Assessing Quality of Novel Plant Proteins For Salmonids Book

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Assessing Quality of Novel Plant Proteins for

Salmonids
THESIS JANUARY 2012

DOI: 10.13140/RG.2.1.1688.0167
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1 AUTHOR:
M A Kabir Chowdhury
University of Guelph
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Assessing Quality of Novel Plant Proteins for Salmonids
by
M A Kabir Chowdhury
A Thesis
Presented to
The University of Guelph
In partial fulfilment of requirements

for the degree of
Doctor of Philosophy
in
Animal and Poultry Science
Guelph, Ontario, Canada
M A Kabir Chowdhury, January, 2012

ABSTRACT
ASSESSING QUALITY OF NOVEL PLANT PROTEINS FOR SALMONIDS
M A Kabir Chowdhury Advisor:

University of Guelph, 2012 Professor D. P Bureau
Approaches for the evaluation of plant protein ingredients for salmonid feeds were
investigated in a series of four trials. The first trial compared the apparent digestibility
coefficients (ADC) of crude protein (CP) and amino acids (AAs) of two novel products -
Indian mustard protein concentrate (IMC, 62% CP) and Indian mustard protein meal (IMM,
42% CP), to a commercially available soy protein concentrate (SPC, 57% CP) for two
salmonid species, rainbow trout and Atlantic salmon. The second trial involved assessment
of relative bioavailability of arginine (Arg) from IMC, IMM and SPC compared to that of a
crystalline Arg (L-Arg) in rainbow trout using slope-ratio assay. In the third trial, the effects
of phytic acid (PA) and lignin on nutrient utilization and partitioning in rainbow trout were
assessed. Finally, a series of experiments was conducted in the final trial to establish the
evaluation criteria for pellet quality assessment. The ADC of CP and most AAs in IMC and
IMM were high (>90%). Differences in the ADCs of some AAs can be attributed to the high
PA intake by fish fed 30%-IMC diet. The significantly higher (P<0.05) bioavailability of
Arg from IMC (123 to 187%) and IMM (116 to 211%) relative to that of L-Arg, as
determined by various regression approaches, reaffirmed the findings of the first trial that
these ingredients are of excellent protein quality and can readily be used in compounded fish
feeds. It can be inferred from the lack of effects of PA, lignin or PA plus lignin on most
indices of physiology, performance, and nutrient utilization in the pair-fed fish, that like any
other animal, controls feed intake when in the presence of one or more dietary ANF. It was
also shown in the pellet quality assessment trial that minor changes in dietary composition
can significantly alter physical properties of aquaculture feed. This study highlighted the
importance of a comprehensive assessment for the effective evaluation of the nutritive value
of plant protein ingredients for use in aquaculture feeds.

Dedication
To my father
Dr. Badiuzzaman Chowdhury (1936-1981)
iv
ACKNOWLEDGMENTS
First and foremost, I would like to thank my advisor Dr. Dominique P. Bureau for his
guidance, caring help and support during this endeavour to shape my ideas and knowledge
around the basic principles of fish nutrition. His straightforward but no stones unturned
approach to science and nutrition in particular, has taught me some very valuable lessons. I have
nothing but utmost respect for you. Thank you, Dom.
Thanks to my advisory committee, Dr. Cornelis F. M. de Lange, Dr. Kendall Swanson, and
Dr. Vernon Osborne for all the help and guidance they have provided me along the way. Thank
you for all your patience and open-door approach to something relatively uncommon. Thank you
Kees for your valuable insights and ideas that pushing me to think differently. Thank you Vern
for the opportunities you have given me, whether it is teaching or discussing new ideas or other
forms of support.
I would also like to thank Professor Richard Moccia and Dr. Albert Tacon for their
contribution to the thesis. As examiners, their broad perspectives and positive attitudes have
made a stressful day more rewarding.
I would like to thank Dr. Margaret Quinton for her help in understanding some difficult
statistical analyses. I would also like to thank Kattia Peciado Iniguez and Thomas Martie for
their significant role in two of my experiments. Thanks to my past and present lab mates Dr.
Andre Dumas, Dr. Katheline Hua, Dr. Guillaume Salze, Jamie Hooft, Patricio Saez, and many
others who have been a true inspiration with their attitude towards building a good team and
helping each other out at difficult times.
v
The study was funded by the BIOEXX Specialty Proteins Ltd. (Toronto, ON, Canada),
Mathematics of Information Technology and Complex Systems (MITACS Inc., Vancouver, BC,
Canada), Martin Mills Inc. (Elmira, ON, Canada), and the Ontario Ministry of Natural
Resources. I would also to recognize DANISCO Animal Nutrition (MO, USA), Evonik
Industries AG (Hanau, Germany) and Pure Lignin Environmental Laboratories (BC, Canada) for
their contribution in analyzing phytic acid, amino acid and providing purified lignin,
respectively.
Finally, I would like to thank my family for their love, patience, support and help during
this study. Without their support, it would have been impossible to pursuit this dream.
vi
TABLE OF CONTENTS
Acknowledgment..................................................................................................................... v
Table of Contents.................................................................................................................... vii
List of Tables........................................................................................................................... xiii
List of Figures......................................................................................................................... xvii
List of Abbreviations.............................................................................................................. xx
Chapter 1 General Introduction.......................................................................................... 1
Chapter 2 Review of Literature........................................................................................... 7
2.1. Introduction................................................................................................................ 7
2.2. Review of Aquaculture Feed Production and Aquaculture Nutrition........................ 9
2.2.1. Evolution of salmonid feed formulation............................................................. 9
2.2.2. Evolution of fish meal usage.......................................................... 15
2.2.3. Desired characteristics of a potential feed ingredient......................................... 15
2.3. Overview of World Feed Ingredients......................................................................... 16
2.3.1. Production of oilseed meals............................................ 18
2.3.2. Production of protein concentrates and isolates............................................. 18
2.3.3. Further improvement of the protein quality............................................ 19
2.3.4. Production approaches................................................ 20
2.3.2. Rapeseed / canola / mustard (Brassicacae)........................................................ 22
2.3.2.1. Indian mustard (Brassica juncea)............................................................... 22
2.3.2.2. Nutritive value of mustard products: issues and challenges....................... 23
2.4. Nutritive Value of Protein Ingredients to Fish........................................................... 24
2.4.1. Overview of methodological approaches........................................................... 25
vii
2.4.2. Digestibility............................................................ 26
2.4.2.1.. Digestive process related to protein and amino acids... 26
2.4.2.2. Assessing digestibility in fish: techniques, issues and limitations......... 30
2.4.2.2.1. Fecal collection methods.................................................................. 30
2.4.2.2.2. Indigestible markers.......................................................................... 31
2.4.2.2.3. In-vitro assessment of digestibility................................................... 32
2.4.2.2.4. Limitations of digestibility of proteins in feed ingredients.............. 33
2.4.3. Bioavailability of amino acids............................................................................ 33
2.4.3.1. Methodological approaches........................................................................ 35
2.4.3.2. Limitations of slope-ratio approaches......................................................... 36
2.4.4. The presence and effects of ANF and other non-nutritive components............. 37
2.4.4.1. General overview........................................................................................ 37
2.4.4.2. Phytic acid................................................................................................... 41
2.4.4.2.1. Interactions with minerals................................................................... 41
2.4.4.2.2. Interactions with protein and amino acids.......................................... 45
2.4.4.2.3. Interactions with proteolytic enzymes................................................. 46
2.4.4.2.4. Effect of PA on nutrient utilization................................................. 46
2.4.4.3. Fibre components and their effects............................................................. 47
2.4.4.4. Lignin.......................................................................................................... 48
2.4.4.4.1. Lignin biosynthesis............................................................................. 48
2.4.4.4.2. Effects of purified lignin..................................................................... 50
2.4.4.4.3. Effects of lignin on minerals and amino acid availability................... 50
2.4.5. Physical characteristics of aquaculture feed...................................................... 51
viii
2.4.5.1. Overview of feed production technology.................................................... 51
2.4.5.2. Physical properties of pellets and their importance.................................... 54
2.4.5.3. Different factors affecting physical properties........................................... 56
2.4.5.4. Components of plant proteins influencing pellet characteristics................ 56
2.5. Conclusion........................................................................................................................ 57
Chapter 3 Digestibility of Amino Acids in Two Novel Ingredients, Indian Mustard
Protein Concentrate and Indian Mustard Meal Compared to That of A
Soy Protein Concentrate in Rainbow Trout and Atlantic Salmon............... 59
Abstract.............................................................................................................................. 59
3.1. Introduction................................................................................................................ 60
3.2. Methods...................................................................................................................... 62
3.2.1. Dietary design..................................................................................................... 63
3.2.2. Experimental conditions..................................................................................... 67
3.2.3. Chemical analyses.............................................................................................. 68
3.2.4. Calculations........................................................................................................ 69
3.2.5. Statistical analysis.............................................................................................. 69
3.3. Results........................................................................................................................ 70
3.3.1. Apparent digestibility of energy and nutrients................................................... 70
3.3.2. Apparent digestibility of amino acids................................................................ 71
3.4. Discussion.................................................................................................................. 75
Chapter 4 Relative Bioavailability of Arginine in Novel Indian Mustard Protein
Concentrate and Indian Mustard Meal Compared to That of A Soy
Protein Concentrate in Rainbow Trout (Oncorhynchys Mykiss) as
ix
Determined by Slope-Ratio Assay................................................................... 81
Abstract............................................................................................................................. 81
4.1. Introduction................................................................................................................ 82
4.2. Methods...................................................................................................................... 85
4.2.1. Experimental diets.............................................................................................. 87
4.2.2. Experimental conditions..................................................................................... 87
4.2.3. Chemical analyses.............................................................................................. 91
4.2.4. Calculations........................................................................................................ 92
4.3. Results........................................................................................................................ 93
4.3.1. Growth performance and nutrient utilization..................................................... 94
4.3.2. Relative bioavailability of arginine.................................................................... 94
4.4. Discussion.................................................................................................................. 103
Chapter 5 Effect of Dietary Phytic Acid and Purified Lignin and Their Interactions
on Physiological Indices, Growth Performance, Nutrient and Energy
Partitioning of Rainbow Trout, Oncorhynchus Mykiss................................. 107
Abstract............................................................................................................................ 107
5.1. Introduction................................................................................................................ 108
5.2. Methods...................................................................................................................... 111
5.2.1. Experimental diets.............................................................................................. 112
5.2.2. Experimental conditions and feeding management............................................ 112
5.2.3. Physiological indices.......................................................................................... 113
5.2.4. Chemical analysis............................................................................................... 113
x
5.2.5. Calculations........................................................................................................ 115
5.3. Results........................................................................................................................ 120
5.4. Discussion.................................................................................................................. 125
Chapter 6 Assessing The Effects of Lignin on Physical Properties of Steam-Pelleted
Experimental Trout Feed................................................................................. 130
Abstract............................................................................................................................. 130
6.1. Introduction................................................................................................................ 131
6.2. Methods...................................................................................................................... 132
6.2.1. Oil and water absorption by the mash................................................................ 135
6.2.2. Physical properties of the pellets........................................................................ 135
6.3. Results........................................................................................................................ 137
6.3.1. Oil and water absorption.................................................................................... 137
6.3.2. Physical properties............................................................................................. 137
6.4. Discussion.................................................................................................................. 142
6.4.1. Oil and water absorption.................................................................................... 142
6.4.2. Pellet quality....................................................................................................... 143
6.4.3. Conclusion.......................................................................................................... 145
Chapter 7 General Discussion............................................................................................ 146
7.1. Evaluation of A Novel Plant Protein Ingredient: Why is It Important? .................... 146
7.2. Digestibility of Amino Acids from Plant Protein Sources......................................... 147
7.3. Bio-availability of Amino Acid and Slope-ratio Assay............................................. 149
xi
7.4. Effect of Phytic Acid and Ligin on Nutrient Utilization............................................ 151
7.3. Beyond Tradition........................................................................................................ 153
Bibliography............................................................................................................................ 157
xii
LIST OF TABLES
Table 2.1 A chronological account of the evolution of salmonid feed type, feed
ingredients, crude protein and lipid contents since
1920................................................................................................................ 14
Table 2.2 Major co-products from grain, oilseed and animal production industries
available for compounded feed productidson (estimated global output, in
MMT)............................................................................................................. 17
Table 2.3 Major enzymes in fish for the digestion of protein, peptides, amino acids
and other non-nitrogenous compounds. 29
Table 2.4 Major anti-nutrients in soybean meals and their effects on salmonid fishes. 38
Table 2.5 Total phosphorus and phyate-P content in the major plant ingredients
currently utilized in compounded aquafeed.................................................. 44
Table 2.6 Indication of final pellets bulk density in relation to floating or sinking
properties........................................................................................................ 55
Table 2.7 Final fat contents (%) in the pellets of different aquaculture species in
relation to floating or sinking properties........................................................ 55
Table 3.1 Ingredient composition of the basal diet (%)................................................ 64
Table 3.2 Proximate chemical composition, gross energy, essential amino acid
profiles, total phosphorus and phytic acid content of the test ingredients
(as is basis).................................................................................................... 65
Table 3.3 Proximate analysis and, gross energy, essential amino acid profiles, total
phosphorus and phytic acid concentrations of the diets (as is basis)............ 66
Table 3.4 ADC of nutrients, phosphorus, gross energy and essential amino acids in
xiii
the reference diet (diet 1) and the three test diets for rainbow trout and
Atlantic salmon............................................................................................. 72
Table 3.5 ADC of nutrients and gross energy in Indian mustard protein concentrate
(IMC), Indian mustard meal (IMM) and soy protein concentrate (SPC) for
two rainbow trout and Atlantic salmon......................................................... 73
Table 3.6 ADC of eight essential amino acids in Indian mustard protein concentrate
(IMC), Indian mustard meal (IMM) and soy protein concentrate (SPC) for
rainbow trout and Atlantic salmon................................................................ 74
Table 4.1 Proximate chemical composition, gross energy, essential amino acid
profiles, total phosphorus and phytic acid content of the test ingredients... 86
Table 4.2 Ingredients composition of the diets (g kg-1)............................................... 89
Table 4.3 Nutrient, gross energy and essential amino acid (EAA) composition of the
test diets (DM basis)..................................................................................... 90
Table 4.4 Weight gain, TGC and feed intake and feed efficiency of rainbow trout fed
diets with increasing levels of arginine supplied by L-Arg or three protein-
bound sources (mean SD)............................................................................ 95
Table 4.5 Protein (PD) and lipid (LD) deposition, recovered energy (RE), N- (NRE)
and energy (ERE) retention efficiencies for rainbow trout fed diets with
increasing levels of arginine (mean SD)..................................................... 96
Table 4.6 Slopes (b) and confidence intervals (CI) of thermal-unit growth
coefficients (TGC) and protein deposition (PD) in fish fed diets from
different sources of arginine (L-arginine, Indian mustard protein
concentrate - IMC, Indian mustard protein meal - IMM, soy protein
xiv
concentrate - SPC) as determined by linear regressions using two and one
level of arginine (%) and arginine intake (g fish-1)...................................... 97
Table 4.7 Bioavailability of arginine from Indian mustard protein concentrate - IMC,
Indian mustard protein meal - IMM, soy protein concentrate - SPC in
rainbow trout in compare to L-arginine based on thermal-unit growth
coefficients (TGC) and protein deposition (PD) as determined by linear
regressions using two and one level of arginine (%).................................... 98
Table 5.1 Basal mix for the test diets............................................................................ 117
Table 5.2 Ingredient and proximate chemical composition of the test diets (DM
basis).............................................................................................................. 118
Table 5.3 Analyzed essential and non-essential amino acid composition of the test
diets (digestible AA, % DM)........................................................................ 119
Table 5.4 Performance and physiological indices of fish (initial weight = 40 g fish-1)
fed the experimental diets for 84d at 15C. Significance between the
diets............................................................................................................... 122
Table 5.5 Whole carcass nutrient deposition and retention efficiency of rainbow
trout fed test diets with various levels of phytic acid and lignin................... 123
Table 5.6 Nutrient deposition and retention efficiencies in dressed carcass and
viscera of rainbow trout fed the experimental diets...................................... 124
Table 6.1 Ingredient composition of the basal mix prepared for assessing oil and
water absorption capacity with the addition of lignin. 16.0% fish oil was
added before mixing with kaolin and lignin to prepare three diets of 0%,
1.5% and 3% lignin....................................................................................... 134
xv
Table 6.2 Oil (fish oil and vegetable oil) and water absorption capacity (in %) of the
diet mash with various levels of lignin (Mean SE).................................... 138
Table 6.3 Sinking velocity (cm s-1) and hardness (Newton) of the diet pellets with
various levels of lignin (mean SE)............................................................. 139
Table 6.4 Parameter estimates (b) of the effect of lignin levels (0%, 1.5% and 3%),
pellet size, duration in water (time) and their interactions on pellet water
stability (%), parameter estimates (b) of the effect of the three lignin levels
on sinking velocity and hardness of the pellets, and oil and water
absorption of the mash (lignin levels 0%, 1.5%, 3%, 6%, and 12%)........... 141
xvi
LIST OF FIGURES
Figure 2.1 The world production of fish and shrimp (*excluding filter feeding
fishes, dark grey), and the fraction produced fed compounded feed (in
%, light grey) since 1995.......................................................................... 10
Figure 2.2 Global output of farmed salmonids in weight (MMT) and value (billion
USD)........................................................................................................ 11
Figure 2.3 Production of farmed salmonids (in thousand metric tonne) in
continental America, Asia, and Europe in 2008 and 2009....................... 12
Figure 2.4 Flow-chart of soybean processing showing the techniques used to
produce various soy products for consumption........................................ 21
Figure 2.5 Digestion and absorption of dietary proteins in the intestine of fish....... 28
Figure 2.6 Dietary fibre components......................................................................... 40
Figure 2.7 The structure of phytic acid (myo-inositol hexaphosphate, IP6)............. 43
Figure 2.8 Structure of three monolignol precursors to lignin (adapted from
Hatfield and Vermerris, 2001).................................................................. 49
Figure 2.9 Steps in a typical aquaculture feed manufacturing process...................... 53
Figure 3.1 Apparent digestibility coefficients (%) of arginine - Arg (pKa 12.5),
lysine Lys (pKa 10.5) and histidine - His (pKa 6.1) in Indian
mustard protein concentrate (IMC), Indian mustard meal (IMM) and
soy protein concentrate (SPC) for rainbow trout and Atlantic salmon
and their relationships to the dietary phytic acid intake. Dietary phytic
acid intake in 30%-IMC, 30%-IMM and 30%SPC diets were 29
mg.fish-1.d-1, 15 mg.fish-1.d-1 and 14 mg.fish-1.d-1 for rainbow trout and
xvii
10 mg.fish-1.d-1, 6 mg.fish-1.d-1 and 5 mg.fish-1.d-1 for Atlantic salmon,
respectively............................................................................................. 79
Figure 3.2 Broken line regression of ADC (%) of phenylalanine in Indian mustard
protein concentrate (IMC), Indian mustard meal (IMM) and soy
protein concentrate (SPC) for rainbow trout and Atlantic salmon with
the dietary phytic acid intake (R2=0.83). Dietary phytic acid intake in
30%-IMC, 30%-IMM and 30%SPC diets were 29 mg.fish-1.d-1, 15
mg.fish-1.d-1 and 14 mg.fish-1.d-1 for rainbow trout and 10 mg.fish-1.d-1,
6 mg.fish-1.d-1 and 5 mg.fish-1.d-1 for Atlantic salmon,
respectively............................................................................................. 80
Figure 4.1 Weight gain (g fish-1) and thermal-unit growth coefficient (TGC) of
rainbow trout in response to dietary arginine intake.............................. 99
Figure 4.2 Protein (PD) and lipid (LD) deposition in rainbow trout in response to
dietary arginine intake (g fish-1)............................................................. 100
Figure 4.3 Thermal-unit growth coefficient (TGC) and protein deposition (PD) of
rainbow trout in response to arginine level (%) in diets from four
arginine sources (L-Arg, IMC - Indian mustard protein concentrate,
IMM -Indian mustard protein meal, SPC -soy protein concentrate).
Solid and dotted lines represent response based on two and one level of
arginine, respectively............................................................................ 101
Figure 4.4 Thermal-unit growth coefficient (TGC) and protein deposition (PD) of
rainbow trout in response to arginine intake (g fish-1) from four
arginine sources (L-Arg, IMC - Indian mustard protein concentrate,
xviii
IMM -Indian mustard protein meal, SPC -soy protein concentrate).
Solid and dotted lines represent response based on two and one level of
arginine, respectively............................................................................ 102
Figure 5.1 Total feed intake of fish fed experimental diets when fed to satiety
during the first two weeks of the trial. Diets 1, 2, 3 (0% lignin), diets 4,
5, 6 (1.5% lignin) and diets 7, 8, 9 (3% lignin)..................................... 121
Figure 6.1 Effect of lignin inclusion rate (0%, 1.5% and 3%) on dry matter loss
and nitrogen leaching from pellets of two sizes (2-mm and 3-mm)
following immersion in shaking water bath at 100 rpm min-1 for >1-
min, 10-min and 20-min of durations...................................................... 140
Figure 7.1 A flow-diagram for characterization of the compoenents of a plant
protein ingredient..................................................................................... 155
Figure 7.2 A frame-work for evaluation of a plant protein
ingredient.................................................................................................. 156
xix
LIST OF ABBREVIATIONS
AA Amino acid
ADC Apparent digestibility coefficient
ADF Acid detergent fibre
ADL Acid detergent lignin
ANF Anti-nutritional factors
ATP Adenosine tri-phosphate
BBM Brush border membrane
BBS Bobesin
CART Cocaine- and amphetamine-regulated transcript
CCK Cholecystokinin
CGM Corn gluten meal
CI Confidence interval
CP Crude protein
CRF Corticotrophin-releasing factor
DDGS Dried distillers grain solubles
DEI Digestible energy intake
DHA Docosahexaenoic acid
DM Dry matter
DNI Digestible nitrogen intake
EAA Essential amino acid
EFA Essential fatty acid
EPA Eicosapentaenoic acid
ERE Energy retention efficiency
EU European Union
FA Fatty acid
FAS Fatty acid synthetase
FBW Final body weight
FCI Fultons condition index
FCR Feed conversion ratio
xx
FE Feed efficiency
FO Fish oil
G Guaicyl
GDAS Gastric dilation air sacculitis
GE Gross energy
GIT Gastro-intestinal tract
GLP Glucagon like peptide
GRP Gastrin releasing peptideds
IBW Initial body weight
H Hydroxyphenyl
IMC Indian mustard protein concentrate
IMM Indian mustard protein meal
IP6 Myo-inositol hexaphosphate
IPF Intra-peritoneal fat
LD Lipid deposition
LPL Lupoprotein lipase
LRE Lipid retention efficiency
MBM Meat and bone meal
MBW Metabolic body weight
MUFA Mono-unsaturated fatty acid
MMT Million metric tons
NA North America
NDF Neutral detergent fibre
NEAA Non-essential amino acid
NPY Neuropeptide-Y
NRC National Research Council
NRE Nitrogen retention efficiency
PA Phytic acid
PBM Poultry by-product meal
PD Protein deposition
PDI Protein dispersibility index
xxi
PUFA Poly-unsaturated fatty acid
RE Recoevred energy
S Syringyl
SBM Soybean meal
SPC Soy protein concentrate
TAG Tri-acyl glyceraldehyde
TGC Thermal-unit growth coefficient
USDA United States Department of Agriculture
VO Vegetable oil
WRO World Renderers Organization
xxii
CHAPTER 1
GENERAL INTRODUCTION
Aquaculture, the fastest growing food-producing sector in the world, has been supplying
49% of the worlds food fish since 2010 (FAO, 2011). A majority (22.2 million metric tons
MMT) of this is comprised of various carp species mainly produced in China and Indian sub-
continent followed by 3.2 MMT of tilapia (FAO, 2011). The significant production increase of
these two groups of fish species in the past few years is mainly attributable to the culture
intensification resulting in higher growth in the worldwide demand for compounded aquaculture
feed than that of the production (Naylor et al., 2009; Tacon and Metian, 2008). Global
production of various salmonid species is third after carps and tilapias at 2.5 MMT in 2009, an
increase of 67% from its 2000 production of 1.5 MMT (FAO, 2011). Salmonids are generally fed
high protein and lipid diets (nutrient-dense), which can support high growth performance and
feed efficiency, and low waste outputs (Bureau and Hua, 2010). Fish meal has traditionally been
the main protein source in nutrient-dense feeds (Bakke et al., 2011; Bureau and Hua, 2010).
Salmonid feeds are increasingly formulated to contain high levels of economical plant
protein ingredients due to the rising demand, stagnating supply, and price volatility of fish
meal. Improved understanding of the nutritional requirements of salmonids has allowed
formulation of successful feeds based on plant protein ingredients. But the inclusion of many
plant protein ingredients in salmonid feed formulations is limited because of their low protein
and high fibre contents, and/or the presence of various anti-nutritional factors (ANF)
(Dabrowska and Wojno, 1977; Dabrowski et al., 1989; Ketola, 1975; Page and Andrews, 1973;
Refstie et al., 2000; Wee and Shu, 1989). Growth and fish health problems have frequently been
1
reported for salmonid fish fed diets formulated with very high levels of plant proteins (Gaylord
et al., 2010).
In recent years, application of various extraction, fractionation, and processing techniques
resulted in innovative high quality plant protein products (>45% CP) low in fibre and starch and
ANF from oilseed and grain industries. Processing addressed many of the limitations associated
with feeding diets with very high levels of plant ingredients. Protein concentrates of soybean and
canola, for example, are of very high nutritive value to salmonids and very valuable ingredients
for nutrient dense diets formulation (Drew et al., 2007; Higgs et al., 1994; Mambrini et al., 1999;
Mwachireya et al., 1999; Storebakken et al., 1998b; 2000; Thiessen et al., 2004). These
ingredients are more widely available and increasingly economical, and their use in commercial
salmonid feed formulations is growing steadily. However, the cost of many of these ingredients
is still often prohibitive and many studies have shown that growth and feed efficiency of
salmonids can suffer when high levels of these ingredients are used in diets.
Increasing use of a wide variety of plant protein ingredients in aquaculture feeds has
highlighted the need for better characterization and evaluation of the nutrients of these
ingredients (Bureau, 2008; Gatlin et al., 2007). Glencross et al. (2007b) reviewed strategies
employed in evaluating aquafeed ingredients, and described five key components including
ingredient characterization (proximate, macro- and micro-nutrients composition), digestibility of
nutrients, palatability, nutrient utilization, and functionality. Characterization of a plant protein
ingredient should include information on proximate composition, its amino acid profile, macro-
and micro-mineral contents, composition of various fibre components and anti-nutritional
factors.
2
The determination of nutrient digestibility is the first step to evaluate the potential of an
ingredient for use in an aquaculture diet. The digestibility of hundreds of different feed
ingredients to different aquaculture species has been estimated in the past (NRC, 2011).
However, most digestibility studies have focused on apparent digestibility of proximate analysis
components (crude protein, lipids, gross energy) and very limited effort has been invested in
estimating the digestibility or availability of individual nutrients (e.g. amino acids) in feed
ingredients (Bureau, 2008; De Silva et al., 2000; Gaylord et al., 2010; Kaushik and Seiliez, 2010;
NRC, 2011). Fish feeds are increasingly formulated on digestible amino acid basis and more
effort should be invested in determining the amino acid digestibility of different protein sources
(El-Haroun et al., 2009; Poppi et al., 2011; Yamamoto et al., 2005).
Digestibility is a measure of disappearance of nutrients from the gastrointestinal tract and
estimates of digestibility may not necessarily reflect the amount of nutrient useable
(bioavailable) to the animal. More direct assessment of proportions of ingested dietary nutrients
that is potentially available for metabolism or synthesis is important (Ammerman et al., 1995;
Batterham, 1992; Lewis and Baily, 1995; Stein et al., 2007). Among several nutrient bio-
availability protocols, slope-ratio assays have been shown to provide direct and practical
assessment of the level of of individual nutrients in feed ingredients, and have been used to
assess the bioavailability of individual amino acids in dfferent ingredients for livestock (Lewis
and Bailey, 1995). Application of slope-ratio assays to determine bioavailability of amino acids
in fish is relatively recent, and significant variations exist among the methods adopted (El-
Haroun and Bureau, 2007; Li et al., 2008, 2009; Poppi et al., 2011; Zarate and Lovell, 1997).
Another important step in evaluation of plant protein ingredients is to assess the effects of
ANF and fibre components on utilization of nutrients, and on health and well-being of the
3
species. Although fibre components and most ANF are either reduced or removed during
processing of ingredients, some are difficult to remove and their concentration may increase
during fractionation of protein from fibre components. Increasing inclusion of novel plant
proteins in compounded fish feed has accentuated the need for comprehensive species-specific
evaluation of the effect of ANF and fibre components of individual ingredients.
Phytic acid is probably one of the most studied ANF in fish nutrition because of its
effects on availability of phosphorus, and other divalent minerals such as Ca2+ and Mg2+. Proton
disassociation sites on the phytic acid molecules have variable pKa values between 1.5 and
>10.0. Beside their well- known effects on digestibility of minerals (Cowieson et al., 2004;
Maenz et al., 1999; Torre et al.; 1991), increasing evidence suggests that phytic acid may affect
digestibility of amino acids (Cowieson et al., 2004). Phytic acid may become negatively charged
when a proton is disassociated attracting positively charged molecules at appropriate pH (Adeola
and Sands, 2003). Phytic acid also interacts with protein and creates binary (phytic acid-protein)
or ternary (phytic acid-mineral-protein) complexes that occur only at optimal pH conditions in
the gastro-intestinal tracts (Cheryan and Rakis, 1980; Kies et al., 2001; Maenz, 2001). The -
NH2 terminal group and the -NH2 group of lysine, the imidazole group of histidine, and the
positively charged guadinyl group of arginine have been implicated as possible protein binding
sites for phytate at low pH (Cosgrove, 1966). Ternary complexes are rare but usually formed in
slightly alkaline environment in the intestine (Riche et al., 2001).
Most studies on dietary fibre from various plant sources dealt mainly with inclusion
levels and their effects on nutrient utilization and growth of an animal such as swine (Laplace et
al., 1989; Mitaru et al., 1984; Souffrant, 2001), poultry (Slominski and Campbell, 1990;
Villamide and San Juan, 1998), or fish (Anderson et al., 1984; Bromley and Adkins, 1984;
4
Hemre et al., 1995; Kraugerud et al., 2007). Recent studies have shown that soluble such as
pectin and insoluble fibre components such as cellulose and hemicellulose affect digestive
processes of an animal (Amirkolaie et al., 2005; Glencross, 2009; Ovrum-Hansen and
Storebakken, 2007; Zahedifar et al., 2002; Zhu et al., 2005).
An ingredient could have low ANF contents, excellent digestible amino-acid profile, and
the availability of the amino acids could be very good but would not be appropriate for inclusion
if it negatively affects the physical quality of the manufactured feed pellets. Physical properties
of feed pellets are of great importance in aquaculture. Bulk density, hardness, fines, undersized
pellets, sinking velocity and water stability are all pellet quality parameters important to
aquaculture feed manufacturers and fish producers (Aas et al., 2011).
Water stability of a pellet is a very important characteristic (Cho and Bureau, 2001;
Obaldo et al., 2002). Water stability is known to be affected by feed composition, notably by
soluble fibre (e.g., guar gum, Brinker, 2007; Brinker and Reiter, 2011; Storebakken, 1985) or
insoluble fibre (e.g., cellulose, Bromley and Adkins, 1984; Hansen and Storebakken, 2007;
Lupatsch et al., 2001) composition playing important roles. Level and composition of the fibre
components in the plant protein ingredients alter the physical properties of the mash and pellets
during processing. However, few studies have systematically examined the effect of different
various fibre components on physical properties of aquaculture feed (e.g., Baeverfjord et al.,
2006; Glencross et al., 2007; Hansen and Storebakken, 2007; Kraugerud et al., 2011), and
criteria for evaluation of these properties are not well-established (e.g., Glencross et al., 2007;
2010).
1.1. Objectives
5
The main objective of this thesis is to set up criteria for comprehensive evaluation of
plant protein sources encompassing amino acid digestibility and bioavailability, effects of anti-
nutritional factors and fibre components on nutrient utilization, and assessment of physical
properties of feed pellets with inclusion of a novel ingredient. Two novel plant protein
ingredients, Indian mustard protein concentrate and Indian mustard protein meal with variable
fibre and phytic acid contents were selected as test ingredients and compared with a commercial
soy protein concentrate commonly used in commercial rainbow trout and Atlantic salmon feeds.
Bioavailability of arginine from these three ingredients to rainbow trout was evaluated because
of the high concentration of this amino acid in these ingredients. Phytic acid and lignin,
respectively were chosen to assess the effects of ANF and a fibre component, and their
interactions on growth, utilization and partitioning of nutrients, and physiological response of
rainbow trout because these two types of ANF were present at variable concentrations (from low
to very high) in the test ingredients. Finally, to establish criteria to assess physical properties of
aquafeed, graded levels of a purified lignin were used to determine its effect on oil and water
absorption of a dietary mash, and physical properties of steam-pressed pellet including the water
stability.
6
CHAPTER 2
REVIEW OF LITERATURE
2.1. Introduction
Aquaculture is the fastest growing food-producing sector in the world supplying more
than half (37 MMT) of the worlds food fish production in 2009. About 56 million metric tons
(MMT) of finfish and shell-fish valued at US$ 105 billion were produced in 2010 (FAO 2011;
Bostock et al., 2010). More than 200 species of fish, crustaceans and molluscs are cultivated
around the world, but only 28 species together contributed more than two-thirds of the world
production in 2010, a majority of which is from six species of carps (about 16 MMT), followed
by tilapia (2.5 MMT) and salmonid (2.4 MMT) species (FAO, 2011).
The share of farmed fish and crustacean production fed compounded feed worldwide has
risen from about 44% in 1995 to 68% in 2010 (Tacon and Metian, 2008). Fishmeal, long been
considered as major component in compounded feed, has transformed from the major protein
source to a strategic protein ingredient. Currently, fish meal is still used as a major protein source
in larval feed and in some marine fish feed, contribution of which gradually decreases or
diminishes as the fish grow. However, despite decreasing inclusion in the feed of marine shrimp,
marine fish and salmonids over the past few decades, they remain the major consumers of the
fish meal (Naylor et al., 2009; Tacon and Metian, 2008).
The rising demand for compounded feed, and limited supply and rising cost of fish meal
have encouraged investigation on the nutritive value to fish of different protein-rich ingredients
over the past few decades. Various factors such as amino acid composition, anti-nutrients, and
non-nutritional components can affect the nutritive value of protein-rich ingredients and the
7
amount of these ingredients that can be used in the diets of various fish species (Bell 1993;
Bureau et al., 1998; Francis et al., 2001; Higgs et al., 1995; Mwachireya et al., 1999; Refstie et
al., 2001; Sanden et al., 2005).
Known nutritional limitations such as poor amino acid profile, heat-induced chemical
damage to some amino acids during processing, the presence of ANF and non-nutritional fibre
components are major challenges for increasing the inclusion of many economical protein
sources. Lack of information on available amino acids and other nutrients in ingredients forces
fish feed formulators to rely on high safety margins increasing the economical and
environmental costs. Accurate information on chemical composition, digestibility and
bioavailability of amino acids would support more economical feed formulation. Another
challenge is associated with the anti-nutritional compounds in plant proteins (Francis et al., 2001;
Tacon, 1997). Although many are destroyed or inactivated during feed processing, some remain
and could negatively affect digestibility of nutrients in fish. These include phytic acid,
glucosinolates, saponins, tannins, soluble NSP, and gossypol. In addition, the physical
characteristics, such as water stability and pellet durability, of feed pellets are very important to
prevent the economical and environmental loss caused by the increased amount of feed needed to
be used and wastes (Cho and Bureau, 2001; Obaldo et al., 2002).
The main focus of this literature review is to provide an overview of the state of
aquaculture nutrition and feed production, introduce some common and novel plant protein
ingredients potentially valuable for aquaculture feed production and to review strategies for
evalution of plant protein ingredients, with a focus on digestibility and bioavailability of
nutrients in fish, and the physical quality of feed pellets.
8
2.2 Review of Aquaculture Feed Production and Aquaculture Nutrition
World industrial feed output for farmed terrestrial and aquatic animals has exceeded 700
MMT in 2009 from 600 MMT in 2003 (Best, 2009). The share of aquafeed to overall production
is very low (~4%) but has increased more than 60 percent during this period. Global industrial
aquafeed output has increased from 14 MMT in 2001 to about 17 MMT in 2008, an estimate that
doesnt account for the production by feed manufactures (feed mills) with annual capacity of less
than 2400 MT (Best, 2009). Considering this output, the real global aquafeed production is
closer to 37 MMT (Rana, 2009). Several factors such as intensification of the existing farming
systems, increasing culture of high trophic level and high market value finfish species, and
growing reliance on compounded aquafeed could be responsible for the phenomenal growth
experience for aquafeed production (Tacon and Metian, 2008; Tacon et al., 2010). This is
particularly evident in that since 1995, the amount of compounded feed used in finfish farming
has been increasing at a higher rate (2.5 times higher) than that of the growth of the aquaculture
industry (Tacon and Metian, 2008; Figure 2.1).
2.2.1. Evolution of salmonid feed formulation
Global output of salmonids in 2009 was 2.5 MMT, an increase of 67% from its 2000
production of 1.5 MMT (FAO, 2011; Figure 2.2). Atlantic salmon, Salmo salar, are the most
widely farmed salmonid species globally (1.44 MMT), followed by rainbow trout, Oncorhynchus
mykiss (0.7 MMT), Chinook salmon, O. tshawytscha and Coho salmon, O. kisutch (0.19 MMT).
More than half (1.4 MMT) of the worlds salmonid production comes from the Europe (mainly
Norway and Scotland) and about a third is produced in the Americas (mainly Chile and Canada)
(Figure 2.3).
9
Figure 2.1 The world production of fish and shrimp (*excluding filter feeding fishes, dark
grey), and the fraction produced fed compounded feed (in %, light grey) since 1995 (data from
Tacon and Metian, 2008).
80 Production of fish and shrimp (MMT)*

