
Thioredoxin peroxidase 9, 11, 83, 85
Thioredoxin reductase 9, 12, 14, 19, 21, 23, 50, 51, 82-89, 103, 1106, 107, 168, 253, 258,
374, 418, 716, 724, 857, 877, 887, 893, 928
Tyrosine hydroxylase 12
Xanthine oxidase 2, 10, 26, 65, 597
Epididymis 89, 409, 427, 433
Epilepsy 52, 81, 289, 290, 291, 296, 299
Erythrocyte 51, 52, 55, 56, 57, 80, 84, 93, 105, 165, 166, 169, 170, 172, 177, 179, 181, 182,
183, 230, 296, 299, 405, 409, 449, 452, 498, 531, 535-537, 547, 549, 557, 560, 592, 595,
603, 609, 624, 683, 742, 746, 813
Essential oils 26, 858, 885
Ethanol 8, 56, 57, 610, 738, 870, 871, 888
Ethoxyquin 26
Exudative diathesis 102, 104, 105, 172, 174, 183, 256, 364-366, 367, 385, 617
Faeces 162, 164, 180, 197, 448, 524, 525
Fatty acids
Arachidonic (20:4n-6) 5, 280, 282, 370, 371, 372, 375, 379, 380, 601, 645, 652, 727,
860
Docosatetraenoic acid (22:4n-6) 280, 282
Docosapentaenoic acid (DPA, 22:5n-3) 280, 282, 859
Eicosapentaenoic acid (EPA, 20:5n-3) 650, 652, 859, 860
Linoleic (LA, 18:2n-6) 370, 371, 372, 651, 727,825, 890
-Linolenic (ALA, LNA, 18:3n-3) 371, 372, 651, 654, 818, 825, 843, 860
Monounsaturated 648, 840
n-3 (omega-3) 615, 646, 651-654, 815, 818, 827, 835, 840-843, 946
n-6 (omega-6) 646, 651-654, 894
Polyunsaturated 4, 25, 78, 259, 279, 280, 341, 374, 375, 377, 397, 492, 546, 601, 616,
620, 649, 653, 660, 809, 817, 840, 924, 938
Saturated 4, 25, 399, 449, 646, 817, 819, 820, 840
Favism 8
Fenton reaction 3, 12, 863
Ferritin 12, 863
Fertility, see spermatozoa
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962
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Index 963
Glucomannan 344, 245
Glucose 24, 101, 183, 184, 192, 226, 609, 612, 649, 743, 872, 882
Glutaredoxin 9, 14, 52, 82
Glutathione
Antioxidant 15-16
In semen 286
Glutathione peroxidase, see Antioxidant enzymes
Glutathione reductase, see Enzymes
Guinea fowl 280, 292, 293, 933
Gull 417, 947
Haber-Weiss reaction 3
Haemagglutination inhibition (HI) 229
Haemoglobin 475, 504, 863
Haptoglobin 12
Hatchability 105, 302, 322, 323, 367- 370, 402-404,415, 416, 421-423, 935, 936
Heavy metals 8, 30, 96, 173, 177, 182, 602,709, 717-718, 725, 858, 865-867, 874, 888, 896,
942, 943, 948
Hemopexin 12
Hemorrhages 104, 365, 370, 448, 450
Heart
Antioxidants 380
Fatty acids 380
Heat shock protein 25, 410, 888, 925
Hemaglutination (HA) 222
Hepatotoxicity 319, 320, 752
Herbicides 30
Heterophils 30, 335, 399
Hormones 49, 89, 219, 287, 498, 538, 542, 558, 603, 752, 944
Hydrogen peroxide 2, 3, 6, 11, 53, 60, 72, 73, 76,77,80, 87, 110, 217, 228, 249, 290, 291,
329, 376, 546, 663, 866, 868, 879
Hydroperoxides 13, 14, 15, 31, 52, 53, 54, 69, 70, 73, 74, 79-82, 87, 102, 107, 115, 173,
254, 286, 288, 290, 859, 860, 883
Lipid hydroperoxides 12-13, 67, 7682, 96, 376, 709, 725, 859-863, 874, 890
5-hydroxyeicosatetraenoic acid 254
15-hydroxyeicosatetraenoic acid 372
Hypertension 651, 652, 720, 730
Hypochlorous acid 2, 15, 16,217, 613
Ileum 163, 889
Immune system 214-262
Immunity
Adaptive (acquires, specific) 214-262
Cell mediated 214-262
Humoral 214-262
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964