% produced fed compounded feed 68%
70
61%
60 54%
50 44%
40
31.6
30 22.3
20 14.9
9.1
10
0
1995 2000 2005 2010
Year
10
Figure 2.2 Global output of farmed salmonids in weight (million metric tons MMT) and
value (billion USD) (FAO 2011).
3 Production (MMT) Value (in billiion dollars) 14

World production of salmonids
World salmonid production in

12
2.5
value (billion $)
10
(MMT)
2 8
6
1.5
4
1 2
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
Year
11
Figure 2.3 Production of farmed salmonids (in thousand metric tonne) in continental America,
Asia, and Europe in 2008 and 2009 (FAO, 2011).
1600
1400 2008 2009
Production ('000 tonne)
1200
1000
800
600
400
200
0
Americas Asia Europe
Continents
12
In 2006, annual world production of salmonid feeds was estimated at about 2.6 MMT
worldwide (Tacon and Metian, 2009) up from 1.9 MMT in 2000 (Hardy and Tacon, 2006). Over
a period of six decades, formulations of salmonid diets have evolved from wet-mixture of locally
available ingredients such as salmon eggs, fresh or frozen fish, livestock offal, and oilseed meals
(from 1850 to 1950) to the modern compounded extruded feed used today (Grassl, 1956; Hardy
and Barrows, 2002). Table 2.1 provides a chronological overview of the changes in ingredient
composition, advances in manufacturing practices and nutrient composition of salmonid feeds.
Diet formulation and preparation are the process of combining feed ingredients to form a
mixture that will meet the specific goals of production. It is often a compromise between ideal
and practical considerations. The primary objectives are to produce a mixture that (is):
1. Nutritionally balanced (to support maintenance, growth, reproduction, health)
2. Economical, Palatable, Water stable
3. Minimizes waste output & effect on water quality
4. Produces desirable final product (attractive & safe)
5. Practical considerations:
i. Ingredients price and availability
ii. Anti-nutritive factors
iii. Pelletability of mixture
iv. Storage and handling requirements
13
Table 2.1 A chronological account of the evolution of salmonid feed type, feed ingredients,
crude protein and lipid contents since 1920.
Year Feed type Major ingredients Crude Crude Reference

Protein lipid
1920- Moist pellets Salmon eggs; slaughter house <10% <10% Hardy and
1940 (>50% water) waste, beef-hog liver, salmon Barrows, 2002
viscera etc.
1940- Semi-moist Meat-meal mixtures, blend of 10-20% 10-15% Phillips et al.,
1955 pellets slaughter-house wastes and 1940
commercial ingredients.
1956- Dry-sinking Mostly fish meal, animal by- 35-45% <15% Grassl, 1956;
1980 steam pressed products, soybean meal, corn gluten Hilton et al.,
pellets meal, vitamin and/or mineral 1981
premix.
1990- Dry-floating Mostly fish meal, animal by- 35-50% >30% Hardy and
2000 extruded products, plant protein meals, salmon Barrows, 2002
pellets vitamin and mineral premix. >20%
trout
2000- Dry-floating Reduced fishmeal content, soybean 35-50% >35% Rana et al., 2009
extruded meal and protein concentrate, maize salmon
pellets and wheat gluten, blood meal, meat >30%
meal, gelatin, brewers yeast. trout
14
2.2.2. Evolution of fish meal usage in aquaculture feed
Fish meal has long been considered preferred sources of proteins in the feed of most
farmed crustaceans, most marine fishes, and other intensive farming practices that need a high
quality diet (De Silva et al., 2011). Fish meals are high in small bioactive compounds such as
choline, taurine, anserine etc. These compounds play important roles such as stabilizing protein
structures, protecting cells against osmotic stresses or preventing oxidative damage and
stimulating immune system in swine, poultry and fish (Kuzmina et a;., 2010; Yun et al., 2011).
Despite the effort to decrease fishmeal inclusion rate, aquafeeeds shall utilize about 68% (3.7
MM) of reported world production of fish meal (Tacon and Metian, 2008).
Feed for shrimp (27%), marine fish (20%) and salmonids (14%) account for significant
proportion of fish meal consumption worldwide (Tacon and Metian, 2009). Improved
understanding of the nutritional requirement of farmed fish and shrimp, and better
characterization of alternative ingredients have allowed a significant reduction in fish meal and
fish oil usage in the diets of shrimp, salmon and marine fish over the past decade (Naylor et al.,
2009; Tacon and Metian, 2009). Today, fish meal has become more of a strategic ingredient.
Starter and fry feeds for salmonids and other marine species are generally formulated to supply
appropriate feed intake of the animals. However, fish meal levels are progressively reduced in
feed formulation once once fish reach juvenile and grow-out stages (Naylor et al., 2009)
2.2.3. Desired characteristics of a potential feed ingredient
An increasingly wide variety of feed ingredients are playing a role in the formulation of
compounded fish feeds (Gatlin et al., 2007; Hardy, 2010; Naylor et al., 2009; Sinha et al., 2011;
Tacon and Metian, 2008). Inclusion of any feed ingredient demands adequate supply, consistent
15
quality, competitive price per unit protein, accurate characterization of its nutrient and anti-
nutrient profiles, evaluation of the desired nutrients available to an animal (Bureau, 2008;
Glencross, 2009; Hardy, 2010; Kaushik and Seiliez, 2010), and its effect on physical properties
of the manufactured pellets (Sapkota et al., 2007).
2.3. Overview of World Feed Ingredients
Commonly used animal and plant protein products in fish feed are fish meal, poultry by-
product meal (PBM), meat and bone meal (MBM), soybean meal (SBM), soy protein
concentrates (SPC), and corn gluten meal (CGM). Rendered animal proteins such as poultry by-
product meal, meat and bone meal, feather meal and blood meal products are readily available
and economical sources of protein that have shown to be of relatively high nutritive value to fish
(Tacon et al., 2006). Table 2.2 reports the global or North American (NA) output of major plant
or animal protein-rich feedstuffs. A large variety of oilseeds and grains are cultivated worldwide.
Estimated production of protein meals from soybean, corn and Brassica oilseed (canola-mustard-
rape) globally in 2009-10 was about 300 MMT with soybean meal representing about 175 MMT
worldwide. Worldwide production of rendered animal protein ingredients is somewhat limited,
around ~12 MMT (WRO, 2010; Table 2.2).
16
Table 2.2 Major co-products from grain, oilseed and animal production industries available for
compounded feed production (estimated global output, in MMT).
Projected
Ingredient Year Reference
production
(MMT1)
Major Plant Proteins
Soybean meal 174.9 2010-11 USDA, 2011
Canola/rapeseed/mustard meal 33.5 2010-11 USDA, 2011
DDGS2 (in EU3) 6.0 2010 Ziggers, 2007
Corn DDGS (In NA4) 40.0 2010 OBrien, 2010
Corn Gluten Meal 30.5 2009 USDA, 2011
Major Rendered Proteins 13.0 2010 WRO, 2010
Poultry by-product meal (US) 1.5 2010 Swisher, 2007
Meat and bone meal (US) 2.5 2010 Swisher, 2007
Feather meal (US) 0.4 2010 Swisher, 2007
Porcine meal (US) 0.8 2010 Swisher, 2007
Aquatic Animal Protein (World) 11.0 2010 Tacon, 2010