Natural 214-262
Immunoglobulin 214-262
Immunomodulation 51, 102, 262, 410, 608, 612, 718, 733, 754
Immunosuppression 213,225,259, 260, 261, 320, 323, 324, 330, 331, 340,341, 516, 707,
713, 931
Industrial solvents 2
Infertility 247, 263, 285, 289, 303, 488, 495, 505, 512, 729
Inflammation 2,3,8, 30, 83, 108, 216, 223, 245, 254, 255, 259, 488, 499, 504, 505, 506,
597, 608, 688, 703, 712, 724, 731, 732, 733, 869-873, 889,890, 896
Interferon (IFN) 20, 215, 253, 255, 256, 331, 340, 712
Intestine
Antioxidant-prooxidant balance 857-897
Interleukin (IL) 215, 253, 341
IL1 65, 218, 227, 261, 603, 790
IL2 237, 242, 335, 747
IL6 20, 218, 227
Iodothyronine deiodinases, see Enzymes
Iron 8, 55, 57, 155, 379,380, 449, 453, 468, 475, 476, 528, 546, 612, 621, 689, 812, 814,
815, 834, 862,-864, 866, 891, 896
Lipid peroxidation inducer 12, 260, 413, 422, 492, 615, 874, 882, 889, 931
Isoflavonoids 858
Jejunum 164, 521, 889
Kidney
Antioxidants 380-381
Fatty acids 380-381
Klebsiella pneumoniae 247
Lactoferrin 12
Lameness 247, 367, 475, 488, 493, 512, 591
Laying hens 105, 184, 365, 375, 387, 391, 404, 405, 412, 416, 417, 418, 419, 423, 821,
822, 835, 840, 843, 935, 936, 946
Leukocytes 3, 106, 215, 217, 224, 234, 235, 255, 365, 498, 499, 614, 705, 710, 730, 747,
872
Leukotrienes 76, 77, 217, 218, 254
Linseed oil 372, 451
Lipase 9, 17, 82, 384, 398, 473
Lipid classes
Phospholipid 23, 82, 727, 879
Chicken embryo 375, 377, 379, 380, 381
Egg yolk 817
Free fatty acids 817
Semen 280, 281, 282, 284, 285, 932
Triacylglycerol 280, 860
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Index 965
Lipid peroxidation 3, 4, 27-32 397, 399, 420, 421
Egg 416, 822, 823, 824
Embryo 378-385
GIT 615, 842, 859-874
Horses 597
Human 654, 724, 725, 726, 728, 730, 739
Immune system 241, 248, 250, 253
Initiation 4-5
Meat 411- 415, 832, 834, 835, 892
Mycotoxins 318-330
Petfood 603
Pig 451, 459, 473
Propagation 5
Ruminants 492
Sperm 279, 281-295
Stomach 891
Taurine 609, 610, 611
Lipofuscin 8, 491, 605
Lipoic acid 14, 23, 87
Lipopolysaccharide (LPS) 223, 227, 748
Lipoproteins
HDL 166, 819
Cholesterol 648, 722, 726, 727, 747
LDL 73, 166, 649, 725, 878
Cholesterol 648, 650, 726
Oxidation 62, 648, 721, 725, 726, 884, 885,890, 925, 948
Lymphocyte 18, 58, 113, 114,214,216, 219, 221-260 , 366, 399, 491, 501, 504, 597, 612,
613, 618, 705, 710, 735, 747
B 214, 215, 220-260, 930
T 214, 215, 220-260, 930
Mycotoxins 326- 342
Lymphocyte proliferation assay 223
Macrophages 4, 55, 214-228, 250- 261, 451, 501, 504, 613, 622, 747, 872, 873, 930
Mycotoxins 331-342
Macular degeneration 7, 8, 644, 665, 940
Maize 446, 863
Diet 389
Mycotoxins 318, 320, 321, 322, 323, 869
Malabsorption 213, 659, 842, 875
Malaria 8, 243
Malondialdehyde (MDA) 16, 822-824, 860-866
Chicken embryo 378, 396, 397
Human 730, 734, 737
Muscles 412, 413, 414, 835
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Mycotoxins 325-346
Semen 283-298
Taurine 609, 610, 611
Manganese 10
Mareks disease (MD) 243, 250, 410
Mast cells 215, 216, 222
Meat quality 26, 103, 107, 411-415, 473-476, 833-835
Membrane fluidity 233, 281, 337
Metallothionenin 717, 866