1
MMT Million metric tons; 2DDGS Dried distillers grain solubles; 3EU European Union;
4
NA North America
17
2.3.1. Production of oilseed meals
Oilseeds are an important agriculture commodity that are generally processed in
vegetable oils and high-protein meals for food, feed, and industrial uses. The economic outcome
of oilseeds begins with separating oil and protein meal. The technology of oil separation has
progressed from primitive manual methods to chemical and mechanical methods using organic
solvents and extrusion technologies, respectively.
Solvent extraction is by far the most widely used method to extract oil where seeds are
prepared by cleaning, drying, tempering, cracking, dehulling, conditioning, and flaking. Oil is
extracted from the flakes using an organic solvent, usually a petroleum distillate high in n-
hexane followed (Johnson and Lusas, 1983; Woerfel, J.B., 1995). The advantage of this process
is high extraction efficiency, with the residual oil in the meal potentially reduced to <2%.
Detailed information of the various solvent extraction processes and the types of solvents has
been summarized by Johnson and Lusas (1983) and Woerfel (1995).
Two mechanical processes, extruding-expelling (EE) and screw-pressing methods are
also used separately or in combination for the extraction of oil (Wijeratne et al., 2004). The
extruding process involves shearing, compressing and heating of the flakes where heat is
provided only by the dissipation of the mechanical energy. The average residance time of the
material within the extruder barrel is less than 30 s. In combination, the extrudate is conveyed
continuously into a crew press, where the the residual oil is pressed out (Wijeratne et al., 2004).
2.3.2. Production of protein concentrates and isolates
Protein concentrates and isolates are produced by further processing of the protein meals.
Concentrates (65-70% CP) are primarily produced after removing the water soluble
18
carbohydrates from the meal. Isolates are highly purified form of proteins (>90% CP) made by
removing most of the non-protein components, residual fats, and carbohydtrates from the meal.
Plant proteins are composed mostly of three types of proteins: albumins (water soluble),
globulins (salt soluble) and glutelins (alkali soluble) (Prakash and Rao, 1986). Water soluble
albumins represent about 45-50% of the total proteins in brassica seeds where as in most other
oil seeds, including soybean, the most common protein is the globulins (Appleqvist, 1972).
Albumins can be isolated by an aqueous extraction method by mixing the sample with
water for 30-120 min and at low temperatures (e.g. 4oC) to prevent proteolysis (Neves and
Lourenco, 1995; Sathe et al., 2002). After centrifugation to remove insoluble materials,
supernatant is dialyzed to eliminate residual salt and precipitate salt-soluble proteins (e.g.,
globulins). A low molecular weight cut-off membrane (<8000) may be used during dialysis to
prevent the loss of proteins of interests (Sathe et al., 2002). The precipitate (salt-soluble
globulins) is then extracted using dilute solutions of NaCl, followed by centrifugation and
dialysis (at 4oC) of the supernatant agains watar.
Aqueaous alcoholic solutions (usually 50-60% ethanol) are also used to separate the
solubles e.g., sugars, odour and flavour substances from the insoluble residues (Uzzan, 1988).
The residue is desolventized at temperatures not exceeding 50-55oC in inert gas environment or
under vacuum to maintain protein quality. Proteins are also isolated using buffer solution of their
isoelectric points (pI), at which they are least soluble.
2.3.3. Further improvement of the protein quality
Further processing of plant proteins are often needed for improving their functional
properties such as protein solubility and removing toxic or inhibitory compounds (Panyam and
19
Kilara, 1996). The isolated proteins can be subjected to chemical, enzymatic or thermal
treatments in order to improve their functional and nutritional properties.
Chemical treatments involving alkylation, oxidation, acylation, esterification and amide
formation have shown to improve the solubility, and the emulsifying and foaming properties of
the proteins (Shih, 1987; Shahidi and Wanasundara, 1998; Sitohy et al., 1999). Enzymatic
hydrolysis such as trypsin treatment of protein bonds can improve protein solubility (Jones and
Tung, 1983). In addition, treatments using pepsin, papayin, tripsin or phytase have been reported
to remove some anti-nutritional factors such as protease inhibitors and phytic acid (Kaushik et
al., 1995; Moure et al., 2006; Murai et al., 1989).
2.3.4. Production approaches
Driven by market demands, the current trend in plant protein production is to develop
tailor-made products destined for a specific industry or for a particular species or life stage of a
species. Various techniques such as roasting, toasting, aqueous alcohol extraction, enzymatic
hydrolysis, thermal processing have been employed to produce various products including
protein meals, concentrate, isolate, and various specialty products texturized and low-antigen
protein concentrate, or enzyme modified protein isolate (Figure 2.4).
20
Figure 2.4 Flow-chart of oil-seed processing showing the techniques used to produce various soy products for consumption (using
soybean as an example).
21
2.3.2. Rapeseed/canola/mustard (Brassicacae)
World production of canola/rapeseed/mustard of the Brassicacae family is currently at 58
MMT contributing about 15% to the worlds oilseed production, second only to soybean at 211
MMT (USDA, 2011). Six Brassica species are cultivated worldwide, three of which contain
diploid genomes: Brassica rapa (Turnip), Brassica nigra (Black mustard), and Brassica
oleracea (broccoli, cauliflower, cabbage). The other three species were inter-specifically bred
from the three diploid species. They are: Brassica juncea (Indian mustard) crossed between B.
rapa and B. nigra, Brassica napus (rapeseed) between B. rapa and B. oleracea, and Brassica
carinata (Ethiopian mustard) between B. oleracea and B. nigra.
Combined world production of protein meals from the Brassicacea seeds have recently
reached 34 MMT in 2010-11 (Table 2.2), and mainly utilized in animal feed (Newkirk et al.,
1997; Thacker and Petri, 2010). New cultivars of Brassica napus (dubbed as canola) and B. rapa
are low in glucosinolate (<30 mol g-1 of oil-free dry matter) and erucic acid (<2% of total fatty
acids in the oil) (Higgs et al., 1982).
2.3.2.1. Indian mustard (Brassica juncea)
Cultivation of another Brassica species, Indian mustard (B. juncea), is gaining popularity
worldwide because its drought resistance attributes are superior to those of the canola cultivars
(Chevre et al., 2008; Oram et al., 1999; Wijesundera et al., 2008). Some Indian mustard varieties
yield more seed per pod, produce heavier seeds, and contain higher dry matter (DM) in the seeds
than the canola varieties in drier conditions (Chauhan et al., 2007; Gunasekera et al., 2006;
Wright et al., 1995). Low drought susceptibility index (a combination of growth, maturation
22
(days), number of pods and seed yield per pod, and DM content), and low-glucosinolate and
erucic acid levels of the newer Indian mustard varieties developed in Australia, India and North
America have raised the potential of growing this crop in the drought-prone regions of the world.
2.3.2.2. Nutritive value of mustard products: issues and challenges
The development of B. juncea cultivars with oil and nutritional characteristics similar to
canola has enhanced the potential of the co-products of this oilseed in animal feeds (Burton et al.,
2004). Amino acid profiles of Indian mustard protein meals and concentrates are very good and
comparable to similar soybean products. Macro-minerals such as calcium and phosphorus are
also in high concentration. The nutritive value of low glucosinolate Indian mustard meals (Jia et
al., 2008; Meng et al., 2006; Newkirk et al., 1997) and Indian mustard protein concentrates and
isolates (Thacker and Petri, 2010) have been evaluated in the diet of poultry. These studies
indicated that the nutritional value of meals and concentrates from low glucosinolate Indian
mustard was equal or superior to that of canola meal samples derived from B. napus and B. rapa
cultivars.
The lower protein and high levels of fibre (indigestible carbohydrates) of Brassica
cultivars by-products limit the potential of these ingredients in high digestible nutrient density
salmonid fish feeds (Carter and Hauler, 2000; Hardy, 2010). Different processing techniques
have been developed to reduce the levels of indigestible carbohydrate and to produce high
protein ingredients from a variety of plant co-products (Bureau and Cho, 1997; Glencross et al.,
2010; Hardy, 2010).
23
2.4. Nutritive Value of Protein Ingredients to Fish
Accurate assessment of nutritive value of feed ingredients is extremely important for the
formulation of cost-effective feeds. Better characterization of the nutrient composition of
feedstuffs is essential to improve their valorization by least-cost feed formulation programs.
More efforts need to be invested in systematically investigating the effect of numerous factors
that can affect the nutritive value of feed ingredients.
Diets produced with poor quality raw materials may have inferior nutritive value and
have adverse effects on production and fish health. Quality criteria for the ingredients must be
respected to insure that the final product is of consistent quality and that deleterious effects are
avoided. The chemical composition (nutrients, anti-nutritional factors, contaminants) usually
provides a snapshot of the potential nutritional value of an ingredient, but the biological aspects,
such as digestibility and availability of nutrients are highly important and yet often overlooked.
Nutritional quality of any ingredient can vary significantly by the sources of raw
materials and processing equipment, techniques, and conditions. Processing techniques such as
expeller or solvent extraction (Glencross et al., 2004b), extraction temperature and duration
(Fenwick et al., 1986), and drying conditions (Glencross et al., 2007a; Stewart et al., 2003) may
affect the nutritive values of oilseed processing co-products. For example, during alkaline
treatment, lysinoalanine is created by the addition of the -NH2 group of lysine to the double
bond of a dehydroalanine residue (Friedman, 1992). Racemization of some amino acids also
occurs during alkaline processing. Heat treatment required during processing may damage some
amino acids through oxidation of sulphahydryl bonds (-SH) to disulfide bonds (S-S), non-peptide
cross linking, Maillards reactions, and oxidative degradation (Moughan and Rutherfurd, 1996;
Moughan and Rutherford, 2008).
24
2.4.1. Overview of methodological approaches
Successfully formulating more economical feeds with cheaper protein sources requires
careful consideration of the nutritional quality of these ingredients to ensure that the performance
and health of the animals and final product quality are not being sacrificed. Digestibility of
nutrients is currently the standard in ingredient and diet evaluation. One option is to simply
assay proximate components and their respective digestibility. However proximate components,
such as crude protein, are not nutrients per se. Crude protein is only a measure of the total
nitrogen (N) content of the feed and is not necessarily an indication of that components
usefulness to the animal. A few studies have focused on the composition and digestibility of
amino acids in common fish feed ingredients (Bureau, 2008; De Silva et al., 2000; Kaushik and
Seiliez, 2010).
Digestibility of amino acids is not always a reliable indicator of the availability of these
nutrients to animals, notably for feed ingredients processed under high heat and pressure
conditions, such as rendered animal protein ingredients. More direct methods of assessment of
the available nutrient contribution of different ingredients to the diet should also be used. One
method of assessing this is through the use of one of a number of bioavailability assays
whereby the effect on growth, protein or nutrient deposition, or other parameters of interest, are
examined with inclusion of the test ingredient at a number of levels and compared to the
response of animals fed diets containing corresponding levels of the component of interest (e.g.
the studied amino acid in a free form (crystalline amino acid) in the case of amino acid
bioavailability studies). In this way, a more accurate assessment of the proportion of an
ingredient which is able to be effectively utilised by the animal can be made.
25
2.4.2. Digestibility
Morrison (1936) stated that digestibility trials provide the general basis for our
knowledge concerning the amounts of digestible nutrients furnished by the many different
feeding stuffs. Measurement of digestibility provides, in general, a good indication of the
availability of energy and nutrients, thus providing a rational basis upon which diets can be
formulated to meet specific standards of available nutrient levels.
2.4.2.1. Digestive process related to proteins and amino acids
Proteins are digested, absorbed and metabolized through a series of processes. In
vertebrates, proteins and polypeptides are broken down to mono-, di-peptides and amino acids
through reactions catalyzed by enzymes mostly secreted in the stomach. The polypeptides and
amino acids are then absorbed in the intestinal brush-border membranes by several active
transport systems and then, to the hepatic portal system through passive diffusion mechanisms
(Bender, 2008). Besides dietary sources, some amino acids and peptides in the intestinal chime
are also derived from the degradation of proteins. These degraded proteins are either reabsorbed
or excreted as components in faeces along with the unutilized dietary amino acids (Figure 2.5).
A detailed definition of the digestibility of AA was provided by Fuller (2003) as a
mechanism that reflects enzymatic hydrolysis and microbial fermentation of ingested proteins
and peptides and absorption of AA and peptides from the gastrointestinal lumen. Digestion and
absorption of nutrients follow closely similar pathways in the digestive system of all vertebrates.
All vertebrates have a digestive tract and associated glands but different parts are not necessarily
directly comparable among the groups. In fish, length and structures of the gastro-intestinal tract
26
(GIT) vary from species to species depending on their food and habitat types (Horn and
Gawlicka, 2001).
Functions of the stomach involve grinding and breaking down large feed particles by
mechanical (contraction-expansion) and chemical processes (pepsinogen- and acid-secreting
functions). In the stomach of fishes, only one secretory cell type exists within the gastric glands
performing both pepsinogen- and acid-secreting functions unlike the differentiation into separate
cells by functions observed in mammals (Wilson and Castro, 2011).
In the intestine, polypeptides are digested to smaller peptides and by several enzymes
derived from pancreas or secretory cells of the intestinal epithelium in slightly alkaline
environment achieved by pancreatic secretion of bicarbonates and bile acids from the gall
bladder (Deguara et al., 2003; Fard et al., 2007) (Table 2.3). The absorption of nutrients occurs in
the intestine by optimizing the intestinal surface area within the constraints of the coelomic
cavity. Mucosal folding and amplification of apical plasma membrane aid considerably to
increase the intestinal surface area. The major cell types in intestine of all fishes are enterocytes,
mucous cells, enteroendocrine cells, rodlet cells (in most teleosts), and ciliated and zymogen
cells (in elasmobranchs) (Wilson and Castro, 2011).
27
Figure 2.5 A conceptual diagram of digestion and absorption of dietary proteins in intestine of
fish.
Absorbed amino acids available for synthesis
Peptides Free amino acids
DIETARY FAECES
PROTEINS
Endogenous
Absorption
Loss
Endogenous Amino Acids
Proximal Intestine Distal Intestine
28
Table 2.3 Major enzymes in fish for the digestion of protein, peptides, amino acids and other
non-nitrogenous compounds.
Source Enzyme Substrate Specificity or products

Stomach Pepsins (pepsinogens) Proteins and Peptide bonds adjacent to
polypeptides aromatic AA
Exocrine Trypsin (trypsinogens) Proteins and Peptide bonds adjacent to
pancrease polypeptides arginine or lysine
Chymotrypsins Proteins and Peptide bonds adjacent to
(chymotrypsinogens) polypeptides aromatic AA
Elastase (proelastase) Elastin, some Peptide bonds adjacent to
other proteins aliphatic AA
Carboxypeptidase A Proteins and Carboxy terminal AA with
(procarboxypeptidase A) polypeptides aromatic or branched aliphatic
side chains
Carboxypeptidase B Proteins and Carboxy terminal AA with
(procarboxypeptidase B) polypeptides basic side chains
Ribonuclease RNA Nucleotides
Deoxyribonuclease DNA Nucleotides
Intestinal Enterokinase Trypsinogen Trypsin
mucosa
Aminopeptidases Polypeptides N-terminal AA from peptide
Dipeptidases Dipeptides Two AA
Nucleases Nucleic acids Purine and pyrimidine bases
Cytoplasm of Various peptidases Di-, tri- and AA
mucosal cells tetrapeptides
Adapted from: Ganong, 2009; Krogdahl and Sundby, 1999; Lauff and Hofer, 1984;
29
2.4.2.2. Assessing digestibility in fish: techniques, issues and limitations
The loss of indigestible matter from the diet as faeces is one of the primary reasons for
variations in the nutritional value of feed ingredients. Measurement of digestibility provides, in
general, a good indication of the availability of energy and nutrients, thus providing a rational
basis upon which diets can be formulated to meet specific standards of available nutrient levels.
In simple terms, digestibility is the measure of disappearance of nutrients in the digestive tract
usually estimated by the difference between the ingested nutrient and the amount of nutrients in
faeces. Various in-vivo and in-vitro methods to determine digestibility of nutrients in fish have
been developed over the years. The in-vivo methods mainly differ in faecal collection
approaches, use of indigestible marker, diet composition, level of test ingredients etc. (e.g.
Bureau and Hua, 2006; Bureau et al., 1999; Cho and Slinger, 1979; Cho et al., 1982; Forster,
1999).
2.4.2.2.1. Fecal collection methods
Meaningful estimation of digestibility relies on collection of fecal material using
adequate approaches. The most widely used technique is the collection of faeces from the lower
part of the intestine by stripping, suction or intestinal dissection (Austreng, 1978; Nose, 1967).
Each of these fecal collection methods has its own advantages and disadvantages. Major
shortcomings of stripping or dissection are: (a) removal of chime before complete digestion and
absorption; (b) contamination of faeces with blood and sloughed mucosal cells; and (c) handling
stress. It generally leads to an underestimation of the digestibility of nutrients (protein in
particular) because of contamination of feces with endogenous material that would otherwise be
reabsorbed before the feces are excreted (Cho et al., 1982).
30
Passive methods for the collection of faecal material yield more meaningful estimates of
digestibility of nutrients, if used correctly. These include the system of Ogino et al. (1973) in
which the feces are collected by passing the effluent water from the fish tanks through a filtration
column (TUF column), the system of Cho and Slinger (1979) in which a settling column is used
to separate the feces from the effluent water (Guelph system) and that of Choubert et al. (1979)
in which a mechanically rotating-screen is used to filter out fecal material (St-Pee system).
Vandenberg and de la Noe (2001) compared apparent digestibility coefficients (ADC) in
rainbow trout among three different faecal collection methods that include continuous filtration
and collection (St. Pee system, Choubert et al., 1979), settling column (Cho and Slinger, 1979)
and stripping by massaging the ventral surface from the anal fin towards the anus. The authors
reaffirmed that collection of faeces involving physical interactions with the animal such as
stripping results in lower ADC values vs. both passive faecal collection methods.
2.4.2.2.2. Indigestible markers
The standard approach in the indirect digestibility assessment for any fish species is the
incorporation of one or more inert compound as indigestible marker in the test diets. These
compounds are assumed to be homogeneously incorporated into the feed, do not interfere with
the digestive metabolism of the animals, never absorbed or utilized, analysed accurately in feed
and feces, and non-toxic and harmless to animal and human (Austreng et al., 2000).
Chromium oxide (Cr2O3) has been the most commonly used inert marker, but
digestibility values obtained using Cr2O3 are questionable because of shortcomings such as low
recovery in faeces (Fernandez et al., 1999; Lied et al., 1982), effect on intestinal microflora
(Ring, 1993), and different passage rate than the nutrients or the digesta (Dabrowski and
31
Dabrowska, 1981; Tacon and Rodriguez, 1984). Several other inert markers such as titanium
oxide (TiO2 Lied et al., 1982), polyethylene (Tacon and Rodrigues, 1984), hydrolysis resistant
ash and acid washed sands (Atkinson et al. 1984; Tacon and Rodrigues, 1984), have been
evaluated in fish with variable results.
Rare earth metal oxides such as yttrium oxide (Y2O3) or ytterbium oxide (Yb2O3) are
increasingly preferred in digestibility studies. They do not affect nutrient metabolism in fish
because of the very low concentration needed in the diets (at mg g-1 level) and can be easily
detected by plasma emission spectroscopy and by atomic absorption, thus, are measurable at a
very low concentration (Davies and Gouveia, 2006).
2.4.2.2.3. In-vitro assessment of digestibility
In the last few decades, considerable attention has been given towards in-vitro
digestibility assessment with enzymatic or acid digestion (Bassompierre et al. 1998, Carter et al.,
1999; Dimes and Haard, 1994; Dimes et al., 1994; Haard, 1992; Rungruangsak-Torrisen et al.,
2002). These assays are quick and useful but significant variations exist in the source and
number of enzymes, use of chemicals and environmental factors such as pH and temperature.
Another in-vitro method, the everted sleeve technique developed by Buddington et al.
(1987) and adopted in several studies (Berge et al., 1999; Rosas et al., 2008), have become a
useful technique that attempts to imitate the nutrient transport mechanisms in the gastro-
intestinal tracts. A drawback of this method is as solute concentration increases in the tissue, the
rate of uptake decreases or reaches a plateau and fails to provide accurate digestibility values
when large quantity of nutrients are available. Recently, a two-step hydrolysis method is
developed to address this issue using sequentially closed reactor for acid digestion and semi-
32
permeable membrane reactor for alkaline digestion, and at the same time, continuously removing
the solutes from the reactor (Hamdan et al., 2009; Morales and Moyano, 2010).
2.4.2.2.4. Limitations of digestibility of protein in feed ingredients
Digestibility gives a limited view of the nutritional value of an ingredient, and provides
no indication of the proportion of a nutrient used for growth and other metabolic functions (Stein
et al., 2007). Digestibility is not always a reliable indicator of the availability of proteins to
animals, notably for feed ingredients processed under high heat and pressure conditions.
Digestibility calculated by the difference between intake and fecal excretion doesnt
count for the amount from endogenous loss or microbial production. Endogenous loss of AA
depends on dietary ingredients (e.g., composition of soluble and insoluble fibres in the
ingredients, presence of anti-nutritional factors) and physiological state of the animal. In
addition, obtained values are based on the disappearance of nutrients from the digestive tract that
do not reflect the net breakdown or synthesis in the intestinal lumen or the form in which they
are absorbed. This is specifically an issue with ingredients that have undergone extensive thermal
processing. In heat-treated feed ingredients, some AA, such as lysine, may be present in
chemical forms, like Maillard reaction products, that may be absorbed but are metabolically
inactive and preclude their utilization for protein synthesis (Moughan and Rutherfurd, 1996).
2.4.3. Bioavailability of amino acids
Apparent digestibility of any nutrient is a measure of the disappearance of that nutrient
from the digestive tract, defined as that present in the feed but not in the fecal material (Hepher,
1990). This missing component is then considered to have been absorbed and utilized by the
33
animal which could be a misrepresentation of the true value of the ingredient because the
nutrient may be in a form able to be absorbed by the animal but may remain unavailable for
growth and other metabolic processes (Waibel et al., 1977). This has been observed in some
amino acids, such as lysine, the most chemically reactive amino acid, containing an -amino
group that is susceptible to chemical modification during processing and prolonged storage
(Moughan and Rutherfurd, 1996). These groups likely bind to others, forming structures which
resist chemical reaction, making them less available for metabolism.
Simple growth trials, where test ingredients replace fish meal or other established
ingredients in the diet and the subsequent effect on growth is monitored, have similar problems
with their value and replication due to interactions with other ingredients (Lewis and Bayley,
1995). For example, a diet containing a specific ingredient, or level of an ingredient, may meet
the nutritional requirements of an animal when some nutrients are supplied by certain other
ingredients (such as highly digestible essential amino acid content ingredients like fish meal) but
may not show the same effect if the diet was formulated with alternative ingredients. These
alternative ingredients may provide apparently adequate levels of nutrients but in forms which
are less effectively utilised (Regost et al., 1999).
A more relevant approach is to consider the available nutrient contribution of the test
ingredients to the diet. In this way, a more accurate assessment of the levels of each essential
nutrient in that ingredient actually usable by the animal can be undertaken, leading to more
confidence in the nutritional adequacy of the diet and less wastage of expensive ingredients
currently provided in excess to ensure nutrient requirements are met. In order to assess the
nutrient contribution of an ingredient to the diet, one must first determine the level of that
nutrient able to be used by the animal for metabolic processes (termed bioavailability).
34
Bioavailability is defined as the proportion of ingested dietary AA that is absorbed in a chemical
form that these AA potentially suitable for metabolism or synthesis (Ammerman et al., 1995;
Batterham, 1992; Lewis and Baily, 1995; Stein et al., 2007).
2.4.3.1. Methodological approaches
Traditionally, estimates of bioavailability of AA have been obtained using slope-ratio
assays. Slope-ratio assays provide direct and practical assessment of the availability of individual
amino acids and have been used to assess the bioavailability of individual amino acids for
animals (Lewis and Bailey, 1995). Batterham et al. (1979) first proposed the slope-ratio assay to
determine availability of a single amino acid, lysine in various plant proteins for growing pigs
and rats. Major and Batterham (1981) applied similar approach to determine availability of AA
in protein concentrates in chicks. A properly designed slope-ratio assay based on basal diet and
supplementation with a test protein source usually generate meaningful estimate of the
bioavailability of essential AA and other nutrients (Lewis and Bayley, 1995).
Several statistical approaches such as linear and non-linear regression approaches have
been proposed. For example, relative bioavailability of a nutrient from a test source compared to
the standard is determined from the ratio of the slopes of a linear regression or in the case of an
exponential regression, from the slopes of the steepness coefficients.
2.4.3.1.1. Slope-ratio assay linear regression models
Slope-ratios are usually determined by a linear regression model (y = b1 + b2*S + b3*T,
S - standard and T - test diets) that requires pre-validation of three assumptions: linearity of the
responses, common intercepts for all lines, and the equal basal level response to the common
35
intercept (Batterham et al., 1979; Finney et al., 1951, 1964; Littel et al., 1997). Fundamental
requirements for validity of a linear assay as described by Finney (1971) are: (1) common point
of intersection of the regression lines for standard and tests; and (2) the Y value of the common
point of intersection and the Y value of the mean response measured at X = lowest level or blank
level are not significantly different.
Linear models have been extensively used to determine bioavailability of individual
amino acids or minerals in poultry (Adeola, 1998; Batterham et al., 1986; Lumpkins and Batal,
2005; Major and Batterham, 1981; Parsons et al., 1998), swine (Adeola, 1996, 2009; Batterham
et al., 1990; Fanimo et al., 2006; Mateo et al., 2007; Petersen et al., 2011; Shoveller et al., 2009),
and recently, in fish (El-Haroun and Bureau, 2007; Li et al., 2008, 2009; Poppi et al. 2011;
Zarate and Lovell, 1997).
2.4.3.1.2. Non-linear approaches
In case of non-linear responses, an exponential regression model (y = b1 + b2*(1 exp
(b3*S + b4*T) can be used to estimate bioavailability from the ratio of the steepness coefficients
b3 and b4, where b1 (the response to basal diet) and b2 (difference between the lowest and
highest response) have to be equal (Kim et al., 2006; Littel et al., 1997; Liu et al., 2004).
Broken-line models can also be used to determine the RBV of the nutrient of interest where the
slopes of the linear portion of the responses are used (Coon et al., 2007; Wiltafsky et al., 2009).
2.4.3.2. Limitations of slope-ratio approaches
Like other assays, slope-ratio assays also have some limitations and are sensitive to the
experimental artefacts (Poppi et al., 2011). Two important dietary considerations for slope-ratio
36
assays are that the response to changes in test amino acid intake must be predictable and the
observed response must not be influenced by other dietary nutrients in the test feed ingredient
(Batterham, 1992; Littel et al., 1997). The latter condition is difficult to achieve with most
ingredients since most protein sources are complex ingredients containing variable levels of
various components (non-starch polysaccharides, anti-nutritional factors). Besides, the
composition and source of amino acids in the diets also play a role in feed intake and nutrient
utilization. Meaningful comparison of RBV estimates between different sources can be difficult
when variable composition of other nutrients or anti-nutrients in the sources aside from the
amino acid of interest affect feed intake, nutrient digestion and utilization.
2.4.4. The presence and effects of ANF and other non-nutritive components
2.4.4.1. General overview
Anti-nutritional factors (ANFs) are defined as innate components of an ingredient with a
limiting effect on feed intake, digestion, nutrient absorption, and well-being (Francis et al.,
2001). Plant protein ingredients contain a wide variety of ANF, most commonly protease
inhibitors, phytic acid, lectins, gossypol, antivitamins, allergens, saponin etc. (Tacon, 1997).
Among these, phytic acid (PA) and its salts, phytates have been identified as the major anti-
nutritional factors affecting nutrient utilization in farmed fishes with increasing inclusion of plant
protein ingredients in aquafeed (Francis et al., 2001). Common anti-nutritional factors in soybean
meal and their effects on salmonids are presented in the Table 2.4.
37
Table 2.4 Major anti-nutrients in soybean meals and their effects on salmonid fishes
Anti-nutrient Types of effects References
Trypsin inhibitors - bind with trypsin and Krogdahl and Holm, 1983;
chymotrypsin de Haard et al., 1996;
- smaller fish are more sensitive Murai et al. 1989
Lectin or - effects intestinal function Buttle et al., 2001
hemagluttanins - binds to the enterocytes lining the
intestinal villi
Antigenic proteins - effects non-specific defense Rumsey et al., 1994
(glycinin or glycinin) mechanisms
Saponin (glucosides) - palatability, feed intake Bureau et al., 1998
Phytic acid - overall depression in growth and Numerous studies
nutrient availability
Flavonoids (glycinin, - affects the synthesis of estrogens Pelissero et al., 1991
daidzin, genistin) - endocrine disruption, gamete
quality
- may have some positive effects
38
Other non-nutritive components in plant ingredients include indigestible starch and non-
starch polysaccharides. Dietary fibres are the sum of plant polysaccharides and lignin not
hydrolysed by endogenous enzymes (Kritchevsky, 1988; Wenk, 2001). Polysaccharides are
complex polymers of sugars occur in structural variations differing in side-chain types and the
distribution of glycoside linkages in the macro-molecular backbone (Figure 2.6). For example,
two pectins can differ in structure based on their composition of the smooth region composed of
apigalacturonan, homo-galacturonan, and xylo-galacturonan, and hairy chain ramified regions,
such as rhamno-galacturonan-I, and rhamno-galacturonan-II (Paulsen and Barsett, 2005).
39
Figure 2.6 Dietary fibre components (adapted from Thebaudin et al., 1997).
40
2.4.4.2. Phytic acid
Phytic acid (PA) - myoinositol hexaphosphate (IP6), a natural compound usually formed
during maturation of plant seeds, is a common constituent in plant ingredients (Figure 2.7). In
dormant seeds, phytate-P represents 60 to 90 percent of the total phosphorus, and is the primary
storage of both phosphorus and inositol in plant seeds (Reddy, 2001). However, its location
within the seed differs among plants. For example, ninety percent of the PA in corn is found in
the germ portion of the kernel, whereas in wheat and rice it is concentrated in the aleurone layers
of the kernel and the outer bran (ODell and De Boland, 1976). In most oilseeds and grain
legumes, it is associated with protein and concentrated within subcellular inclusions called
globoids that are distributed throughout the kernel; however, in soybean seeds, there appears to
be no specific location (Ravindran et al., 1995). Table 2.4 reports total phosphorus and phytate-P
content in some commonly used plant ingredients.
2.4.4.2.1. Interactions with minerals
Phytic acid has 12 replaceable protons or reactive sites. Six are strongly acidic, with pKa
of 1.5 to 2.0; two are weakly acidic with a pKa of approximately 6.0, and four are very weakly
acidic with pKa of 9.0 to 11.0 (Costello, et al., 1976; Erdman, 1979). This means that at pH
normally encountered in feeds and in the digestive tract, PA will carry a strong negative charge
and is capable of binding di- and trivalent cations such as Zn2+, Cu2+, Ni2+, Co2+, Mn2+, Fe2+, and
Ca2+ in very stable complexes (Wise, 1983, 1995; Persson et al., 1998; Maenz, et al., 1999;
Kaufman and Kleinberg, 1971; Vohra et al., 1965; Maddaiah, et al., 1964), thus reducing the
availability of these complexed minerals to the animal (Pallauf and Rimbach, 1997).
41
The order of potency of selected minerals to inhibit phytate hydrolysis by phytase at pH 7
is Zn2+ >> Fe2+ > Mn2+ > Fe3+ > Ca2+ > Mg2+ (Maenz et al., 1999). The ability of the different
metal ions to inhibit hydrolysis is related to the stability of the complex, the pH of the solution,
and the phytate to mineral molar ratio. A detail review on the interactions of these minerals with
phytic acid could be found in Lopez et al. (2002).
42
Figure 2.7 The structure of phytic acid (myo-inositol hexaphosphate, IP6) (from Reddy, 2001).
OH OH
OH
OH
OH
OH
43
Table 2.5 Total phosphorus and phyate-P content in the major plant ingredients currently
utilized in compounded aquafeed.
Feed ingredients Total P Phytate P Phytate P Source
(% DM) (% DM) (% total P)
Canola meal 0.88 0.67 59 Selle et al., 2003
Corn gluten meal 0.60 0.42 70 Ravindran et al., 1995
Corn protein concentrate1 0.23 0.16 70 This study
Ground corn1 0.28 0.18 64 This study
Indian mustard meal1 1.14 0.80 70 This study
Indian mustard protein conc.1 1.45 1.17 81 This study
Lupin seeds 5.70 3.50 61 Steiner et al., 2007
Soy protein concentrate1 0.80 0.50 63 This study
Soy protein isolate - 1.4-2.1 Reddy, 2001
Soybean meal1 0.68 0.42 62 This study
Wheat gluten1 0.24 0.09 38 This study
Wheat middlings1 0.63 0.46 73 This study

1
Analyses of total phosphorus and phytate-P content in the samples of these ingredients are
conducted by DANISCO Animal Nutrition, MO.
44
2.4.4.2.2. Interactions with proteins and amino acids
Phytic acid also creates binary (PA-protein) or ternary (PA-mineral-protein) complexes
(Kies et al., 2001). Selle et al. (2000) proposed that the de novo formation of binary protein-PA
complexes in the GIT as the key mechanism by which PA influences the AA digestibility.
Several modes of forming these complexes are suggested: (1) the presence of protein-PA
complexes in feedstuffs, (2) the de novo formation of binary and or ternary complexes in the
digestive tract, and (3) the inhibition of proteolytic enzymes (Kies et al., 2001; Selle et al., 2006).
The de novo formation of loose electrostatic association of PA and proteins in the GIT
occurs only at optimal pH conditions (Cheryan and Rackis, 1980; Maenz, 2001). At pH below
the iso-electric point (pKa) of protein, the anionic phosphate groups of PA binds strongly to the
cationic groups of the protein to form binary complexes that dissolve only at pH below 3.5. The
high pKa basic amino acids (Arg, Lys, His) are highly susceptible to chelation as they become
polar and positively charged at pH below their pKa. There are three possible protein-binding
sites for phytate at low pH: (1) the -NH2 terminal group and the -NH2 group of lysine; (2) the
imidazole group of histidine; and (3) the positively charged guadinyl group of arginine
(Cosgrove 1966). Okubo et al. (1976) reported that above the iso-electric point of protein (pH
4.9), glycinin component of soy protein stops binding to phytin. The binding potential increases
as pH decreases but maximizes at pH 2.5 (Adeola and Sands 2003).
Ternary PA-mineral-protein complexes are usually formed at the intermediate pH levels
(pH 5.0 and above), such as in intestinal environment (pH 8.5-8.8) (Riche et al., 2001). They are
formed via histidine, bound by mineral-PA complexes involving mainly Ca, Mg, and Zn. At pH
above 10, these ternary structures are disrupted, the protein is released, and what precipitates out
are the mineral-phytate complexes (Kies and Selle, 1998).
45
2.4.4.2.3. Interactions with proteolytic enzymes
Singh and Krakorian (1982) reported reduced activity of the proteolytic enzyme trypsin
by phytate at pH 7.5 in an in-vitro study that increases with the increase of Na-phytate
concentration. Trypsinogen, an inactive precursor of trypsin, is known to bind Ca-ions at two
sites, where as trypsin has only one binding site. The authors hypothesized that the possible
binding of Ca in trypsin by phytate inhibits the activity of the enzyme. Later, Caldwell (1992)
confirmed this hypothesis, and reported negative effects of phytic acids bound Ca on the
activation of trypsinogen and the stability of trypsin.
2.4.4.2.4. Effect of PA on nutrient utilization
Laining et al. (2010) reported decreasing vertebral deposition of Ca, Mg and Zn in
Juvenile Japanese Flounder (Paralichthys olivaceus) with increasing dietary PA levels. Among
the minerals, Ca and Mg deposition in fish were statistically similar in all diets except for in
those fed highest PA (2%) diets. However, Zn deposition in vertebrae decreased significantly at
even 0.5% PA, from 115 mg kg-1 in control to 100 mg kg-1. Similar effects on Zn bioavailability
were also reported in channel catfish Ictalurus punctatus (Gatlin and Phillips, 1989), tilapia
Oreochromis niloticus (Nwanna, 2004), and rainbow trout (Vielma et al., 2004).
Interactions of PA with protein in fish showed significant decrease in CP digestibility in
several fish species, such as Atlantic salmon Salmo salar (Sajjadi and Carter, 2004a,b), rainbow
trout Oncorhynchus mykiss (Spinelli et al., 1983; Sugiura et al., 2001) or common carp Cyprinus
carpio (Hossain and Jauncey 1991). PA also reported to affect digestibility of amino acids Phe,
46
Ile and Leu in poultry (Cowieson et al., 2006; Newkirk and Classen, 2001), and swine (Kies,
2005).
2.4.4.3. Fibre components and their effects
Physico-chemical properties of different components of dietary fibre vary in their
solubility, gel formation, viscosity, holding and absorption capacity of water and lipid. Although
the effects of these properties are studied rarely, these properties can significantly affect the
digestive processes in an animal (Souffrant, 2001). In farmed animals, most studies on dietary
fibre from various plant sources have dealt with inclusion levels and their effects on protein and
energy utilization, and growth. For example: pig (Laplace et al., 1989; Mitaru et al., 1984;
Souffrant, 2001), poultry (Slominski and Campbell, 1990; Villamide and San Juan, 1998), or fish
(Anderson et al., 1984; Bromley and Adkins, 1984; Hemre et al., 1995; Kraugerud et al., 2007).
One of the earliest studies in fish was by Bromley and Adkins (1984) where the authors
reported a significant increase in the size of the stomach of rainbow trout from 2% to 3.5% of the
body weight with increasing dietary cellulose level from 30% to 40%. They also reported a
significant reduction in protein deposition rate when fish were fed high fibre diets. Recent
studies have shown that not the dietary fibre, but the composition of its components influences
digestive processes in fish (Amirkolaie et al., 2005; Glencross, 2009; Ovrum-Hansen and
Storebakken, 2007; Zahedifar et al., 2002; Zhu et al., 2005). Glencross (2009) reported that the
ADC of DM, CP, and GE in rainbow trout was affected more by lignin than pectin or cellulose.
Considering the limited activity of intestinal microflora in salmonids, Leenhouwers et al. (2008)
hypothesized that the biological activity of various fibre components on the digestive processes
are governed by the composition of the polymer chains of the fibre components.
47
2.4.4.4. Lignin
Lignin, a phenolic plant polymer responsible to provide structural support and the flow of
nutrients, forms an integral part of the secondary cell walls of most plants and algae (Jung and
Fahey, 1983). They are derived from the aromatic amino acid producing shikimate-chorismate
pathway in vascular plants (Lewis et al., 1998), providing rigidity to the plants and rendering the
walls water-impermeable (Whetten and Sederoff, 1995), and constitute some of the
metabolically most expensive products generated by plants (Lewis and Yamamoto, 1989).
A lignin polymer is composed of three monolignols - sinapyl alcohol, coniferyl alcohol,
and p-coumaryl alcohol, which upon incorporation, are referred as syringyl (S), guaiacyl (G), or
hydroxyphenyl (H) units, respectively. The proportion of S, G, and H varies among different cell
types and among species, can substantially affect the physical properties of the lignin polymer
(Boerjan et al., 2003). It is believed that monolignols are stabilized to lignin by -glucosidases
(from the soluble fibre -glucans) and glucosyl transferases (Dharmawardhana and Ellis, 1998).
2.4.4.4.1. Lignin biosynthesis
The lignification process encompasses the biosynthesis of monolignols, their transport to
the cell wall, and polymerization into the final molecule. The types of lignin formed are
dependent upon the release of monolignols, such as p-coumaryl alcohol, coniferyl alcohol, and
sinapyl alcohol into the wall matrix followed by diffusion to the site of incorporation (Figure
2.8). These monolignols, differing only in the substitution pattern on their aromatic rings, are
polymerized into lignin.
48
Figure 2.8 Structure of three monolignol precursors to lignin (adapted from Hatfield and
Vermerris, 2001).
p-Coumaryl alcohol Coniferyl alcohol Sinapyl alcohol
49
2.4.4.4.2. Effects of purified lignin
In nature, lignins form covalent bonds with cellulose and hemicelluloses, and do not exist
in their pure form. Three major lignin products are currently available: 1) ligno-sulphonates
from the sulphuric acid treatment in the wood-pulping industries; 2) kraft lignin from alkaline
treatments of the wood fibres; and 3) purified lignin produced by disrupting chemical integrity
of native lignin-cellulose complexes using aqueous-ethanol extraction process at temperatures
between 1850C and 1950C (Baurhoo et al., 2008). Lora et al. (1993) reported that purified lignin,
in contrast to other commercially available forms, consists of low-molecular weight phenolic
fragments, and may possess some functional properties unavailable in other forms.
Anti-microbial and pre-biotic effects of the phenolic components of purified lignin and
associated lignans are well-known (Davidson and Brenan, 1981). Phenolic acids (lignans) and
flavonoids are used as preservatives to prevent microbial contaminations in food products (Roller
and Sreedhar, 2002; Ultee et al., 2000). Mono-phenolic compounds in lignin, such as carvacrol,
thymol and cinnamaldehyde possess anti-bacterial properties. Carvacrol and thymol disintegrate
bacterial cell-membranes by lowering the intra-cellular pH. The acidic condition depletes the
intra-cellular ATP concentration eventually killing the bacteria (Baurhoo et al., 2008). Baurhoo
et al. (2007a,b) also suggested some prebiotic effects of lignin increasing the concentration of
beneficial microflora, Lactobacilli and Bifidobacteria in the intestine of broiler chickens at level
<2.5% in the diets.
2.4.4.4.3. Effect of lignin on minerals and amino acid availability
Studies on some terrestrial animals suggest deleterious effects of lignified fibres on
mineral, amino acid and energy digestibility (Wenk et al., 1998). Wenk et al. (1998) reported
50
significant reduction in energy digestibility with the diets containing all three forms of lignins
than the lignin-free control diet.
In vitro studies showed significantly higher binding capacity of lignin with bi-valent ions
of minerals (e.g., Fe+2, Cu+2 and Zn+2) at intestinal pH (e.g., pH ~5) than pectin and cellulose
(Platt and Clydesdale, 1987). In a study, Fernandez and Philipis (1982) reported hydroxyl and
methoxy groups of lignin react with Fe to form highly stable hexadentate complexes, and
concluded that lignin, but not cellulose or low methoxy pectin, is a potential binder of Fe+2 under
pH conditions similar to those in the proximal intestine of animals. The authors suggested two
active sites in lignin molecule that bind with these minerals in the order of Fe > Cu > Zn.
Lignin also forms protein complexes under oxidative conditions that are generally
resistant to microbial fermentation in ruminant (Fernandez and Philipis, 1982; Zahedifar et al.,
2002). In a study, Whitmore (1982) reported high binding capacity of lignin with proline
followed by serine and glycine. The author hypothesized that proteins, specifically those
containing hydroxyproline, could be very susceptible to bonding with dehydrogenation polymer
of lignin under oxidizing conditions. In fish, Glencross et al. (2008) reported significant
deleterious effects of lignin on CP, lipid and GE digestibility in rainbow trout.
2.4.5. Physical characteristics of aquaculture feeds
2.4.5.1. Overview of feed production technology
Feed manufacturing is the physical process of forming ingredient mixtures in to particles
used to feed fish or shrimp. Any aquaculture feed needs to be stable enough to hold the nutrients
together for the time to be ingested. It also needs to withstand stresses occurred during handling
and transportation. Fines or undersized feed particles are not consumed by fish and contribute to
51
increased nitrogen and phosphorus in the water, reduced water quality in the culture units, and
increased feed cost. The basic steps of manufacturing feed involve grinding, mixing,
conditioning, pelleting, cooling, top-dressing, sacking, storing, and shipping (Figure 2.9).
Grinding, mixing, conditioning and expansion all are important critical steps before
pelleting (Hardy and Barrows, 2002). The main purpose of grinding is to reduce the particle size
using various mechanical processes such as impact (e.g., hammer mills), cutting (e.g., rotary
cutters), and crushing (e.g., roller mills). Ground particles are then passed through sieves to
achieve a uniform particle size. All ingredients are mixed to produce a homogenous blend. Some
or all the oil can be added to the mixture at this time.
After mixing, feed mixtures are conditioned in a chamber with steam and physical
agitation. Conditioning prepares a feed mixture for pelleting by increasing the moisture content,
heating the mixture, and adding energy to activate the gluten proteins (NRC, 2011). Feed
mixtures can be conditioned for short time (~30 secs) for compressed pellets or up to 5 minutes
before extrusion pelleting. The longer conditioning period also results in a higher degree of
starch gelatinization improving overall carbohydrate digestibility (Aas et al., 2011).
Fish and shrimp require different types of pellets such as extruded slow sinking or
floating pellets or compressed sinking pellets, respectively, to maximize intake and to reduce
wastes from unused feed. Different types of feed can be produced by compression that produces
feed with very high bulk density (0.5-0.6 g cm-3) or by extrusion, a more versatile process that
can be used to produce floating pellets or slow sinking pellets.
52
Figure 2.9 A generic diagram of the steps in a typical aquaculture feed manufacturing process.
53
2.4.5.2. Physical properties of pellets and their importance
Physical properties of an ingredient are a measure of its effect on the characteristics of a
feed (Aas et al., 2011). An ingredient may have an excellent nutritional profile but its value
diminishes if cannot be incorporated effectively into a feed (Refstie and sgrd, 2009).
Physical properties include fluid absorption capacity, bulk density and hardness, sinking
velocity, viscosity and water stability of the pellets. Sinking or floating properties of the pellets
can also be determined by bulk density (Table 2.6). Besides bulk density, pellets of different
species of fish and shrimp require different floating or sinking properties that also largely
correspond to the final total fat content in the pellet (Table 2.7). Durability of a pellet can be
determined by hardness and water stability. Water stability of a pellet is usually determined by
the time it takes to disintegrate and may include the determination of the dry matter loss. Rapidly
disintegrating feeds are highly undesirable in commercial farming practices causing significant
environmental and economical losses (Cho and Bureau, 2001; Obaldo et al., 2002).
54
Table 2.6 Indication of final pellets bulk density in relation to floating or sinking properties
(Durand, 2011)
Feed characteristics Fast sinking Slow sinking Neutral Floating
floatability
Bulk density (g cm-3) >640 540 - 600 480 540 <450
Table 2.7 Final fat contents (%) in the pellets of different aquaculture species in relation to
floating or sinking properties (Durand, 2011)
Species Salmon Trout Carp/tilapia/catfish Shrimp
Final pellet total fat, % >35 15-35 5-15 <5
Texture bulk density Slow sinking Slow sinking Floating Fast sinking
55
2.4.5.3. Different factors affecting these physical properties
Type of feedstuffs (Olsen et al., 2006; Refstie et al., 2006), feedstuff components
(Hansen and Storebakken, 2007; Hilton et al., 1981), and processing and manufacturing
conditions (Sorensen et al., 2005; Sorensen et al., 2009) have substantial effects on the stability
of a feed requiring to adjust processing and manufacturing parameters that include physical,
chemical and thermal treatments (e.g., moisture, temperature, pressure, flow, torque, screw
speed) (Draganovic et al., 2011; Thomas, 1996).
Besides increasing the waste-outputs (Cho and Bureau, 2001), rapid disintegration of a
feed after ingestion appeared to cause oil-belching in salmonids fed high-fat diets often
coinciding with gastric dilation air sacculitis (GDAS) (Baevefjord et al., 2006; Forgan and
Forster, 2007; Lumsden et al., 2010).
2.4.5.4. Components of plant proteins influencing the pellet characteristics
Pellet water stability is highly vulnerable to the changes in ingredient composition of the
diet, physical and chemical properties of the ingredients, and processing and manufacturing
conditions. Water stability can be improved by adding starch and soluble (e.g., guar gum,
Brinker, 2007; Brinker and Reiter, 2011; Storebakken, 1985) or insoluble (e.g., cellulose,
Bromley and Adkins, 1984; Hansen and Storebakken, 2007; Lupatsch et al., 2001) fibres as
binders in fish feed.
Level and composition of the fibre components in the plant protein ingredients alter the
physical properties of the mash and pellets during processing often requiring modifications of the
manufacturing parameters. Careful attention to modification of processing and manufacturing
conditions is needed for producing highly stable pellets.
56
2.5. Conclusion
Long-term sustainability of aquaculture operations depends on improving cost-
effectiveness of feed and increasing the reliance on economical protein sources to replace or
supplement conventional protein ingredients. Salmonid feeds are formulated with an increasingly
wide variety of ingredients of plant, animal or microbial origins.
Plant protein ingredients significantly differ from their animal counter-parts in terms of
AA compositions, fatty acid and lipid profiles, starch and fibre contents, and presence of some
ANF. They are usually deficient in one or more EAA that need to be supplemented to meet the
requirement of farmed fishes. Further research is needed to investigate the effects of various
ANFs and fibre components, individually and in combinations, on nutrient digestibility,
utilization and metabolism as well as on the intestinal function, structure, defense mechanisms
and gut micro-flora. This will add to the ability to predict how a novel plant ingredient as well as
combinations of plant ingredients may affect the fish and to identify steps needed to
reduce/prevent adverse health effects.
Specific effects of alternative protein sources on the digestive physiology of fish have
been most closely studied in the case of soybean products in feeds for farmed salmonids. The
components in soybeans that lead to reduced nutrient digestibility and the inflammatory response
in the distal intestine are still largely unknown, but affect many specific digestive processes,
from feed intake to enzyme activities to nutrient transport to gut histology. Such details are often
lacking in studies focusing on protein sources that are already in use in formulated feeds for
various fish species. Thus the long-term implications they may have on fish production, health,
57
and product quality are questionable, and this topic deserves substantial investments in further
research to preserve and enhance the sustainability of the aquaculture industry.
Evaluation of a plant-protein ingredient for its inclusion in aquafeed therefore, needs
comprehensive species-specific studies starting with characterization of physical and chemical
(amino acids, fatty acids, and mineral compositions) properties followed by digestibility and
bioavailability of amino acids, and growth and health implications of the ANFs and fibre
components of the ingredient.
58
CHAPTER 3
DIGESTIBILITY OF AMINO ACIDS IN TWO NOVEL INGREDIENTS, INDIAN
MUSTARD PROTEIN CONCENTRATE AND INDIAN MUSTARD MEAL COMPARED TO
THAT OF A SOY PROTEIN CONCENTRATE IN RAINBOW TROUT AND ATLANTIC
SALMON
Abstract
Digestibility trials were carried out to determine the apparent digestibility of amino acids
to rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) of two novel plant
protein ingredients, Indian mustard protein concentrate (IMC, 62% crude protein) and Indian
mustard meal (IMM, 42% crude protein) produced through a proprietary low temperature
process, and a commercially available soy protein concentrate (SPC, 57% crude protein). The
apparent digestibility coefficient (ADC) of crude protein (CP) in IMC was 90% and 97% for
trout and salmon, respectively. The ADCs of CP in IMM and SPC were also very high (>95%)
for both species. The ADCs of lysine (95%), isoleucine (91%), leucine (86%) and phenylalanine
(90%) in IMC were slightly lower (P<0.05) than those in IMM and SPC for rainbow trout.
Conversely, in Atlantic salmon, ADCs of isoleucine in both IMC (94%) and IMM (92%) were
lower than those found in SPC (97%). Despite small differences, ADC of most AA in these
novel products were very good (>90%). These results suggest that low glucosinolate Indian
mustard protein concentrate and Indian mustard meal are ingredients of high nutritive value.
However, it is hypothesized that the high phytic acid level in the Indian mustard protein
concentrate may negatively affect the digestibility of CP and certain amino acids when this
ingredient is used at high levels in the feed of fast growing fish.
59
3.1. Introduction
Plant protein sources are increasingly used to satisfy the growing demands of the
aquafeed industry for economical protein sources (Hardy, 2010). Meals and protein concentrates
produced from rapeseed (Brassica napus) and canola (Brassica rapa) cultivars with low
glucosinolates (<30 mol g-1 of oil-free dry matter of seed) and erucic acid (<2% of total fatty
acids in the oil) have been widely studied and are used in aquaculture feeds worldwide (Cheng et
al., 2010; Drew et al., 2007b; Shafaeipur et al., 2008; Thiessen et al., 2003; 2004; Zhou and Yue,
2010). Cultivation of another oilseed from the Brassicacae family, Brassica juncea, known as
Indian or oriental or brown mustard, is gaining popularity in some regions of North America,
India and Australia since it is generally more tolerant to heat, drought and fungal infection than
rapeseed or canola (Chevre et al., 2008; Oram et al., 1999; Wijesundera et al., 2008; Woods et
al., 1991). Some Indian mustard varieties yield more seeds per pod, produce heavier seeds, and
contain higher dry matter in the seeds than the canola varieties (Chauhan et al., 2007;
Gunasekera et al., 2006; Wright et al., 1995). Low drought susceptibility index (a combination of
growth, maturation (days), number of pods and seed yield per pod, and dry matter content) of
some Indian mustard varieties is further increasing the potential of growing this crop in the
drought-prone regions of the world.
The development of B. juncea cultivars with oil and nutritional characteristics similar to
canola has enhanced their potential in animal feeds (Burton et al., 2004). The nutritive value of
low glucosinolate Indian mustard meals (Jia et al., 2008; Meng et al., 2006; Newkirk et al.,
1997), and Indian mustard protein concentrates and isolates (Thacker and Petri, 2010) have been
evaluated in the diets of poultry. These studies indicated that the nutritional value of meals and
concentrates from low glucosinolate Indian mustard was equal or superior to that of canola meal
60
samples derived from B. napus and B. rapa cultivars. The lower protein and high levels of fibre
(indigestible carbohydrates) in the Brassica protein meals limit the potential of these ingredients
in highly digestible nutrient dense salmonid fish feeds (Carter and Hauler, 2000; Hardy, 2010).
Different processing techniques have been developed to reduce the levels of indigestible
carbohydrate and to produce high protein ingredients from a variety of plant co-products (Bureau
and Cho, 1997; Glencross et al., 2010; Hardy, 2010). Studies have indicated that protein
concentrates of soybean (Bureau and Cho, 1997; Kaushik et al., 1995; Storebakken et al., 1998a,
b) and canola (Higgs et al., 1994; Mwachireya et al., 1999) are highly digestible and can be
included at high level in the diets of salmonid fish species. Nutritional quality of these plant
products can vary significantly by the sources of raw materials and processing equipment,
techniques, and conditions. Processing techniques such as expeller or solvent extraction
(Glencross et al., 2004b), extraction temperature and time (Fenwick et al., 1986), and drying
conditions (Glencross et al., 2007a; Stewart et al., 2003) may play important roles in modifying
nutritive values of oilseed processing co-products.
Fish, like any other animal, have a dietary requirement for essential amino acids (EAA)
and non-specific amino group sources. Fish feeds are increasingly formulated on a digestible
amino acid basis (El-Haroun et al., 2009; Gaylord et al., 2010; Poppi et al., 2011; Yamamoto et
al., 2005). However, very limited effort has been invested in estimating the digestibility or
availability of individual amino acids in fish feed ingredients (Bureau, 2008; De Silva et al.,
2000; Kaushik and Seiliez, 2010). Poor characterization of the digestibility or bioavailability of
nutrients results in the need to use broader safety margins when formulating feeds, reducing the
ability to formulate feeds on a least-cost basis, and limiting the confidence of feed formulators in
the nutritive value of many ingredients (Bureau, 2006). Accurate information on digestibility of
61
amino acids of traditional and novel ingredients is therefore, essential to improve the
cost-effectiveness of aquaculture feeds.
Subtle differences in the digestibility of some nutrients appear to exist between Atlantic
salmon and rainbow trout - the two most widely cultured salmonid species (Azevedo et al., 2004;
Glencross et al., 2004a; Hua and Bureau, 2009; Krogdahl et al., 2004; Refstie et al., 2000).
However, limited information is available on potential differences in the digestibility of amino
acids between these two species. This study investigated the apparent digestibility of crude
protein and amino acids in novel Indian mustard meal and a novel Indian mustard protein
concentrate produced using a proprietary low temperature (~60oC)extraction process using
florine based solvent. The digestibility of these two novel ingredients was compared to that of a
commercially available soy protein concentrate in rainbow trout (Oncorhynchus mykiss) and
Atlantic salmon (Salmo salar).
3.2. Methods
The digestibility trials were conducted at the UG/OMNR Fish Nutrition Research
Laboratory (Department of Animal and Poultry Science, University of Guelph, Ontario, Canada)
using the standard protocol of Cho et al. (1982) and Bureau et al. (1999). Indian mustard protein
concentrate (IMC) and meal (IMM) were obtained from Bio-Exx Specialty Proteins Ltd.
(Toronto, ON, Canada) and the soy protein concentrate (SPC HP 300) was obtained from
Hamlet Protein A/S (Aarus, Denmark). The Indian mustard protein concentrate and meal are
produced using a proprietary low temperature extraction process while the soy protein
concentrate is produced through a proprietary enzymatic treatment and aqueous extraction
62
process. Digestibility of nutrients in these ingredients for rainbow trout and Atlantic salmon was
determined in two consecutive trials carried out in the same facilities, same aquatic systems,
protocols and staff.
3.2.1. Dietary design
A high quality reference diet (basal diet, Table 3.1) was prepared and combined with each
test ingredient at 70:30 ratio (as is basis) to produce three test diets (one for each ingredient), as
per the indirect digestibility protocol of Cho et al. (1982).
An inert marker, yttrium oxide-Y2O3 (Sigma-Aldrich Inc., MD, USA), was added to the
basal diet at 100 mg kg-1 as digestion indicator. The diets were mixed (using a Hobart mixer
Hobart Ltd., Don Mills, Ontario), pelleted (using a laboratory steam pellet mill California
Pellet Mill Co., San Francisco, California), and dried in forced air at room temperature for 24h.
Dried pellets for each diet were kept at 4oC until used. The amount of diet needed was weighed
weekly and kept at room temperature. The proximate composition, gross energy and amino acid
profiles of three test ingredients and four diets are reported in the Tables 3.2 and 3.3,
respectively.
63
Table 3.1 Ingredient composition of the basal diet (%)
Ingredients %
Fish meal, herring, 70% crude protein 25
Blood meal, porcine, spray-dried 11
Corn gluten meal, 60% crude protein 25
Wheat middlings, 17% crude protein 10
Corn, grain ground 10
Vitamin premix (Martin Mills, PM1-83) 1
Mineral premix (Martin Mills, 9504) 0.5
Dicalcium phosphate 0.5
Fish oil, herring 17
Total 100
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Table 3.2 Proximate chemical composition, gross energy, essential amino acid profiles, total
phosphorus and phytic acid content of the test ingredients (as is basis)
Ingredients
Proximate composition (as is) Indian mustard Indian mustard Soy protein
concentrate meal concentrate
Dry matter, % 95 95 93
Organic matter, % 87 88 86
Crude protein, % 62 44 57
Lipid, % 0.2 1.9 2.2
Total carbohydrate, % 30 48 33
Neutral detergent fibre, % 13 17 7
Acid detergent fibre, % 11 13 6
Ash, % 8 7 8
Gross energy, kJ.g-1 19 19 19
Essential amino acids (% in the weight of ingredient as is)
Arginine 4.1 2.8 3.9
Histidine 1.6 1.1 1.4
Isoleucine 2.4 1.7 2.5
Leucine 4.2 2.9 4.1
Lysine 2.6 1.9 2.7
Methionine+Cystine* 2.8 1.9 1.3
Phenylalanine 2.3 1.6 2.7
Threonine 2.2 1.6 1.8
Tryptophan* 1.0 0.6 0.6
Valine 3.0 2.1 2.7
Phosphorus and phytate-P (as is)
Total phosphorus, % 1.5 1.1 0.8
Phytate-P, % 1.2 0.8 0.5
Phytate-P is calculated as phytic acid*0.282; ; * manufacturer data