Metal-binding proteins 9, 12, 25, 924
Mieloperoxidase, see enzymes
Migration inhibitory factor (MIF) 236, 240, 249, 872
Mitochondria 63,70, 71, 112, 228, 286, 366, 368, 450, 451, 491, 492, 617, 660, 714, 739, 868
A source of free radicals 2, 26, 589, 923
CoQ 877, 878, 879
Electron transport chain 4
GSH-Px 52, 59, 60, 67, 68, 69, 72, 75, 289, 728, 933
MCS 98-99
MsrA 20
ROS 329, 861
Semen 296, 304
SOD 10, 857
TPx 85
TR 46, 86, 88, 89
Trx 83, 84
Mitogen 28, 86, 223, 225, 226, 233-243, 250, 260, 333,335, 336, 338, 339, 341, 612, 712,
714, 747, 931, 945
Molting 30
Moulds 213, 319
Multiple sclerosis 8
Mutation 11, 61, 100, 219, 251, 252, 262, 671, 708-713, 742, 744, 932
Mycosorb 344-347
Mycotoxicosis 107, 318, 330, 342, 346
Mycotoxins
Aflatoxin 230, 317, 318, 319, 322, 323, 327, 331, 332, 341, 343, 344, 362, 602, 604,
868-870
Aurofusarin 326-328
Deoxynivalenol (DON) 317, 318, 320-344, 870
Fumonisins 317-347
Ochratoxin A 317 347, 602, 869
Patulin 870
T-2 toxin 317, 318, 320, 323-331, 337, 341, 344, 345-347, 870
Trichothecenes 318, 320, 331, 340
Zearalenone 317-347, 870
Myoglobin 10, 12, 863, 864
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Index 967
Natural killer cells (NK cells) 214, 216, 221, 222, 239, 253, 259, 260, 931
Neutrophils 15, 214-262, 338-339, 383, 499, 501-508, 614, 873, 930
Newcastle disease/virus (NDV) 229, 230
Nitric oxide 2, 6, 28, 101, 217, 255, 337, 726, 872, 973, 887
Nitrogen dioxide 2, 865
3-nitrotyrosine 6
Nitrous acid 2
Oats 175, 176, 186, 190, 225, 236
Mycotoxin 319, 322
Ochratoxin A, see Mycotoxins
Ocular haemorrhage 8
Oedema 104, 321, 364, 490, 741
Oestrogens 650
Oligoasthenospermia 69
Olive oil 114, 644-648, 894
Oxidative stress 10-11, 20-32, 56, 62-65, 72-116,252-262, 289, 320, 341, 345, 372-383,
409, 445-470, 495, 539, 589, 595, 597, 602-627, 649, 708, 730, 731, 733, 734-750, 859891, 924, 925, 930, 932, 944
3-oxohistidine 6
Oxidised fat 859
Oxygen 1, 4-25
Radicals 382
Ozone 2, 56, 342
Pancreas 105, 164, 170-175, 395, 448, 461-473, 612, 658, 667-687
Pancreatic atrophy 184, 366, 367, 373
Parkinsons disease 8, 82, 739
Penicilium species 317-319
Perforins 221
Peritoneal exudate cells 240, 333
Peroxidability Index 280
Peroxidation, see lipid peroxidation
Peroxinitrite 15, 54, 217
Peroxisomes 2, 11, 59
Peroxyl radical, see free radicals
Pesticides 15, 30-31, 867-868
Phagosome 217, 218, 257, 340, 924
Phenolic acids 858
Phospholipids, see lipid fractions
Phosphatidylcholine 285, 379, 381, 879
hydroperoxide 54, 71, 73, 74, 82
Phosphatidylinositol (PI) 285
Phosphatidylserine (PS) 285
Phosphatidylethanolamine (PE) 285
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968
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Index 969
Availability 49, 87, 88, 154, 155, 172, 174, 175, 182, 191, 287, 405, 446, 527, 528, 538,
606, 626, 627, 824, 833, 837, 843, 926, 929, 938
Brain function 738-741
Cancer 671-729
Case-control studies 682-682
Epidemiology 672-682
Human intervention trials 694-707
Laboratory animal studies 687- 693
Molecular mechanisms 708-720
Secondary lymphedema 704-705
Treatment 705-708
Cow