65
Table 3.3 Proximate analysis and, gross energy, essential amino acid profiles, total phosphorus
and phytic acid concentrations of the diets (as is basis)
Diets
Proximate composition (as is) 1 2 3 4

70%-Ref + 70%-Ref + 70%-Ref +
Ref
30%-IMC 30%-IMM 30%-SPC
Dry matter, % 96 95 96 97
Organic matter, % 88 89 89 89
Crude protein, % 51 53 48 53
Lipid, % 19 13 14 14
Total carbohydrates, % 21 23 29 24
Neutral detergent fibre, % 8 9 11 7
Acid detergent fibre, % 4 6 7 4
Ash, % 6 7 6 7
Gross energy, kJ g-1 24 23 23 23
Essential amino acids (% in the weight of diet)
Arginine 2.9 3.4 2.7 3.1
Histidine 1.9 1.8 1.5 1.6
Isoleucine 2.0 2.2 1.8 2.1
Leucine 6.7 5.9 5.2 5.8
Lysine 2.8 2.9 2.4 2.5
Methionine+Cystine 1.6 2.0 1.7 1.6
Phenylalanine 2.9 2.8 2.4 2.8
Threonine 1.9 2.0 1.7 1.8
Valine 3.5 3.3 2.8 3.1
Phosphorus and phytate-P
Total phosphorus, % 1.0 1.2 1.1 1.0
Phytate-P , % 0.3 0.6 0.4 0.3
Ref reference diet, IMC Indian mustard protein concentrate, IMM Indian mustard meal, and SPC soy protein
concentrate. Phytate-P is calculated as phytic acid*0.282.
66
3.2.2. Experimental conditions
Rainbow trout (Oncorhynchus mykiss, Ontario domestic fall-spawning strain) and
Atlantic salmon (Salmo salar, Lahave River strain) fingerlings were obtained from the Alma
Aquaculture Research Station of the University of Guelph (Elora, ON, Canada) and the
Normandale Fish Culture Station of the Ontario Ministry of Natural Resources (Normandale,
ON, Canada), respectively. Fish were stocked in individually aerated fibreglass tanks (60-L),
where water temperature and photoperiod were maintained at 15oC and 12h L 12h D cycle,
respectively. Fish were acclimatized to the experimental conditions for seven days before
starting the experiments. During that time, fish were fed a high quality commercial salmonid diet
(Profishent High Energy Feed, Martin Mills, Elmira, ON, Canada).
Rainbow trout (average weight 71g) and Atlantic salmon (average weight 65g) were
stocked in 24 tanks at 13 and 15 fish tank-1, respectively. The four experimental diets were
randomly allocated to two collection units each. Each unit has three tanks with a column for
faeces collection. The fish were acclimatized to the experimental diets for four days before the
initiation of faeces collection. Fish were hand-fed to near-satiety three times daily between 0930
and 1600 h. Feed residues and faeces were removed everyday an hour after the last daily meal.
One-third of the water in the tanks was drained during the process to ensure complete removal of
faeces and uneaten food. At 0900 h of the following day, the settled faeces and surrounding
water were gently withdrawn into a large centrifuge bottle (250 ml). Collected samples were then
centrifuged at 4,000 x g for 15 min (Eppendorf Centrifuge 5810R, Eppendorf AG, Hamburg,
Germany), separated from supernatant, and stored in a container at -20oC till the end of the trial.
A total of five pooled samples for rainbow trout and four pooled samples for Atlantic salmon
67
were collected for each diet over the experimental periods. At the end of the trials, samples were
lyophilized, finely ground, and stored at -20oC for proximate and amino acid analysis.
3.2.3. Chemical analyses
Diets, ingredients, and faeces samples were analyzed for dry matter (DM) and ash
according to AOAC (1995), crude protein (CP, %N x 6.25, Dumas method) using a
LECO Analyzer (LECO Corporation, St. Joseph, MI), lipids with petroleum ether
extraction at 90oC and 150 PSI for 60 min using ANKOM XT-20 analyzer (ANKOM
Technology, Macedon, NY), and gross energy (GE) using an automated oxygen bomb
calorimeter (Model # 1271, Parr Instruments, Moline, Illinois). Neutral detergent fibre
(NDF) and acid detergent fibre (ADF) contents of the ingredients and the diets were
determined according to the methods of Van Soest et al. (1991) using an Ankom 200
Fibre Analyzer (ANKOM Technology, Macedon, NY). Phosphorus and Y2O3 was
analysed in a contracting laboratory (Laboratory Services Division, University of Guelph,
ON, Canada) using inductively coupled plasma optical emission spectroscopy technique
(ICP-OES). Phytic acid content in the ingredients and diets were analyzed using high
performance liquid chromatography (HPLC, Waters Corporation, Millford, CA)
according to the method of Lehrfeld (1989) at the Danisco Animal Nutrition laboratory,
Danisco, St. Louis, MO. Amino acid profiles of the test ingredients, diets and faeces were
analyzed at the Amino Acid Laboratory of the Toronto Hospital for Sick Children (ON,
Canada) using an acid hydrolysis method. For the quantification of amino acids,
approximately 0.05 g of dried sample was hydrolysed in 15 ml test tube with 450 L of
6N HCl and 1% phenol for 24 hrs at 110oC under pre-purified nitrogen atmosphere. After
68
24 hrs, tubes were removed from oven, cooled, capped, vortex mixed and centrifuged at 2500
rpm. Then, 5 L of the supernatant was derivatized following PICO-Tag method (Cohen et al.,
1989). After derivatization, supernatant were re-dissolved in 150 L phosphate buffer and 2 L
of this solution was injected into the column of an ACQUITY Ultra-Performance LC machine
(Waters Corporation, Millford, CA) for quantification of all amino acids except tryptophan,
methionine and cysteine which are partially destroyed in the hydrolysis process.
3.2.4. Calculations
The apparent digestibility coefficients (ADC) for the nutrients and energy of the test and
reference diets were calculated as follows (Cho et al. 1982):
ADC = 1-(F/D x Di/Fi), where D = % nutrient (or kJ.g-1 GE) of diet, F = % nutrient (or
kJ.g-1 GE) of feces, Di = % digestion indicator of diet (Y2O3), Fi = % digestion indicator of feces
(Y2O3). ADC of the test ingredients (ADCingr) and individual amino acids were calculated based
on the digestibility of the reference diet and the test diets using the equation proposed by Forster
(1999) as mathematically simplified by Bureau and Hua (2006):
ADCtest ingredient = ADCtest diet + [(ADCtest diet - ADCref. diet) x (0.7 x Dref / 0.3 x Dingr)] where,
Dref = % nutrient (or kJ.g-1 GE) of reference diet mash (as is), Dingr = % nutrient (or kJ.g-1 GE) of
test ingredient (as is). Total carbohydrate content is calculated as 100-CP-lipid-ash and organic
matter content is calculated from the difference between dry matter and ash.
3.2.5. Statistical analysis
Data were analysed using SAS v9.2 (Cary, NC, USA). ADC of nutrients and amino acids
of the three test ingredients were compared using Factorial ANOVA under Proc GLM.
Significant differences were determined by Tukey's HSD (honestly significant difference) test at
69
P<0.05 level. Significance of the species-ingredient interaction effects were determined
by the probability values of their least square means.
3.3. Results
3.3.1. Apparent digestibility of energy and nutrients
Table 3.4 reports the ADC of proximate components, GE and EAA of the
reference and the test diets. ADC of most nutrients and GE were significantly different
(P<0.05) between rainbow trout and Atlantic salmon except those for total carbohydrates
(51% for rainbow trout and 49% for Atlantic salmon) and ash (60% for rainbow trout
and 55% for Atlantic salmon) (Table 3.5). ADC of all proximate components,
phosphorus and GE were significantly different among the ingredients (P<0.05). ADC of
CP in the SPC (99%) was higher (P<0.05) than the Indian mustard products (93% and
97% for IMC and IMM, respectively). The ADC of lipid was high, exceeding 100% in
some instances, for all three ingredients because of very low lipid content in the
ingredients (i.e., 0.2, 1.9 and 2.2% in IMC, IMM and SPC, respectively).
Significant species-ingredient interaction effect was observed on the ADC of DM,
OM, CP, P and GE. Least square means of the ADC of DM and GE in IMM for Atlantic
salmon were significantly lower (P<0.0001 and P<0.001, respectively) than those in
IMM for rainbow trout. However, among the six species-ingredient interactions, ADC of
CP in all test ingredients for Atlantic salmon and in IMM and SPC for rainbow trout were
higher than the ADC of CP in IMC for rainbow trout (P<0.001). Lower ADC of CP in
70
IMC for rainbow trout may be attributable to the slightly lower digestibility of four EAA,
isoleucine, leucine, lysine and phenylalanine compared to other amino acids (Table 3.6).
3.3.2. Apparent digestibility of amino acids
ADC of eight EAA and the effects of species, ingredient and species-ingredient
interactions are reported in Table 3.6. ADC of all EAA were not significantly different between
rainbow trout and Atlantic salmon except for those of arginine (P<0.05), histidine (P<0.01) and
valine (P<0.05). Conversely, ADCs of all EAA were significantly different (P<0.05) among the
ingredients except for that of arginine (Table 3.6). None of the six possible species-ingredient
interaction scenarios were significant for the ADC of arginine and threonine. Despite the lack of
significant interaction effects, ADC of histidine and valine in IMC for rainbow trout were
significantly lower than their ADC in SPC for Atlantic salmon (P<0.05 and P<0.01,
respectively). Significant species-ingredient interaction effects were observed in the ADC of four
EAA, lysine, isoleucine, leucine and phenylalanine (P<0.05, Table 3.6).
All NEAA in the ingredients showed high ADC for both the species except for the
glycine in Atlantic salmon. Apparent digestibility of glycine in IMM (93%) was significantly
lower than their ADC in SPC (97%). No species-ingredient interaction effect was observed in the
ADC of any of these NEAA. However, ADC of aspartic acid, glycine, and tyrosine were
significantly different between the two species. Two of these NEAA, aspartic acid and glycine
were also significantly different among the ingredients.
71
Table 3.4 ADC of nutrients, phosphorus, gross energy and essential amino acids in the reference diet (diet 1) and the three test diets
for rainbow trout and Atlantic salmon (mean SD)
Rainbow trout Atlantic salmon
Diet 1 2 3 4 1 2 3 4
70% Ref 70% Ref 70% Ref 70%Ref 70%Ref 70% Ref
ADC (%) Ref Diet 30%IMC 30%IMM 30%SPC Ref Diet 30%IMC 30%IMM 30%SPC
Dry matter 70.3 2.6 71.5 0.4 74.2 1.4 75.4 1.2 76.4 1.4 76.2 1.4 75.3 0.3 78.6 0.4
Organic matter 72.0 2.7 73.8 0.6 75.8 1.3 76.9 1.1 78.3 1.4 79.1 1.1 77.0 0.4 80.2 0.4
Crude protein 87.8 2.6 88.6 0.7 90.7 0.6 91.2 0.6 91.9 1.6 93.6 0.7 93.2 0.7 94.3 0.3
Total carbohydrates 33.6 4.9 34.6 4.5 44.8 4.1 41.6 1.2 30.2 2.1 32.7 2.9 38.9 6.7 40.5 1.8
Ash 43.9 2.5 45.2 3.4 48.8 2.7 54.4 2.9 47.7 1.6 44.9 2.5 48.1 2.3 57.6 1.8
Phosphorus 54.0 1.8 45.9 1.2 46.2 3.1 52.0 2.9 54.6 1.6 44.0 1.6 49.4 2.3 57.9 1.2
Gross energy 72.6 3.1 75.4 0.7 77.3 1.3 78.1 1.0 82.2 1.1 82.6 1.1 81.0 0.4 83.5 1.1
ADC (%) of essential amino acids
Arginine 93.1 1.4 94.8 0.4 94.5 0.3 95.0 0.9 96.1 1.0 97.1 0.5 96.8 0.4 97.4 0.2
Histidine 92.2 1.2 92.6 0.5 93.0 0.3 93.5 1.4 95.2 1.5 95.8 0.8 96.0 0.4 96.6 0.1
Lysine 94.9 0.6 95.0 1.1 96.0 0.3 97.3 0.4 96.2 0.8 97.1 0.5 96.5 0.2 97.4 0.1
Threonine 89.0 1.9 89.4 0.6 89.3 0.3 90.3 1.5 94.4 1.4 94.4 0.8 93.3 0.5 94.8 0.2
Valine 90.0 2.0 89.9 0.6 90.8 0.2 91.3 1.4 94.5 1.5 94.4 0.8 94.1 0.6 95.5 0.3
Isoleucine 86.6 2.8 88.1 0.8 89.3 1.1 90.1 1.3 93.7 1.3 93.8 0.9 93.2 0.8 94.7 0.4
Leucine 88.2 2.7 87.8 1.0 90.2 1.1 90.0 1.6 94.9 1.7 94.7 1.0 94.8 0.6 95.3 0.4
Phenylalanine 88.4 2.5 88.8 1.0 91.5 1.8 92.5 1.0 94.2 1.8 94.8 0.8 94.6 0.7 95.4 0.3
72
Table 3.5 ADC1 of nutrients and gross energy in Indian mustard protein concentrate (IMC), Indian mustard meal (IMM) and soy
protein concentrate (SPC) for two rainbow trout and Atlantic salmon (mean SD).
Apparent Digestibility Coefficients (%)

Species Ingredient Organic Crude Total
Dry matter matter protein carbohydrates Ash Phosphorus Gross energy
(%) (%) (%) (%) (%) (%) (%)
IMC 74.1b 1.5 76.7b 1.5 b
90.1 2.0 a
36.4 12.4 47.7b 9.8 32.2ab 3.3 83.1b 2.6
Trout IMM 83.0a 3.4 85.1a 4.3 98.6a 2.3 56.6a 18.8 59.5ab8.6 28.2b 10.2 90.4a 5.2
SPC 87.1a 3.9 88.5a 3.6 98.2a 2.1 55.0a 8.4 73.3a 8.0 45.6a 12.2 93.4a 3.8
IMC 75.9ab 4.0 80.9a 3.8 96.9a 2.0 37.8b 9.0 39.9b 7.2 25.9b 4.3 83.8ab 4.0
Atlantic
salmon IMM 72.7b 1.0 74.1b 1.3 96.8a 2.4 49.5ab14.8 48.9b 7.2 37.1b 7.8 77.8b 1.5
SPC 83.8a 1.1 84.8a 1.4 99.3a 1.0 60.7a 5.4 75.4a 4.9 68.3a 5.1 87.1a 4.1
Effect (P- value)2
Species P<0.01 P<0.01 P<0.05 NS NS P<0.05 P<0.001
Ingredients P<0.001 P<0.001 P<0.001 P<0.01 P<0.001 P<0.001 P<0.01
Species X Ingredient P<0.01 P<0.01 P<0.01 NS NS P<0.01 P<0.01
1
Means of the ADC of a nutrient for ingredients in a species followed by a common superscript are not significantly different (Tukeys HSD, P>0.05).
2
Effects of species, Ingredient and species-ingredient interactions for individual nutrient are deemed significant at P<0.05 (in bold).
73
Table 3.6 ADC1 of eight essential amino acids in Indian mustard protein concentrate (IMC),
Indian mustard meal (IMM) and soy protein concentrate (SPC) for rainbow trout and Atlantic
salmon (mean SD).
Apparent Digestibility Coefficients (%)

Essential amino acids2
Species Ingredient
Arg His Lys Thr Val Ile Leu Phe
a a b a a b b
97.4 93.5 95.2 90.4 89.5 90.8 86.3 90.0b
IMC 1.0 2.0 2.1 2.2 2.6 2.6 5.7 4.6
97.5a 95.8a 98.0ab 90.3a 92.2a 95.8a 100.0a 100.0a

Trout
IMM 0.9 1.6 4.3 1.5 1.4 4.3 7.9 9.2
97.1a 95.0a 100.0a 91.4a 92.8 95.1a 94.2ab 100.0a

SPC 1.0 3.5 0.6 2.8 2.0a 1.0 2.5 2.1
98.9a 97.3b 99.0a 94.3a 94.oab 94.2ab 93.8a 96.5a

IMC 1.2 2.6 1.6 2.1 3.2 2.7 5.0 3.2
Atlantic 98.6a 98.8b 97.5a 90.1b 92.7b 91.6b 94.3a 96.6a

salmon IMM 1.3 1.6 1.1 2.0 2.9 3.1 4.5 3.6
99.6a 100.0a 99.7a 94.3ab 98.4a 96.6a 97.2a 98.4a

SPC 0.5 0.5 0.5 2.5 1.1 1.1 1.9 1.2
Effect (P-value)3
Species P<0.05 P<0.01 NS NS P<0.05 NS NS NS
Ingredients NS P<0.05 P<0.01 P<0.01 P<0.01 P<0.05 P<0.05 P<0.01
Species*Ingredient NS NS P<0.05 NS NS P<0.05 P<0.05 P<0.05
1
Means of the ADC of a nutrient for ingredients in a species followed by a common superscript letter are not
significantly different (Tukeys HSD test, P>0.05).
2
Arg arginine; His histidine; Lys lysine; Thr threonine; Val valine; Ile isoleucine; Leu leucine; Phe
phenylalanine.
3
Effects of species, Ingredient and species-ingredient interactions for individual nutrient are deemed significant at
P<0.05 (in bold).
74
3.4. Discussion
Apparent digestibility of nutrients and amino acids of two novel ingredients, a meal and a
protein concentrate produced from Indian mustard, compared to those of a commercially
available soy protein concentrate for rainbow trout and Atlantic salmon were assessed in this
study. IMC and IMM used in this study were produced by a proprietary low-temperature (<50oC)
oil-extraction process that prevents protein from denaturing leaving more soluble forms of
protein than the more commonly used high-temperature (>65oC) extraction processes.
Crude protein and the EAA in the three test ingredients were highly digestible (ADC
>90%) for both rainbow trout and Atlantic salmon. The observed ADC of crude protein in IMC
(90%) was similar to the protein digestibility of two rapeseed (canola) protein concentrates (89-
90%) fed to rainbow trout (Higgs et al., 1995; Thiessen et al., 2004). However, ADC of crude
protein (90%) and four essential amino acids i.e. lysine (95%), isoleucine (91%), leucine (86%)
and phenylalanine (90%) in IMC were slightly lower (P<0.05) than their ADC in IMM and SPC
for rainbow trout. Newkirk et al. (1997) examined the digestibility of crude protein of different
oilseed meals (four Indian mustard meals, two canola meals and one low-fibre soybean meal) in
broiler chickens and reported lower CP digestibility for high fibre oilseed meals compared to low
fibre oilseed meals. Total carbohydrate content (calculated by difference) in the IMC (30%) used
in this study was similar to that in the SPC (33%) but much lower than in the IMM (48%). NDF
content was also higher in IMM (17%) than in IMC (13%) and SPC (7%). In this study,
differences in ADC of CP did not appear to be related to the total carbohydrate or to the NDF
and ADF contents of the diets and ingredients. However, the ADC of threonine, valine and
isoleucine of the IMM were significantly lower (P<0.05) than those of the IMC and SPC for
Atlantic salmon, and that could be attributable to the enhanced loss of endogenous proteins in
75
IMM fed fish. Both threonine and valine are reported to present in higher concentration (5.1%
and 4.8%, respectively) than other EAA in the ileal endogenous proteins in swine (Jansman et
al., 2002). Mucins produced by intestinal cells are rich in threonine and high threonine losses
have been reported in swine (De Lange et al. 1989; Zhu et al., 2005) and poultry (Cowieson et
al., 2008) fed high fibre diets.
It can be hypothesized that the low ADC of nutrients in IMC for rainbow trout could be
related to the high phytic acid (PA) content (4.2%) compared to IMM (2.8%) and SPC (1.6%). In
this study, PA intake by rainbow trout fed 30%-IMC diet was significantly higher (37 mg fish-1
d-1) than that of by fish fed 30%-IMM (19 mg fish-1 d-1) or 30%-SPC (25 mg fish-1 d-1) diets. A
relationship may exist between the ADCs of crude protein observed in this study and the PA
intake of the animals. Spinelli et al. (1983) reported a negative effect of PA on the apparent
digestibility of CP in canola meal for rainbow trout. The authors also reported significantly lower
growth and feed efficiency in rainbow trout fed diets containing 0.5% PA compared to those fed
PA-free diets. In this study, feeding the 30% IMC diet (2.0% PA) resulted in significantly lower
apparent digestibility of CP compared to the basal diet (0.9% PA) and the diets containing 30%
IMM (1.5% PA) and 30% SPC (1.1% PA). At pH-values below the isoelectric point of protein,
the anionic phosphate groups of PA bind strongly to the cationic groups of the protein to form
binary insoluble complexes at acidic pH (e.g., in the stomach, pH 2-3), and become unavailable
for intestinal (pH 8-9) absorption (Sugiura et al., 2006).
Among the EAA, the ADC of histidine ( = -0.228; P<0.05) and lysine ( = -0.175;
P<0.05) were inversely correlated with the dietary PA intake in this study (Figure 3.1). The ADC
of aspartic acid ( = -0.352; P<0.01), valine ( = -0.294; P<0.05), leucine ( = -0.465; P<0.05),
and phenylalanine ( = -0.367; P<0.05) were also significantly correlated with the phytic acid
76
intake. Broken-line regression analysis of ADC of Phe and PA intake showed that ADC of the
amino acid was not affected below the PA intake of 17 mg-1 fish d-1 (Figure 3.2). Histidine,
lysine, and arginine are highly susceptible to chelation in both stomach and intestine as they
become polar and positively charged at pH below their pKas (6.1, 10.5 and 12.5, respectively;
Cosgrove, 1966). High dietary PA intake appears to have more significant impact on the ADC of
histidine than lysine and arginine, which agrees well with the differences in pKa between these
amino acids. The -NH2 terminal group and the -NH2 group of lysine and the imidazole group
of histidine and the positively charged guadinyl group of arginine have been implicated as
possible protein binding sites for phytate (Selle et al., 2000). Kinetic and thermodynamic studies
with human serum albumin indicate that PA forms salt-like linkages combining first with the -
NH2 terminal group, followed by the -NH2 of lysine, then histidine and guanidyl groups of
arginine (Cheryan and Rackis, 1980).
No significant differences in the ADC of EAA were observed between rainbow trout and
Atlantic salmon except for those of arginine, histidine and valine. Differences in digestibility of
these three amino acids between the two species were small (less than 5%) and probably
negligible. The large differences in growth rates (TGC between 0.184 and 0.237 for rainbow
trout and between 0,045 and 0.085 for Atlantic salmon) and feed intakes that exist between the
species used in this study make it difficult to meaningfully determine if the small differences in
the digestibility of EAA in different ingredients is determined by species or feed intake
differences. The relationships between PA intake and the digestibility of certain amino acid
observed here are interesting but circumstantial and many other factors may explain these results.
The effect of dietary PA on digestibility of protein and amino acids deserves to be systematically
investigated in future trials.
77
In conclusion, the novel Indian mustard protein concentrate and Indian mustard meal
evaluated in this study appear to be excellent sources of digestible EAA to rainbow trout and
Atlantic salmon tested in this study. The amino acid profile of these ingredients is very good
when compared to essential amino acid requirements of teleost fishes. However, additional
studies are needed to determine if digestibility of CP and certain amino acids can be affected by
dietary PA intake. Small differences in amino acid digestibility were observed between species
but these differences are probably negligible.
78
Figure 3.1 Apparent digestibility coefficients (%) of arginine - Arg (pKa 12.5), lysine Lys
(pKa 10.5) and histidine - His (pKa 6.1) in Indian mustard protein concentrate (IMC), Indian
mustard meal (IMM) and soy protein concentrate (SPC) for rainbow trout and Atlantic salmon
and their relationships to the dietary phytic acid intake. Dietary phytic acid intake in 30%-IMC,
30%-IMM and 30%SPC diets were 29 mg.fish-1.d-1, 15 mg.fish-1.d-1 and 14 mg.fish-1.d-1 for
rainbow trout and 10 mg.fish-1.d-1, 6 mg.fish-1.d-1 and 5 mg.fish-1.d-1 for Atlantic salmon,
respectively.
100
Apparent digestibility coefficient (%)
98
pKa (Arg) = 12.5
R2 = 0.86
96
pKa (Lys) = 10.5
Arginine R2 = 0.78
94 Lysine
pKa (His) = 6.1
Histidine
R2 = 0.94
92
0 5 10 15 20 25 30
Dietary phytic acid intake (mg fish-1 d-1)
79
Figure 3.2 Broken line regression of ADC (%) of phenylalanine in Indian mustard protein
concentrate (IMC), Indian mustard meal (IMM) and soy protein concentrate (SPC) for rainbow
trout and Atlantic salmon with the dietary phytic acid intake (R2=0.83). Dietary phytic acid
intake in 30%-IMC, 30%-IMM and 30%SPC diets were 29 mg.fish-1.d-1, 15 mg.fish-1.d-1 and 14
mg.fish-1.d-1 for rainbow trout and 10 mg.fish-1.d-1, 6 mg.fish-1.d-1 and 5 mg.fish-1.d-1 for Atlantic
salmon, respectively.
100
ADC of phenylalanine (%)
95
90
85
0 5 10 15 20 25 30
Dietary phytic acid intake (mg fish-1 d-1)
80
CHAPTER 4
RELATIVE BIOAVAILABILITY OF ARGININE IN NOVEL INDIAN MUSTARD
PROTEIN CONCENTRATE AND INDIAN MUSTARD MEAL COMPARED TO THAT OF
A SOY PROTEIN CONCENTRATE IN RAINBOW TROUT (ONCORHYNCHYS MYKISS)
AS DETERMINED BY THE SLOPE-RATIO ASSAY
Abstract
Relative bioavailability (RBV) of arginine (Arg) from two novel ingredients, Indian
mustard protein concentrate (IMC, 62% crude protein) and Indian mustard meal (IMM, 42%
crude protein), and a soy protein concentrate (SPC, 57% crude protein) was compared to that of
crystalline L-arginine (L-Arg) in rainbow trout using slope-ratio assay. A basal diet highly
deficient in Arg (1.23%) was formulated using corn gluten meal and skim milk powder as the
main protein sources. Eight other isoproteic and isoenergetic diets were formulated to contain
increasing amount of IMC, IMM, SPC, and L-Arg (~1.35% and ~1.5% Arg). The experimental
diets were fed for 16 weeks using a standard growth assay protocol. Growth rate (expressed as
thermal-unit growth coefficients, TGC), weight gain (g fish-1), protein (PD, g fish-1) and lipid
(LD, g fish-1) of fish increased linearly with increasing level of Arg for all test ingredients. RBV
was determined by linear regression analysis using TGC and PD as the response variables and
Arg intake (g fish-1) and level (%) as predictor variables. In general, ARG availability from
protein-bound sources were higher than those from L-Arg. RBV estimates of Arg from IMC and
IMM were ranged from 123% to 187% and from 116% to 211%, respectively than that from L-
Arg (assumed as 100% bio-available) (P<005). The findings suggest that the RBV of Arg of
IMC and IMM are very high and comparable to that of the commercial SPC used in this study.
81
4.1. Introduction
Cultivation of oilseeds from the Brassicacae family has gained in popularity over the past
several decades. Various protein-rich feedstuffs (meals and protein concentrates) produced from
rapeseed (Brassica napus) and canola (Brassica rapa) are increasingly used in aquaculture feeds
worldwide. The nutritive value of these ingredients has been the focus of significant number of
studies (Cheng et al., 2010; Drew et al., 2007; Shafaeipur et al., 2008; Thiessen et al., 2003;
2004; Zhou and Yue, 2010). Cultivation of another oilseed from the Brassicacae family,
Brassica juncea, known as Indian or oriental or brown mustard, is gaining popularity in some
regions of North America, India and Australia since it is generally more tolerant to heat, drought
and fungal infection than rapeseed or canola (Chvre et al., 2008; Oram et al., 1999; Wijesundera
et al., 2008; Woods et al., 1991). Protein meals and concentrates produced from Indian mustard
cultivars have excellent amino acid (AA) profile and appeared to be of high nutritive value to
poultry and swine (Jia et al., 2008; Meng et al., 2006; Newkirk et al., 1997; Thacker and Petri,
2010).
Accurate characterization of AA composition and bio-availability of feed ingredient is
important to improve cost-effectiveness of feeds. Lack of accurate information force feed
formulators to rely on high safety margins for AA and other nutrients in the feed leading to high
production costs (Bureau, 2006, 2008; Cho and Bureau, 2001). Fish feeds are increasingly
formulated on digestible AA basis (El-Haroun et al., 2009; Gaylord et al., 2010; Poppi et al.,
2011; Yamamoto et al., 2005) but very limited effort has been invested in estimating the
digestibility or availability of individual AA in fish feed ingredients (Bureau, 2008; De Silva et
al., 2000; Kaushik and Seiliez, 2010). Bioavailability is defined as the proportion of ingested
dietary AA absorbed in a chemical form potentially suitable for metabolism or synthesis
82
(Ammerman et al., 1995; Batterham, 1992; Lewis and Baily, 1995; Stein et al., 2007). Among
several approaches, slope-ratio assays offer direct and practical assessment of the availability of
individual AA and have been used to assess the bioavailability of AA for farmed animals (Lewis
and Bailey, 1995). In this assays, graded levels of the test ingredient are included in diets with a
known nutritional composition. These diets, formulated to be first limiting in the nutrient (e.g.,
amino acid) of interest, are then fed to experimental animals and the growth, and other responses
of interest, of the animals are recorded in response to increasing levels of the test ingredient. A
regression line is then fitted to the data and the slope of this line is compared to that of the data
from animals fed diets containing graded levels of a purified form, or well defined source, of the
nutrient of interest.
Relative bio-avalaibility is determined by comparing the slopes of the response of fish to
increasing level of test nutrient supplied by the test ingredients with the slope of the response of
those fed diets where the test nutrient is provided by the reference source of this nutrient. The
slope-ratio is usually determined by linear regression model (y = b1 + b2*S + b3*T, S - standard
and T - test diets) that needs pre-validation of three assumptions: linearity of the responses,
common intercepts for all lines, and the equal basal level response to the common intercept
(Batterham et al., 1979; Finney et al., 1951, 1964; Littel et al., 1997). Linear models have been
extensively used to determine bioavailability of individual AA or minerals in poultry (Adeola,
1998; Batterham et al., 1986; Lumpkins and Batal, 2005; Major and Batterham, 1981; Parsons et
al., 1998), swine (Adeola, 1996, 2009; Batterham et al., 1990; Fanimo et al., 2006; Mateo et al.,
2007; Petersen et al., 2011; Shoveller et al., 2009), and recently, in fish (El-Haroun and Bureau,
2007; Li et al., 2008, 2009; Poppi et al. 2011; Zarate and Lovell, 1997). In case of non-linear
responses, an exponential regression model (y = b1 + b2*(1 exp (b3*S + b4*T) can be used to
83
estimate bioavailability from the ratio of the steepness coefficients b3 and b4, where b1 (the
response to the basal diet) and b2 (the difference between the lowest and highest response) have
to be equal for all sources (Kim et al., 2006; Littel et al., 1997; Liu et al., 2004). Another method
is the broken-line models where the slopes of the linear portion of the responses are used to
determine the RBV of the nutrient of interest (Coon et al., 2007; Wiltafsky et al., 2009). Like
other assays, slope-ratio assays also have some limitations and are sensitive to the experimental
artefacts (Poppi et al., 2011). In these assays, the desired levels of the nutrient in question in the
diets are achieved by adding the test ingredients. This approach allows for the concentration of
various other components such as trace-minerals and fibres to vary, and results can be affected
by the concentration of test nutrient and dietary intake.
Indian mustard protein concentrates and meals are very high in crude protein (62% and
44%, respectively) and in most EAA of which Arg constitutes about 4.1% and 2.8%,
respectively. Accurate estimation of biologically available AA in these high protein ingredients
would allow feed manufactures to further diversify the protein sources while enabling them to
attain greater degree of accuracy in formulation using these feedstuffs. In fish, RBV of Lys (El-
Haroun and Bureau, 2007) and Arg (Poppi et al., 2011) from blood meal and feather meal,
respectively in rainbow trout, and MHA-FA (Li et al., 2009) in juvenile striped bass have been
determined by linear regression method. Rainbow trout has high dietary Arg requirement (1.5%,
NRC, 2011), and responds very well and predictably to small changes in the dietary levels of Arg
(Cho et al., 1992; Rodehutscord et al., 1995). Since Indian mustard protein concentrate and
protein meals are rich in Arg (4.1% and 2.8%, respectively), dietary concentrations of this AA
can be manipulated by simply adding the ingredients to the Arg deficient basal diet. Information
of the bioavailability of Arg could potentially be a reliable indicator of the availability of other
84
AA. The major focus of this study was to determine the bioavailability of Arg from novel Indian
mustard protein concentrate and meal, and a commonly used soy protein concentrate (SPC)
compared to a crystalline source (98.5% pure L-Arg) in rainbow trout using a slope-ratio assay.
The efficacy of different regression approaches such as with two-level versus one-level of Arg
from the test and standard sources, and with level (% diet) and intake (g fish-1) as predictors was
assessed.
4.2. Methods
The growth trial was conducted at the UG/OMNR Fish Nutrition Research Laboratory
(Department of Animal and Poultry Science, University of Guelph, Ontario, Canada) using
standard research protocol. (1999). Indian mustard protein concentrate (IMC) and meal (IMM)
were obtained from Bio-Exx Specialty Proteins Ltd. (Toronto, ON, Canada), and the soy protein
concentrate (SPC HP 300) was obtained from Hamlet Protein A/S (Aarus, Denmark). The
Indian mustard protein concentrate and meal are produced using a proprietary low temperature
extraction process (<60oC) while the soy protein concentrate is produced through a proprietary
enzymatic treatment and aqueous extraction process. The proximate composition, gross energy
(GE), and amino acid profiles of three test ingredients are reported in Table 4.1.
85
Table 4.1 Proximate chemical composition, gross energy, essential amino acid profiles, total
phosphorus and phytic acid content of the test ingredients (as is basis).
Ingredients
Proximate composition (as is) Indian mustard Indian mustard Soy protein
concentrate meal concentrate
Dry matter, % 95 95 93
Organic matter, % 87 88 86
Crude protein, % 62 44 57
Lipid, % 0.2 1.9 2.2
Total carbohydrate, % 30 48 33
Neutral detergent fibre, % 13 17 7
Acid detergent fibre, % 11 13 6
Ash, % 8 7 8
Gross energy, kJ.g-1 19 19 19
Essential amino acids (% in the weight of ingredient as is)
Arginine 4.1 2.8 3.9
Histidine 1.6 1.1 1.4
Isoleucine 2.4 1.7 2.5
Leucine 4.2 2.9 4.1
Lysine 2.6 1.9 2.7
Methionine+Cystine* 2.8 1.9 1.3
Phenylalanine 2.3 1.6 2.7
Threonine 2.2 1.6 1.8
Tryptophan* 1.0 0.6 0.6
Valine 3.0 2.1 2.7
Phosphorus and phytate-P (as is)
Total phosphorus, % 1.5 1.1 0.8
Phytate-P, % 1.2 0.8 0.5
Phytate-P is calculated as phytic acid*0.282; * manufacturers data