Practical application of Se nutrition 538-546
Route of Se supplementation 537-538
Se-yeast vs selenite 546-559
CVD 720-729
Deficiency
Chickens 91, 183, 364- 374
Cow 244, 487, 491-559
Dogs 604
Fish 616
Goats 236
Horse 589
Human 250, 659, 660-661
Mouse 22, 100, 237
Pig 446-454
Rats 89, 180
Diabetes 733-736
Egg freshness 415-424
Enriched eggs 153, 390, 418, 424, 754, 809-843
Feathering 407, 414, 423
Human health 665-753
Immunomodualtion 213-262
Metabolism
Cats and dogs 606
Effect of mycotoxins 345
Horses 593
Pigs 471
Plants 155, 161
Ruminants 521-526
Mood enhancement 738-741
Mycotoxins 317-347
Pancreatitis 736-738
Radiation 741-746
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970
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Index 971
Selenosis, see Se toxicity
Sel-Plex 88, 151, 168, 194, 197, 259, 301, 346, 388-424, 461-478,528-561
Seminal plasma
GSH-Px 291, 292, 293, 297, 299, 933
Se 287, 291, 297, 298, 729, 933
Selenoproteins 303
Taurine 612
Sheep red blood cells (SRBC) 222, 229, 332, 334, 336-339
Signal transduction 15, 19, 20, 28, 33, 76, 77, 83, 255, 303, 714, 751, 878, 883
Singlet oxygen 2, 15, 71, 217
Soya 174, 446, 645, 649
Soybean meal 173, 174, 190, 225, 403, 446, 450, 460, 471, 626
Soybean oil 653
Sperm
Abnormality 284
Axoneme 286, 291
Capacitation 28, 303, 608, 924
Freezing 286, 304
Midpiece 68, 69, 75, 99, 284, 288, 289, 296, 301, 302, 933
Sperm-oocyte fusion 286
Spermatozoa 68, 279
Antioxidants 286
Avian 280
Boar 295-298, 299, 449
Bull 284
Chicken 280, 301-303
Dogs 280
Duck 280
Fertilizing capacity 28, 103, 304
GSH-Px 292, 294
Guinea fowl 292
Human 281, 284, 729
Lipid peroxidation 284, 286
Mammalian 281
PH-GSH-Px 68, 69, 77, 99, 289
Rat 299
Se 933
Selenoproteins 286
Turkey 284, 285
Sperm storage tubules (SST) 303
Spinach 867, 874, 880
Spleen 12, 80, 84, 88, 93, 164, 166, 170, 226-241, 323-339, 490, 658, 747, 876, 877
Splenocytes 335, 337, 742
Stomach 448, 450, 615, 667-696, 752, 858-892
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Index 973
Urine 163, 164, 167, 171, 173, 180, 185, 197, 334, 340, 472, 524-526, 594, 685, 869, 878,
884, 927, 929, 939
Uterus 74, 90-96, 104, 322, 454, 494, 498-501, 514
Vaccination 30, 220-249, 332, 334, 383, 399, 614, 713, 748
Vaccine 30, 221, 229-250, 334, 488, 612, 613, 748
Vegetables 185-190
Human health 644-655, 720, 811-895, 942-948
Oils 653, 874, 876
Vero cells 326
Viability
Calf 487, 540, 559
Cell 62, 86, 111, 114, 194, 242, 610, 866
Chicks 370, 385, 422, 935
Embryo 385, 397, 398
Fish 627
Foal 597
Lymphocyte 234, 237
Macrophage 225, 228
Piglet 477
Spermatozoa 103, 283-298, 729
Vitamin E 58
Antioxidant 13, 23
Antioxidant properties 70-77
Companion animals 601-615
Cow 488-559
Deficiency 104-106, 364-374,
Egg yolk 312-316, 393, 818-827, 841-842, 893
Embryo 378-385
Excess 102
Fish 616- 627
Gene expression 108
GIT 874- 875
Horses 589-599
Human 648-753
Diet 816-817
Immunity 224-262
Meat 832-836, 892
Mycotoxin 326, 343-347
Pig 449-478
Recycling 14, 23,24, 83, 87, 102
Se interactions 103-109, 446
Spermatozoa 292-305
Stress 27-33
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