86
4.2.1. Experimental diets
A practical basal diet was formulated (Diet 1, Table 4.2) to be highly deficient in arginine
(1.23% Arg) using a combination of corn gluten meal and skim milk powder. The diet contained
approximately 40% crude protein (CP) and 20% crude lipids (Table 4.3). Eight other isoproteic
and isoenergetic (on a digestible energy basis) experimental diets were prepared with graded
levels of arginine (~1.35% and ~1.5% of the diets) with increasing amount of three test
ingredients (IMC, IMM, and SPC) or crystalline L-Arg (98.5% pure; USB Corporation, OH,
USA) (Table 4.3). Levels of all nutrients (except Arg) met the requirements of rainbow trout
according to NRC (2011) recommendations. Ingredients and diets were mixed using a Hobart
mixer (Hobart Ltd., Don Mills, ON, Canada), steam-pelleted using a laboratory scale steam-
pellet mill (California Pellet Mill, San Francisco, CA, USA), and dried under forced-air at room
temperature for 24 hrs. Dried diets were sieved to appropriate size and stored at 4C until used.
The amount of feeds needed were weighed on a weekly basis and kept at room temperature.
4.2.2. Experimental conditions
Rainbow trout (Oncorhynchus mykiss, Ontario domestic fall-spawning strain) fingerlings
were obtained from the Alma Aquaculture Research Station of the University of Guelph (Elora,
ON, Canada). The experimental system, consists of four blocks of nine 60-L fibreglass tanks in
each, were individually aerated and supplied with filtered well water. Water flow, temperature
and photoperiod were maintained at ~3 L min-1, 13oC, and 12h L-12h D cycle, respectively. Fish
were pre-acclimatized to the experimental conditions for seven days, when they were fed a high
quality commercial salmonid diet (Profishent High Energy Feed, Martin Mills, Elmira, Ontario,
Canada).
87
Nine diets were allocated to each experimental block following a completely randomized
design. Fifteen fish, an average weight of 16 g, were distributed to each tank and hand-fed to
near-satiety three times a day on weekdays and once daily on weekends for 112 days. Feed
consumption was recorded weekly and the fish were weighed in every four weeks. The animals
were treated complying with the guidelines of the Canadian Council on Animal Care (CCAC,
1984) and the University of Guelph Animal Care Committee.
88
Table 4.2 Ingredients composition of the diets (g kg-1).
Ingredients (g kg-1 diet) Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8 Diet 9
Skim milk powder 250 250 250 235 210 235 210 235 210
Corn gluten meal 260 259 258 215 167 215 167 215 175
Indian mustard protein conc. - - - 70 140 - - - -
Indian mustard meal - - - - - 100 200 - -
Soy protein conc. - - - - - - - 75 150
Blood meal 40 40 40 35 30 35 30 35 30
Gelatinized starch 117 116.5 116 112 120 82 60 107 102
Wheat gluten meal 70 70 70 70 70 70 70 70 70
Fish oil 180 180 180 180 180 180 180 180 180
1
Vitamin premix 10 10 10 10 10 10 10 10 10
1
Amino acid premix 10 10 10 10 10 10 10 10 10
Lysine, BioLys (51% L-Lys) 35 35 35 35 35 35 35 35 35
DL-Met 3 3 3 3 3 3 3 3 3
L-Arg (98.5% pure) - 1.5 3 - - - - - -
Mineral premix3 10 10 10 10 10 10 10 10 10
NaH2PO4 15 15 15 15 15 15 15 15 15
Total (g) 1000 1000 1000 1000 1000 1000 1000 1000 1000
1
Provides per kg of diet: retinyl acetate (vitamin A), 3750 IU; cholescalciferol (vitamin D), 3000 IU; dl-a-tocopheryl
acetate (vitamin E), 75 IU; menadione sodium bisulphite (vitamin K), 1.5 mg; ascorbic acid polyphosphate (Stay
CTM 25% ascorbic acid), 75 mg; choline chloride, 1500 mg; cyanocobalamine (vitamin B12), 0.03 mg; biotin, 0.21
mg; inositol, 450 mg; folic acid, 1.5 mg; niacin, 15 mg; Pantothenic acid, 30 mg; pyridoxineHCl, 7.5 mg;
riboflavin, 9.0 mg; thiaminHCl, 1.5 mg.
2
Provides per kg of diet: tryptophan, 2000 mg; threonine, 3000 mg; histidine, 1500 mg.
3
Provides per kg of diet: sodium chloride (NaCl, 39% Na, 61% Cl), 3077 mg; ferrous sulphate (FeSO47H2O, 20%
Fe), 65 mg; potassium iodide (KI, 24%K), 11 mg; manganese sulphate (MnSO 4, 36% Mn), 89 mg; zinc sulphate
(ZnSO47H2O, 40% Zn), 150 mg; copper sulphate (CuSO45H2O, 25% Cu), 28 mg; di-sodium selenite (Na2SeO3),
10 mg.
89
Table 4.3 Nutrient, gross energy and essential amino acid (EAA) composition of the test diets
(DM basis).
Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8 Diet 9
Proximate Composition (Determined)
Dry matter (%) 95 93 94 93 93 96 95 94 94
Crude protein (%) 40 41 40 42 41 40 40 39 40
Lipid (%) 19 20 19 19 19 20 19 20 20
Ash (%) 5 5 5 5 5 5 5 5 6
1
NDF (%) 2.2 2.3 2.3 3.7 4.3 4.1 5.1 3.1 3.1
ADF (%)2 1.9 1.9 2.0 2.6 3.0 3.8 4.7 2.1 2.4
Phosphorus (%) 0.8 0.9 0.9 1.0 1.0 0.9 0.9 0.9 0.9
Phytate-P (%)3 0.1 0.1 0.1 0.2 0.3 0.2 0.3 0.1 0.1
Available P (%) 0.7 0.8 0.7 0.8 0.6 0.7 0.7 0.7 0.7
-1
GE (kJ g ) 22.3 22.3 22.2 22.2 21.9 22.1 22.1 22.2 21.9
Digestible Nutrient Composition (Calculated)
Digestible Protein (%) 95 95 95 95 94 96 96 96 96
Digestible Energy (kJ g-1) 21.5 21.7 21.4 21.4 21.4 20.8 21.1 21.4 21.2
Essential amino acid composition (Analyzed)4
Arginine 1.23 1.37 1.54 1.36 1.50 1.34 1.49 1.38 1.53
Histidine 0.98 1.04 0.99 1.04 1.01 1.04 1.02 1.01 1.04
Isoleucine 1.39 1.45 1.39 1.33 1.39 1.39 1.40 1.46 1.44
Leucine 3.95 4.11 3.85 3.56 3.31 3.57 3.42 3.64 3.37
Lysine 2.70 2.73 2.68 3.03 2.77 2.88 2.77 3.07 3.15
Methionine + Cysteine* 1.46 1.46 1.45 1.50 1.53 1.50 1.52 1.44 1.42
Phenylalanine + Tyrosine 2.98 3.08 2.91 2.79 2.62 2.81 2.78 2.90 2.87
Threonine 1.31 1.36 1.29 1.27 1.30 1.31 1.34 1.40 1.38
Tryptophan* 0.54 0.54 0.53 0.51 0.48 0.51 0.49 0.60 0.65
Valine 1.75 1.79 1.71 1.73 1.71 1.73 1.74 1.75 1.75
1
NDF Neutral Detergent Fibre. 2ADF Acid Detergent Fibre. 3Analyzed as phytic acid (PA). Phytate-P is
calculated as 0.282*PA. 4Methionine, cysteine, and tryptophan were not analyzed. *calculated values.
90
4.2.3. Chemical analysis
At the beginning and end of the experiment, 36 fish (one from each tank) and five fish
from each tank, respectively, were sampled for proximate composition and kept at -20oC until
processed. The pooled whole fish samples were cooked in an autoclave, ground into
homogeneous slurry in a food processor, lyophilized, then finely ground, and stored at -20oC
until analysis.
Diets, ingredients, and whole carcass samples were analyzed for dry matter (DM) and ash
according to AOAC (1995), crude protein (CP, %N x 6.25, Dumas method) using a LECO
Analyzer (LECO Corporation, St. Joseph, MI), lipids with petroleum ether extraction at 90oC
and 150 PSI for 60 min using ANKOM XT-20 analyzer (ANKOM Technology, Macedon, NY),
and gross energy (GE) using an automated oxygen bomb calorimeter (Model # 1271, Parr
Instruments, Moline, Illinois). Neutral detergent fibre (NDF) and acid detergent fibre (ADF)
contents of the ingredients and the diets were determined according to Van Soest et al. (1991)
using an Ankom 200 Fibre Analyzer (ANKOM Technology, Macedon, NY). Phytic acid content
in the ingredients and diets were determined using high performance liquid chromatography
(HPLC, Waters Corporation, Millford, CA) according to Lehrfeld (1989) at the Danisco Animal
Nutrition laboratory, Danisco, St. Louis, MO. Amino acid profiles of the test ingredients and the
diets were analyzed at the Amino Acid Laboratory of the Toronto Hospital for Sick Children
(ON, Canada) using the acid hydrolysis method. For the quantification of amino acids,
approximately 0.05 g of dried sample was hydrolysed in 15 ml test tube with 450 L of 6N HCl
and 1% phenol for 24 hrs at 110oC under purified nitrogen atmosphere. After 24 hrs, tubes were
removed from oven, cooled, capped, vortex mixed and centrifuged at 2500 rpm. Then, 5 L of
the supernatant was derivatized following PICO-Tag method (Cohen et al., 1989). After
91
derivatization, the supernatant were re-dissolved in 150 L phosphate buffer and 2 L of this
solution was injected into the column of an ACQUITY Ultra-Performance LC machine (Waters
Corporation, Millford, CA) for quantification of all amino acids except tryptophan, methionine
and cysteine, which were partially destroyed in the hydrolysis process.
4.2.4. Calculations
Growth rate was expressed as thermal-unit growth coefficient (TGC) and calculated as
[100 x (FBW1/3 IBW1/3) / (Temp (0C) x Number of days)] where FBW is the final body
weight and IBW is the initial body weight of the fish, and expressed as thermal-unit growth
coefficient (TGC) (Cho, 1992). Feed efficiency (FE) was calculated as live weight gain /dry
feed intake. Protein (PD) and lipid (LD) deposition, nitrogen retention efficiency (NRE),
recovered energy (RE), and energy retention efficiency (ERE) were also assessed. PD, LD and
RE were calculated from the difference between final and initial carcass content. NRE and ERE
were calculated as: NRE, % = [[FBW x Nfinal) (IBW x carcass Ninitial)]/DNI] x 100 and ERE, %
= [[FBW x GEcarcassfinal) (IBW x GEcarcassinitial)]/DEI] x 10, where: FBW = final body
weight (g) where IBW = initial body weight (g), DNI = digestible nitrogen intake, and DEI =
digestible energy intake.
All data were subjected to Levenes tests (Levene, 1960) of equality of variance followed
by regression analysis against dietary Arg levels using the SAS general linear model procedure
(PROC GLM, SAS v9.2, SAS Institute, Cary, NC, USA). Group means of feed intake, weight
gain, TGC, FE, PD, LD, NRE and ERE were compared using least square means (LSmeans
92
statement in the mixed model analysis) and considered significant at the Bonferroni adjusted
level of P<0.0125. Effects of dietary Arg levels, source, and their interactions were compared
among all sources, and between protein-bound (IMC, IMM, and SPC) and free (L-Arg) sources
of Arg were also analyzed using the PROC GLM procedure and considered significant at
P<0.05.
The responses to Arg level from all four sources were tested for linearity for the
compliance with the criteria necessary for linear slope-ratio model using non-orthogonal
polynomial contrast separately for each source and considered significant at the Bonferroni
adjusted level of P<0.0125. The x, y values for the intercept was calculated from the response to
the basal diet (1.23% Arg) satisfying the requirement for a common intercept (Littel et al., 1997).
RBV estimates of arginine of the test ingredients were derived with one and two levels of
arginine by comparing the slopes of IMC, IMM and SPC with that of L-Arg with either Arg level
(% diet) or intake (g fish-1) as predictors. The difference between the slopes was determined
using 95% confidence intervals and considered significant at P<0.05.
4.3. Results
Fish accepted the basal diet well, and feed intake and growth rate were good for all diets
and comparable to those obtained in other feeding trials at the University of Guelph. Weight gain
of fish fed the basal diet (1.23% Arg) was significantly lower (P<0.05) than those fed high
arginine diets (~1.5% Arg) formulated with IMM, IMC and SPC. Protein, lipid and energy gains
of fish fed these three diets were also significantly higher than those of fish fed all the other
diets.
93
4.3.1. Growth performance and nutrient deposition
Growth rate and protein deposition (PD, g fish-1) in response to intake are presented in
the Figures 4.1 and 4.2, respectively. Fish fed the basal diet had significantly lower (P<0.05)
feed intake, weight gain (Table 4.4), and PD and NRE (Table 4.5) than the fish fed diets with
~1.35% or ~1.5% levels of Arg. Feed intake, weight gain, TGC, PD and LD but not FE, RE,
NRE and ERE were significantly different (P<0.05) between free and protein-bound sources.
Energy retention efficiency also increased linearly (P<0.0125) with increasing level of IMC but
decreased significantly with increasing levels of IMM in the diets (P<0.0125).
4.3.2. Relative bioavailability of arginine
TGC and PD increased linearly with increasing level of Arg and were subjected to the
slope ratio-assay. In general, bioavailability of Arg was higher from all protein bound sources
than those from L-Arg. The RBV of arginine from IMC calculated with PD as response variable
were 178% and 123% higher than that from the standard source, L-Arg (assumed as 100%) for
the predictor variables Arg level (%) and Arg intake (g fish-1), respectively (P<0.05; Tables 4.6,
4.7; Figures 4.3 and 4.4). RBV of Arg from IMM was also significantly higher than that from L-
Arg for Arg level as predictor variable but not for the Arg intake (Tables 4.6, 4.7). On the other
hand, RBV of Arg from SPC was slightly higher but not significantly different than that from the
L-Arg (P>0.05).
94
Table 4.4 Weight gain, TGC and feed intake and feed efficiency of rainbow trout fed diets with
increasing levels of arginine supplied by L-Arg or three protein-bound sources (mean SD).
Feed
Arginine Feed intake Weight gain TGC
Source efficiency
% DM g fish-1 g fish-1 gain:feed
L-Arginine
Diet 1 1.23 85.7 13.7 87.3 14.8 0.168 0.018 1.02 0.05
Diet 2 1.37 102.3 14.5 105.4 13.4 0.187 0.012 1.03 0.05
Diet 3 1.54 103.1 12.4 117.0 16.6 0.200 0.014 1.13 0.04
SEM1 5.7 5.4 0.05 0.04
Linear NS P<0.0125 P<0.0125 P<0.0125
Quadratic NS NS NS NS
Indian mustard protein concentrate
Diet 4 1.36 105.2 201 118.4 19.6 0.201 0.018 1.13 0.03
Diet 5 1.50 115.5 16.0 130.3 11.9 0.212 0.009 1.13 0.06
SEM 8.7 6.9 0.01 0.04
Linear P<0.0125 P<0.0125 P<0.0125 P<0.0125
Quadratic NS NS P<0.0125 NS
Indian mustard meal
Diet 6 1.34 108.7 6.6 117.4 5.3 0.200 0.004 1.08 0.02
Diet 7 1.49 116.5 7.3 124.4 6.4 0.209 0.009 1.07 0.02
SEM 9.3 5.5 0.006 0.02
Linear P<0.0125 P<0.0125 P<0.0125 NS
Soy protein concentrate
Diet 8 1.38 108.5 10.7 115.2 13.5 0.197 0.011 1.06 0.04
Diet 9 1.53 118.8 5.3 127.7 6.5 0.210 0.006 1.07 0.01
SEM 9.8 6.0 0.006 0.02
Linear P<0.0125 P<0.0125 P<0.0125 NS
Effects (P-value, all sources)
Ingredient NS NS NS NS
Level ** * * NS
Ingredient*Level NS NS NS *
Effects (P-value, free & protein-bound)
Ingredient * * ** NS
Level ** * ** **
Ingredient*Level NS NS NS **
1
S.E.M. standard error mean; NS = not statistically significant, * = P<0.05, ** = P<0.01.
95
Table 4.5 Protein (PD) and lipid (LD) deposition, recovered energy (RE), N- (NRE) and
energy (ERE) retention efficiencies for rainbow trout fed diets with increasing levels of arginine
(mean SD).
Arg PD LD RE NRE ERE
Source
%DM g fish-1 g fish-1 kJ fish-1 % %
L-Arginine
Diet 1 1.23 13.1 2.3 14.1 3.2 8.7 1.6 40.4 2.7 30.2 2.7
Diet 2 1.37 16.5 1.4 17.4 3.0 10.9 1.4 42.2 3.3 33.7 3.8
Diet 3 1.54 17.9 2.6 18.9 3.8 11.7 1.3 45.5 2.1 38.3 1.3
1
SEM 0.7 0.8 0.5 1.6 2.3
Linear P<0.0125 P<0.0125 NS NS NS
Quadratic NS NS NS NS NS
Indian mustard concentrate
Diet 4 1.36 18.1 2,8 19.8 3.9 12.2 2.1 44.1 1.5 38.8 1.3
Diet 5 1.50 20.8 2.4 21.6 2.8 13.3 1.2 46.0 3.4 39.7 2.6
SEM 1.2 1.2 0.7 1.0 0.5
Linear P<0.0125 P<0.0125 P<0.0125 NS P<0.0125
Quadratic NS NS P<0.0125 NS NS
Indian mustard meal
Diet 6 1.34 18.4 0.2 19.8 1.4 12.2 0.9 43.6 2.6 40.0 1.6
Diet 7 1.49 19.5 1.3 19.8 1.4 12 0.8 42.9 1.4 36.1 1.9
SEM 0.9 1.0 5.7 0.4 2.0
Linear P<0.0125 P<0.0125 P<0.0125 NS NS
Quadratic NS NS NS NS P<0.0125
Soy protein concentrate
Diet 8 1.38 17.7 2.3 19.0 2.7 11.3 1.3 44.0 2.6 34.4 1.2
Diet 9 1.53 20.1 1.2 20.8 1.2 12.6 0.5 45.6 1.3 35.6 1.3
SEM 1.0 1.0 0.6 0.8 1.1
Linear P<0.0125 P<0.0125 P<0.0125 NS NS
Quadratic NS NS NS NS NS
Effects (P-value, all sources)
Ingredient NS NS NS NS **
Level * NS NS NS NS
Ingredient*Level NS NS NS NS **
Effects (P-value, free & protein-bound)
Ingredient * * NS NS NS
Level * NS NS * NS
Ingredient*Level NS NS NS NS NS
1
S.E.M. standard error mean; NS = not statistically significant, * = P<0.05, ** = P<0.01.
96
Table 4.6 Slopes (b) and confidence intervals (CI) of thermal-unit growth coefficients (TGC)
and protein deposition (PD) in fish fed diets from different sources of arginine (L-arginine,
Indian mustard protein concentrate - IMC, Indian mustard protein meal - IMM, soy protein
concentrate - SPC) as determined by linear regressions using two and one level of arginine (%)
and arginine intake (g fish-1).
Thermal-unit growth coefficient Protein deposition (g fish-1)
Source Two levels One level Two levels One level

Slope Slope Slope Slope
CI CI CI CI
Arginine (% diet)
L-Arg 0.110 0.139 17.2 24.8
0.077 to 0.143 0.071 to 0.208 11.2 to 23.1 15.0 to 34.7
IMC 0.183* 0.260 30.6* 39.0

0.138 to 0.229 0.153 to 0.366 22.3 to 38.8 20.2 to 57.7
IMM 0.179* 0.293* 28.4* 48.2*

0.142 to 0.229 0.265 to 0.322 22.0 to 34.8 43.5 to 52.8
SPC 0.152 0.195 24.9 31.1

0.125 to 0.178 0.138 to 0.252 20.6 to 29.3 20.5 to 41.6
Arginine intake (g fish-1)
L-Arg 0.054 0.05 8.6 8.2
0.048 to 0.061 0.038 to 0.061 7.3 to 10.0 6.1 to 10.4
IMC 0.063 0.076* 10.6* 11.5*

0.055 to 0.070 0.069 to 0.082 9.6 to 11.6 11.0 to 12.1
IMM 0.063 0.077* 10.0 12.4*

0.056 to 0.070 0.071 to 0.084 8.7 to 11.2 11.1 to 13.7
SPC 0.054 0.061 8.9 9.65

0.050 to 0.058 0.056 to 0.066 8.3 to 9.5 8.5 to 10.8
IMC = Indian mustard protein concentrate; IMM = Indian mustard protein meal; SPC = Soy protein concentrate.
Linear model = a+b1*x1+b2*x2+b3*x3+b4*x4; a (TGC) = 0.1675; a (PD) = 13.1. CI = Confidence Interval (lower
limit to upper limit). *Significantly different than L-Arg (P<0.05).
97
Table 4.7 Bioavailability of arginine from Indian mustard protein concentrate - IMC, Indian
mustard protein meal - IMM, soy protein concentrate - SPC in rainbow trout in compare to L-
arginine based on thermal-unit growth coefficients (TGC) and protein deposition (PD) as
determined by linear regressions using two and one level of arginine (%).
Thermal-unit growth coefficient Protein deposition (g fish-1)
Two levels One level Two levels One level
Source Arg availability Arg availability Arg availability Arg availability
(%) (%) (%) (%)
R2 R2 R2 R2
Arginine (% diet)
L-Arg 100 100 100 100
65 62 56 72
IMC 166 187 178 157

71 70 69 63
IMM 163 211 165 194

79 98 76 98
SPC 138 140 145 125

84 82 84 78
L-Arg 100 100 100 100
93 90 89 86
IMC 117 152 123 140

94 99 95 100
IMM 117 154 116 151

94 98 91 97
SPC 100 122 103 118

97 99 97 97
IMC = Indian mustard protein concentrate; IMM = Indian mustard protein meal; SPC = Soy protein concentrate.
Linear model = a+b1*x1+b2*x2+b3*x3+b4*x4; a (TGC) = 0.1675; a (PD) = 13.1.
CI = Confidence Interval (lower limit to upper limit) .
98
Figure 4.1 Weight gain (g fish-1) and thermal-unit growth coefficient (TGC) of rainbow trout in
response to dietary arginine intake.
0.220
Thermal-unit growth coefficient
0.200
0.180
0.160
TGC = 0.06x + 0.11, R = 0.86

0.140
0.50 1.00 1.50 2.00 2.50
99
Figure 4.2 Protein (PD) and lipid (LD) deposition in rainbow trout in response to dietary
arginine intake (g fish-1).
25
Protein deposition (g fish-1)
20
15
PD = 9.3x + 2.9, R = 0.86

10
0.5 1.0 1.5 2.0 2.5
100
Figure 4.3 Thermal-unit growth coefficient (TGC) and protein deposition (PD) of rainbow
trout in response to arginine level (%) in diets from four arginine sources (L-Arg, IMC - Indian
mustard protein concentrate, IMM -Indian mustard protein meal, SPC -soy protein concentrate).
Solid and dotted lines represent response based on two and one level of arginine, respectively.
0.240 L-Arg IMC IMM SPC

L-Arg IMC IMM SPC
Thermal-unit growth
0.220
coefficient
0.200
0.180
(A)
0.160
1.23 1.27 1.31 1.35 1.39 1.43 1.47 1.51
23.0
21.0
19.0
17.0
15.0
(B)
13.0
1.23 1.27 1.31 1.35 1.39 1.43 1.47 1.51
Arginine (% in diet)
101
Figure 4.4 Thermal-unit growth coefficient (TGC) and protein deposition (PD) of rainbow
trout in response to arginine intake (g fish-1) from four arginine sources (L-Arg, IMC - Indian
mustard protein concentrate, IMM -Indian mustard protein meal, SPC -soy protein concentrate).
Solid and dotted lines represent response based on two and one level of arginine, respectively.

L-Arg IMC IMM SPC
0.21
Thermal-unit growth coefficient
0.20
0.19
0.18
0.17
0.16
1.10 1.30 1.50 1.70 1.90
L-Arg IMC IMM SPC

20.0
18.0
16.0
14.0
12.0
1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90
102
4.4. Discussion
Relative bioavailability of Arg from Indian mustard protein concentrate and meal, and a
soy protein concentrate compared to that from crystalline L-Arg in rainbow trout was assessed in
this study. The increase in weight gain, FE and TGC (P<0.05) with increasing dietary Arg
supplied by crystalline L-Arg confirmed that the basal diet was deficient in Arg and the high Arg
levels in the test diets were around the requirement for maximum weight gain currently estimated
to be about 1.5% of dietary DM (NRC, 2011).
Fish fed the crystalline L-Arg based diets showed considerably lower growth
performance such as weight gain, feed efficiency, and nutrient utilization than the fish fed the
protein-bound Arg diets. Various studies earlier have reported lower amino acid utilization by
fish fed crystalline amino acids than those fed protein bound sources (Ambardekar et al., 2009;
El-Haroun and Bureau, 2007; Peres and Oliva-Teles, 2005; Poppi et al., 2011; Zarate and Lovell,
1997). Reasons for the low AA utilization suggested are nutrient leaching and catabolism of free
amino acids being absorbed faster than the protein-bound amino acids (Ambardekar et al., 2009;
Chi et al., 2011; Zarate and Lovell, 1997; Zarate et al., 1999). Earlier studies in growing swine
indicated that increasing the frequency of feeding could improve utilization of free amino acids
(Batterham and Murrison, 1981). Recent studies in fish and shrimp suggested that using coated
amino acids could also possibly improve the utilization of supplemental amino acids (Alam et
al., 2004, 2005; Chi et al., 2011; Lemme, 2010).
Conditions for a valid slope-ratio assay, such as linearity of the responses, common
intercept for standard and tests, and the similar Y values of the common point of intersection,
were met by confirming the linearity of TGC and PD, forcing a common intercept, and the single
data point at the lowest Arg level, respectively. TGC and PD increased linearly with increasing
103
Arg levels in all four protein sources (Tables 4.4 and 4.5). When Arg level is the predictor, RBV
of Arg in IMC and IMM were significantly higher in one-level assay than those in two-level
assays suggesting possible curvilinear or levelling of the responses at higher Arg levels. One
level assays are rare compare to two or more levels (e.g., Batterham et al., 1978; Batterham,
1987). Batterham et al. (1978) compared bioavailability of lysine from cottonseed meal, soybean
meal, meat and bone meal and fish meal for growing swine determined by one (0.3% of the
diets) and seven (0.3% and six other levels with 0.05% increments) levels of Lys and reported no
difference in the availability of lysine among the test ingredients except soybean meal. This
could potentially be a particularity of the experimental design where the maximum level of
dietary Lys experiment (0.6%) was still much lower than the requirement (0.8%) of growing
swine providing similar slopes in both experiments (Martinez and Knabe, 1990).
Recently, Levesque et al. (2010) suggested that maximum intake of the test amino acid
should be set at least 2 SD below the requirement based on the 10% CV estimated by Bertolo et
al. (2005) because of the inter-animal variability of AA requirement. Restricting test amino acid
intake to 80% of its requirement ensures a linear response to the changes in the test amino acid
intake. In this study, the fish were fed to satiety resulting in a variable Arg intake that was
significantly correlated with the protein deposition (R2 = 0.85; P<0.05). In such scenario, AA
intake (g fish-1) appeared to be a better predictor for the determination of bioavailability than the
dietary level (%).
Two important dietary considerations for slope-ratio assays are that the response to
changes in test amino acid intake must be predictable and the observed response must not be
influenced by other dietary nutrients in the test feed ingredient (Batterham, 1992; Littel et al.,
1997). The latter condition is difficult to achieve since most protein sources are complex
104
ingredients with variable levels of non-starch polysaccharides, anti-nutritional factors etc. Like
other farmed animals, non-starch polysaccharides appear to effect nutrient digestion and
absorption by modulating digesta viscosity and rate of passage and by altering gut physiology
and morphology in fish (Brinker and Reiter, 2011; Glencross et al., 2008; Leenhouwers et al.,
2007; Refstie et al., 1999; Storebakken, 1985). Besides, the composition and source of amino
acids in the diets also play a role in feed intake and nutrient utilization. For example, Nang thu et
al. (2007) observed that required amount of lysine intake for protein deposition in rainbow trout
differed significantly between WG and CGM diets suggesting that ingredient matrix may play a
role in amino acid composition. High Arg:Lys ratio or high leucine content in CGM may have
resulted in a reduced feed intake and lysine utilization efficiencies in fish compared to those fed
WG diets. Efficiency of amino acid utilization is usually affected by dietary composition and
source of amino acids, dietary energy level and source, and other components (Encarnao et al.,
2004; Rodehutscord et al., 2000). Meaningful comparison of RBV estimates between different
sources therefore, can be difficult when variable composition of other nutrients or anti-nutrients
in the sources aside from the amino acid of interest affecting the feed intake, nutrient digestion
and utilization.
It can be concluded that the bioavailability of arginine from novel Indian mustard protein
concentrate (IMC) and protein meal (IMM) for rainbow trout is comparable to that of the soy
protein concentrate (SPC) used in this study. Similar performances of fish fed these two novel
plant protein sources compared to those fed commercially available SPC suggest that the quality
of these ingredients is very good, and can be readily utilized in nutrient-dense fish feeds.
Practical basal diets in bioavailability studies in fish have been formulated with either CGM or
WG as the major protein source (Li et al., 2008, 2009; Nang thu et al., 2007; Poppi et al., 2011).
105
Nangthu et al. (2007) reported that imbalance amino acid composition in CGM induced by
excess leucine and high Arg-Lys ratio, may have affected the feed intake suggesting WG could
be a superior candidate as major protein ingredient in basal or reference diets than the CGM. The
difference in the lysine utilization efficiencies observed by Nang thu et al. (2007) could also be
an effect of the variations in digesta viscosity and passage rate because of variable composition
of NSPs in these two ingredients. Besides the variable compositions of other nutrients or anti-
nutrients in the test sources, variations in ingredient compositions of the basal diets may also
hinder from the meaningful comparison of the results among the slope-ratio studies.
Applications of the slope-ratio assay to determine bioavailability of an amino acid could
be very useful tool to assess the quality of novel protein sources in aquafeed. Determination of
appropriate statistical approach (e.g., linear, polynomial or broken-line methods) is essential
before designing a bioavailability trial. Some important points to be considered are: common
intercept of the responses for individual sources, appropriate feeding regime to maintain the level
of test nutrient intake reflecting its level in the diets, and adjusting composition of other
ingredient components such as fibre or anti-nutritional factors by supplementation to nullify their
effects on indicator responses. Further studies are also needed to standardize the practical basal
diets and as well as, to establish a set of criteria for the level of the nutrients of interest in the test
diets for valid, robust and useful slope-ratio responses.
106
CHAPTER 5
EFFECTS OF DIETARY PHYTIC ACID AND PURIFIED LIGNIN AND
THEIR INTERACTIONS ON MORPHOLOGICAL INDICES, GROWTH
PERFORMANCE, NUTRIENT AND ENERGY PARTITIONING OF
RAINBOW TROUT, ONCORHYNCHUS MYKISS
Abstract
This study examined the effects of dietary phytic acid (PA) and purified lignin, and their
interactions on growth performance, physiological indices, nutrient deposition and partitioning in
rainbow trout in a 12 week trial. Nine isoproteic and isoenergetic diets were formulated differing
only in their PA and lignin concentrations. Diets were formulated to be marginally adequate in
five essential amino acids, histidine, lysine, methionine (+cysteine), threonine, and tryptophan.
Fish in six dietary treatments (2 PA X 3 lignin levels) were pair-fed with the treatment with the
lowest feed intake. Fish in three other high dietary PA treatments (2.25%) were fed to satiety,
since their feed intake was much lower (48-58 g fish-1) than those in the pair-feeding treatments
(78 g fish-1). Dietary PA levels affected only the Fultons condition index (FCI) and whole
carcass nitrogen retention efficiency (NRE) (P<0.05) while lignin levels in the diets significantly
affected the whole carcass lipid deposition (LD) and lipid retention efficiency (LRE) among the
pair-fed fish. On the other hand, when fed to satiety, feed intake by fish fed the lignin diets was
significantly reduced contributing to the differences in whole carcass recovered energy (RE) and
ERE and dressed carcass PD, RE, NRE and ERE between the fish fed diets with and without
lignin (P<0.01).
107
5.1. Introduction
Plant protein sources are increasingly used in aquaculture feed to satisfy the growing
need for more economical ingredients. These ingredients contain wide variety of compounds,
some with anti-nutritional properties, such as protease inhibitors, phytic acid, glucosinolates,
saponins, polyphenols, oligosaccharides and non-starch polysaccharides (e.g., mannan, galactan,
pectin, arabinose), lignin, haemaglutanin (e.g., lectins), antivitamins (e.g., anti-pyridoxine,
oxidase, anti-nicotinic acid, cyanogens), or antigenic compounds (e.g., glycinin and beta-
conglycinin (Francis et al., 2001; Tacon, 1997). Various factors such as amino acid composition,
anti-nutrients, and fibre components of a plant protein ingredient can affect its nutritive value
and the amount that can be used in the diets of various fish species (Bell 1993; Bureau et al.,
1998; Francis et al., 2001; Higgs et al., 1995; Mwachireya et al., 1999; Refstie et al., 2001;
Sanden et al., 2005).
The scope of most digestibility and bioavailability studies has been limited to
determining the effect of incorporation of various plant protein ingredients at the expense of fish
meal and other feedstuffs. These diets are formulated to the essential amino acid levels greatly in
excess of the requirement of the animal and result in poor characterization of the test ingredients.
The poor knowledge on the nutrients available to an animal necessitates the use of broader safety
margins when formulating feeds, reducing the ability to formulate on a truly least cost basis, and
forcing feed formulators to impose severe restrictions on the use of poorly characterized
ingredients (Bureau, 2006). Accurate characterization should extend beyond the assessment of
digestibility and bioavailability of nutrients to the effect of anti-nutritional factors and fibre
components on nutrient utilization in an animal.
108
In salmonids, studies with ANF mostly dealt with the effect of their inclusion levels on
growth performance and nutrient utilization (Burel et al., 2000; Cheng and Hardy, 2003; Kaushik
et al., 1995; Pratoomyot et al., 2010; Santigosa et al. 2010; Storebakken et al., 1998b). Growth
and dietary protein and mineral utilization in rainbow trout have been reported to be affected by
various anti-nutritional factors. For example, phytic acid (Kaushik et al., 1995; Spinelli, 1993;
Storebakken et al., 1998b; Teskeredzic et al., 1995; Wang et al., 2009a,b), soyasaponin (Bureau
et al., 1998; Knudsen et al., 2008), glucosinolate (Burel et al., 2000), or soluble and insoluble
non-starch polysaccharides (Amirkolaie et al., 2005; Enes et al., 2008; Glencross et al., 2008;
2011). Phytic acid is probably one of the most studied ANF in fish nutrition because of effects on
availability of phosphorus, and other valuable minerals and amino acids. Proton disassociation
sites on the PA molecules have variable pKa values between 1.5 and >10.0. They become
negatively charged when the proton is disassociated attracting positively charged molecules at
favourable pH (Adeola and Sands, 2003). Bivalent cations of minerals (e.g., Zn2+, Fe2+/3+, Ca2+,
Mg2+, Mn2+ and Cu2+) are particularly vulnerable to this process, creating phytic acid-mineral
binary complexes indigestible to the animal(Denstadli et al., 2006; Laining et al., 2010). Phytic
acid also interacts with protein creating binary (PA-protein) or ternary (PA-mineral-protein)
complexes that occur only at optimal pH conditions in the GIT (Cheryan and Rakis 1980; Kies et
al., 2001; Maenz, 2001). The -NH2 terminal group and the -NH2 group of Lys, the imidazole
group of His, and the positively charged guadinyl group of Arg have been implicated as possible
protein binding sites for phytate at low pH (Cosgrove, 1966). Ternary complexes are rare but
usually formed in slightly alkaline environment in the intestine (Riche et al., 2001).
Dietary fibre is defined as the sum of plant polysaccharides and lignin that are not
hydrolysed by endogenous digestive enzymes (Kritchevesky, 1988; Wenk, 2001). Dietary fibres
109
can also be divided into two physico-chemical groups soluble (pectins, beta-glucans and gums)
and insoluble fibres (cellulose, hemi-cellulose and lignin). Most studies on dietary fibre from
various plant sources dealt mainly with inclusion levels and their effects on nutrient utilization
and growth of an animal such as swine (Laplace et al., 1989; Mitaru et al., 1984; Souffrant,
2001), poultry (Slominski and Campbell, 1990; Villamide and San Juan, 1998), or fish
(Anderson et al., 1984; Bromley and Adkins, 1984; Hemre et al., 1995; Kraugerud et al., 2007).
Recent studies have shown that not the dietary fibre but the type and composition of its
components influences digestive processes in an animal (Amirkolaie et al., 2005; Glencross,
2009; Hansen and Storebakken, 2007; Zahedifar et al., 2002; Zhu et al., 2005).
Lignin, a highly polymerized hydrophobic phenolic plant compound, forms an integral
part of the secondary cell walls of most plants and algae. These polymers are composed mainly
of three different monolignols - sinapyl, coniferyl, and p-coumaryl alcohols, upon incorporation,
which are referred to as syringyl (S), guaiacyl (G), or hydroxyphenyl (H) units of lignin,
respectively. The proportion of S, G, and H varies among plant species, and can substantially
impact the physical properties of the polymer (Baurhoo et al., 2008; Boerjan et al., 2003). It is
hypothesized that monolignols are stabilized to lignin by the enzymes -glucosidases and
glucosyl transferases (Dharmawardhana and Ellis, 1998).
In vitro studies showed a significantly greater binding capacity of lignin with bi-valent
minerals such as Fe+2, Cu+2 and Zn+2 at intestinal pH (e.g., pH ~5) compared to that of pectin and
cellulose (Platt and Clydesdale, 1987). Hydroxyl and methoxy groups of lignin molecules under
pH conditions similar to those in the proximal intestine of animals form highly stable
hexadentate complexes with iron (Fernandez and Philipis, 1982). Affinity of these two binding
sites to minerals are in the order of Fe > Cu > Zn.
110
Under oxidative conditions, lignin also forms highly stable protein complexes resistant to
degradation by microbial fermentation or by acid hydrolysis (Zahedifar et al., 2002). In a study
involving three amino acids, proline, serine and glycine, Whitmore (1982) reported high binding
capacity of lignin with proline followed by serine and glycine. The author hypothesized that
proteins, especially those containing hydroxyproline, could be very susceptible to bonding with
dehydrogenation polymer of lignin under oxidizing conditions. Despite evidences of lignin-
mineral or lignin-protein bonding from in-vitro studies, there is a very limited effort to
understand the effect of lignin on performance of commercially important farmed animals.
A comprehensive understanding of the effect of PA, lignin and lignin-PA interactions on
nutrient utilization and performance of commercially important fishes would provide a strong
basis for the evaluation of novel plant protein ingredients in aquafeed. This study examined the
effects of graded levels of PA and purified lignin inclusion in a practical diet formulated to be
marginally adequate in some essential amino acids, and their interactions on nutrient utilization
in rainbow trout in a 12 weeks growth trial.
5.2. Methods
The growth trial was conducted at the UG/OMNR Fish Nutrition Research Laboratory
(Department of Animal and Poultry Science, University of Guelph, Ontario, Canada) for 12
weeks using standard research protocol. Phytic acid (50 wt% in aqueous solution) was obtained
from Sigma-Aldrich Co. (St. Louis, MO). Purified lignin was obtained from Pure Lignin
Environmental Technology, Kelowna, BC, Canada. The lignin, produced through a proprietary
close-loop process with low-pressure steam, was composed of 76% lignin and 24% cellulose. A
basal mix was prepared composed mainly of animal proteins i.e., herring fish meal, meat and
111
bone meal, regular poultry by-product meal, and spray-dried blood meal and a plant protein, corn
protein concentrate (Empyreal 75, Cargill Corn Milling, Blair, NB) (Table 5.1). Dextrin,
vitamin and mineral premixes, L-lysine, DL-methionine, di-calcium phosphate, and NaCl were
also added to reflect the composition of a typical trout feed. All the ingredients were mixed using
a large Marion mixer (Marion, IA).
5.2.1. Experimental diets
Nine isoproteic and isoenergetic experimental diets were prepared by adding fish oil and
appropriate amount of kaolin, cellufil, phytic acid, and purified lignin to the basal mix (Table
5.2). All diets were formulated to contain 2% cellulose. The concentration of all EAA but Arg,
Ile, Leu, Phe, and Val were formulated to be marginally above the requirement of rainbow
trout in all nine diets (NRC, 2011; Table 5.3). Basal mix and other ingredients were mixed using
a Hobart mixer (Hobart Ltd., Don Mills, ON, Canada), steam-pelleted using a laboratory scale
steam-pellet mill (California Pellet Mill, San Francisco, CA, USA), and dried under forced-air at
room temperature for 24 hrs. Dried diets were sieved to appropriate size and stored at 4C until
used. The amount of feeds needed were weighed on a weekly basis and kept at room
temperature.
5.2.2. Experimental conditions and feeding management
Rainbow trout (Oncorhynchus mykiss, Ontario domestic fall-spawning strain) fingerlings
were obtained from the Alma Aquaculture Research Station (Elora, ON, Canada). The
experimental system consists of three blocks of nine 60-L fibreglass tanks in each. Tanks were
supplied with filtered well water and individually aerated. Water flow, temperature and
112
photoperiod were maintained at ~3 L min-1, 13oC and 12h L 12h D cycle, respectively. Fish
were pre-acclimatized to the experimental conditions for seven days, fed a high quality
commercial salmonid diet (Profishent High Energy Feed , Martin Mills, Elmira, ON, Canada).
Nine diets were allocated to each block following a completely randomized block design.
Fifteen fish, an average weight of 40 g, were distributed to each tank. All treatments were fed to
satiety for the first two weeks to determine the lowest feed intake diet. After two weeks, fish
assigned diets 1, 2, 4, 5, and 7 were pair-fed to those fed diet 8, while continuing satiation
feeding for fish fed diets 3, 6, and 9. Feed consumption was recorded weekly and the fish were
weighed in every four weeks. Canadian Council on Animal Care (CCAC, 1984) and the
University of Guelph Animal Care Committee guidelines were followed to ensure ethical
treatment of the animal.
5.2.3. Morphological indices
At the beginning and the end of the experiment, fork length (mm) and weight (g) of 27
fish and 8 fish tank-1, respectively were measured to determine Fultons condition index (FCI;
Fulton, 1902). Five of these fish were then gutted, and viscera and liver were weighed for
viscera-somatic index (VSI) and hepato-somatic index (HSI), respectively. For the carcass-bones
ratio, eight fish were cooked at 900C for 20 minutes; all bones including the head were carefully
separated from muscle and skins, and weighed in g.
5.2.4. Chemical analysis
At the beginning of the experiment, 27 fish (one from each tank) were euthanized for
proximate composition and kept at -20oC until processed, and at the end of the experiment, a
113
total of ten fish tank-1 (five for whole carcass and five for dressed carcass and visceral
composition) from each tank were sacrificed. The pooled carcass and visceral samples were
autoclaved, ground into homogeneous slurry in a food processor, freeze-dried, then finely ground
and stored at -20oC until analysis.
Diets, ingredients, and carcass samples were analyzed for dry matter (DM) and ash
according to AOAC (1995), crude protein (CP, %N x 6.25, Dumas method) (Leco analyzer,
LECO Corporation, St. Joseph, MI), lipids with petroleum ether extraction at 90oC and 150 PSI
for 60 min using ANKOM XT-20 analyzer (ANKOM Technology, Macedon, NY), and gross
energy (GE) using an automated oxygen bomb calorimeter (Model # 1271, Parr Instruments,
Moline, Illinois). Visceral samples were analyzed only for DM, CP and lipid contents.
Neutral (NDF) and acid detergent fibre (ADF) contents of the ingredients and the diets
were determined according to Van Soest et al. (1991) using an ANKOM 200 Fibre Analyzer
(ANKOM Technology, Macedon, NY). Lignin was determined by reacting ADF residue with
cold 72% sulphuric acid. The sample was then ashed at 5500C for 4 hours and the residue was
measured gravimetrically (Van Soest and Reobertson, 1981). Phytic acid content in the
ingredients and diets were determined colorimetrically using a commercial assay kit (Megazyme
International, Ireland). Amino acid profiles of the test ingredients and the diets were analyzed at
the Amino Acid Laboratory of the Toronto Hospital for Sick Children (ON, Canada) using the
acid hydrolysis method. For the quantification of amino acids, approximately 0.05 g of dried
sample was hydrolysed in 15 ml test tube with 450 L of 6N HCl and 1% phenol for 24 hrs at
110oC under pre-purified nitrogen atmosphere. After 24 hrs, tubes were removed from oven,
cooled, capped, vortex mixed and centrifuged at 2500 rpm. Then, 5 L of the supernatant was
derivatized following PICO-Tag method (Cohen et al., 1989). After derivatization, supernatant
114
were re-dissolved in 150 L phosphate buffer and 1.5 L of this solution was injected into the
column of an ACQUITY Ultra-Performance Liquid Chromatography - UPLC machine (Waters
Corporation, Millford, CA) for quantification of all amino acids except tryptophan, methionine
and cysteine which are partially destroyed in the hydrolysis process.
5.2.5. Calculations
FCI (K) was calculated using the Fultons (1902) formula, where N = 5, BW
= body weight (g), L = fork length (mm). VSI and HSI were calculated as 100 x [viscera weight
(g)/BW (g)] and 100 x [liver weight (g)/BW (g)], respectively. Carcass:bone ratio was calculated
as whole carcass/bone weight.
Growth rate was calculated as thermal-unit growth coefficient (TGC), [100 x (FBW1/3
IBW1/3) / (Temp (0C) x Number of days)], where FBW is the final body weight and IBW is the
initial body weight of the fish (Cho, 1992). Feed efficiency (FE) was calculated as live weight
gain /dry feed intake. Protein (PD) and lipid (LD) deposition, nitrogen retention efficiency
(NRE), recovered energy (RE), and energy retention efficiency (ERE) were also determined. PD
and LD in whole carcass, dressed carcass, and viscera were calculated from the difference
between final and initial values. NRE (%) and ERE (%) were calculated as [[FBW x Nfinal)
(IBW x carcass Ninitial)]/DNI] x 100, [[FBW x carcass GE final) (IBW x carcass GEinitial)]/DEI] x
10, respectively, where DNI = digestible nitrogen intake, and DEI = digestible energy intake.
All data were analyzed using SAS V9.2, SAS Institute, Cary, NC, USA. Pair-feeding and
satiation-feeding treatments were analyzed separately. The difference among the treatments were
115
determined by Tukeys HSD test at P<0.05. The differences in responses of fish in treatments
pair-fed with diet 8 were assessed with Dunetts t-test.
The effects of phytic acid (0%, 1.13%) and lignin (0%, 1.5%, 3%) levels in the diets and
their interactions on final body weight, TGC, feed intake, feed efficiency, fish physiological
indices (VSI, HSI, FCI, and bone:carcass ratio), and nutrient deposition and retention
efficiencies, and PD:LD ratio were analyzed using SAS general linear model procedure (Proc
GLM) for the pair-feeding treatments, while only the effects of lignin were assessed for the
treatments fed high phytic acid diets. Significance of the effects was assessed using non-
orthogonal polynomial (linear and quadratic) contrasts for the pair-feeding treatments.
116
Table 5.1 Basal mix for the test diets.
Ingredient (% in diet)
Herring fish meal, 64% CP 6.8
Meat-bone meal, porcine, 47% CP 10.7
Poultry by-products meal, 66% CP 4.9
Feather meal, 85% CP 2.0
Spray dried blood meal, porcine, 93% CP 2.0
Corn protein concentrate, 77% CP 15.6
Gelatin 9.8
Gelatinized starch 20.1
Wheat middlings 2.0
Vitamin premix1 1.0
Lysine (Bio-lys) 1.3
DL-Met 0.1
Mineral premix2 1.0
Ca(H2PO4)2 0.5
NaCl 0.5
Total (% diet) 78.0
1
acetate (vitamin E), 75 IU; menadione sodium bisulphite (vitamin K), 1.5 mg; ascorbic acid polyphosphate (Stay C
25% ascorbic acid), 75 mg; Choline chloride, 1500 mg; cyanocobalamine (vitamin B12), 0.03 mg; biotin, 0.21 mg;
inositol, 450 mg; folic acid, 1.5 mg; niacin, 12 mg; Pantothenic acid, 30 mg; pyridoxineHCl, 7.5 mg; riboflavin, 9.0
mg; thiaminHCl, 1.5 mg.
2
Provides per kg of diet: sodium chloride (NaCl, 39% Na, 61% Cl), 3077 mg; ferrous sulphate (FeSO47H2O, 20%
Fe), 65 mg; potassium iodide (KI, 24%K), 11 mg; manganese sulphate (MnSO4, 36% Mn), 89 mg; zinc sulphate
10 mg.
117
Table 5.2 Ingredient and proximate chemical composition of the test diets (DM basis).
Pair-feeding Satiation Feeding

0%PA 1.13%PA 0%PA 1.13%PA 0%PA 1.13%PA 2.25%PA 2.25%PA 2.25%PA

0%lignin 0%lignin 1.5%lignin 1.5%lignin 3%lignin 3%lignin 0%lignin 1.5%lignin 3%lignin
Basal mix + composition of ingredients

Basal mix, % 78 78 78 78 78 78 78 78 78
Fish oil, herring, % 16 16 16 16 16 16 16 16 16
Kaolin, % 4.0 3.6 3.3 2.5 2.1 1.8 1.1 0.7 0.4
Cellufil, % 2.0 2.0 2.0 1.5 1.5 1.5 1.0 1.0 1.0
Phytic acid, % 0.00 0.36 0.00 0.36 0.00 0.36 0.75 0.75 0.75
Purified lignin (76% lignin, 24%
0.00 0.00 0.00 1.97 1.97 1.97 3.95 3.95 3.95
cellulose), %
Proximate chemical composition
Phytic acid, % diet 0.06 1.14 0.07 1.11 0.07 1.14 2.21 2.29 2.29
Lignin, % diet 0.0 0.0 1.4 1.4 3.0 3.0 0.0 1.5 3.0
Dry matter, % 95 95 95 97 95 96 95 96 96
Crude protein, % 43 44 45 43 44 42 42 43 43
Lipid, % 19 19 19 19 19 19 18 18 19
Total carbohydrate, % 26 25 27 28 28 30 27 29 28
Neutral detergent fibre, % 5.7 6.2 6.7 6.1 6.2 6.0 5.6 6.4 5.6
Acid detergent fibre, % 5.2 5.3 5.4 6.0 5.6 5.8 5.5 6.3 5.5
Ash, % 11 12 10 10 9 9 13 10 10
Gross energy, kJ g-1 23 22 23 22 23 23 22 22 23
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Table 5.3 Analyzed essential and non-essential amino acid composition of the test diets (digestible AA, % DM).
Pair-feeding Satiation feeding

EAA requirement of rainbow
trout (%), dry matter basis 0%PA 1.13%PA 0%PA 1.13%PA 0%PA 1.13%PA 2.25%PA 2.25%PA 2.25%PA
NRC, 2011 0%lignin 0%lignin 1.5%lignin 1.5%lignin 3%lignin 3%lignin 0%lignin 1.5%lignin 3%lignin
EAA, % in diet (DM basis)

Arginine 1.5 2.9 2.9 3.0 2.8 2.9 3.1 2.9 3.0 3.0
Histidine 0.8 0.9 1.0 1.0 0.9 0.9 0.9 1.0 1.0 0.9
Isoleucine 1.1 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Leucine 1.5 4.6 4.7 4.7 4.5 4.5 4.5 4.7 4.6 4.4
Lysine 2.4 2.4 2.5 2.4 2.5 2.4 2.4 2.5 2.4 2.5
Methionine+cystine1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1
Phenylalanine 0.7 2.1 2.1 2.1 2.0 2.1 2.1 2.1 2.1 2.0
Threonine 1.1 1.2 1.3 1.3 1.3 1.2 1.3 1.3 1.2 1.2
1
Tryptophan 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Valine 1.2 2.1 2.2 2.2 2.1 2.0 2.2 2.2 2.1 2.1
NEAA, % in diet(DM basis)

Alanine 4.1 4.1 4.3 3.9 4.0 4.1 4.1 3.9 4.0
Aspartic acid 2.8 2.9 3.3 2.4 3.1 3.2 2.8 3.2 3.2
Cysteine1 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
Glutamic acid 7.2 7.4 7.5 7.0 7.3 7.5 7.3 7.0 7.1
Glycine 5.3 5.2 5.7 4.9 5.2 5.4 5.2 5.1 5.2
Proline 4.7 4.6 4.9 4.4 4.6 4.8 4.6 4.4 4.6
Serine 1.9 1.9 2.0 1.9 1.9 2.1 1.9 1.9 1.9
Tyrosine 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3
1
Calculated value (digestible AA, % in diet)
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5.3. Results
No mortality occurred over the 12-week trial but a reduced feed intake with increasing
PA levels in the diets was observed during the first two weeks when fish in all treatments were
fed to satiety (Figure 5.1). After that, diets 1, 2, 4, 5, and 7 were pair-fed to the diet 8 while
satiation-feeding continued for fish fed 2.25% PA diets (3, 6, 9).
Final body weight, growth rate, FE, VSI, HSI, FCI, and whole-carcass:bone ratio of fish
in the pair-feeding treatments were slightly higher but not statistically different than those fed the
diet 8 as determined by the Dunnets t-test. None but the FCI among the performance attributes
and physiological indices was affected by PA or lignin or their interactions. Among the pair-
feeding treatments, FCI was significantly higher in fish fed the PA diets (1.05-1.07) than the PA-
free diets (1.02-1.05. In the satiation-feeding treatments, feed intake, TGC and FCI but not FE
and other physiological indices were affected by lignin (Table 5.4).
Among the pair-feeding treatments, whole carcass PD (130 mg fish-1 d-1, Dunetts t test -
P>0.05) and NRE (40%, Dunetts t test - P<0.05) in fish fed the control diet was slightly higher
than the other diets. In contrast, PD and LD in the dressed carcass were not affected but the NRE
of fish fed the control diet was significantly higher than those fed diet 8 (34%, Dunetts t test -
P<0.05). PA levels in the diets significantly affected the whole carcass NRE (P<0.05) (Table
5.5), but did not affect the protein or lipid deposition in the dressed carcass and viscera (Table
5.6). Dietary lignin affected only LD and LRE in the whole carcass (P<0.05).
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Figure 5.1 Total feed intake of the fish fed experimental diets when fed to satiety during the
first two weeks of the trial. Diets 1, 2, 3 (0% lignin), diets 4, 5, 6 (1.5% lignin) and diets 7, 8, 9
(3% lignin).
14 0% phytic acid
1.13% phytic acid
12
2.25% phytic acid
Feed intake (g fish-1)
10
0
1 4 7 2 5 8 3 6 9
Diets
121
Table 5.4 Performance and physiological indices of fish (initial weight = 40 g fish-1) fed the experimental diets for 84d at 15C.
Thermal-unit
Description of Final body growth Feed Feed
treatment weight coefficient intake efficiency Viscero- Hepato- Fulton's Whole
Phytic acid (PA) somatic somatic condition carcass :
g fish-1 g fish-1
Lignin index index index Bone ratio
Pair-feeding
Diet 1 0.0% PA 0.0% Lignin 104.1 1.2 0.120 0.001 77.5 0.2 0.87 0.01 10.0 0.2 1.17 0.03 1.05 0.02 4.7 0.4
Diet 2 1.1% PA 0.0% Lignin 100.7 3.1 0.118 0.003 77.5 1.2 0.83 0.03 12.0 0.6 1.50 0.25 1.05 0.02 4.6 0.2
Diet 4 0.0% PA 1.5% Lignin 100.4 1.0 0.118 0.003 77.5 0.0 0.83 0.02 10.6 0.3 1.27 0.03 1.02 0.01 4.5 0.1
Diet 5 1.1% PA 1.5% Lignin 102.5 4.2 0.118 0.007 77.5 1.9 0.83 0.05 10.8 0.4 1.33 0.09 1.07 0.02 4.4 0.4
Diet 7 0.0% PA 3.0% Lignin 101.1 1.5 0.117 0.003 77.5 0.3 0.83 0.02 10.7 0.3 1.30 0.06 1.02 0.01 4.4 0.1
Diet 8 1.1% PA 3.0% Lignin 95.9 5.8 0.112 0.008 77.5 6.6 0.77 0.02 10.4 0.8 1.27 0.03 1.06 0.01 4.4 0.2
Effects
PA NS NS NS NS NS NS P<0.05 NS
Lignin NS NS NS NS NS NS NS NS
PA x
Lignin NS NS NS NS NS NS NS NS
Satiation feeding
Diet 3 2.2% PA 0.0% Lignin 84.1 2.9 0.092 0.003b 57.7 1.9b 0.81 0.01 11.8 0.4 1.22 0.06 1.10 0.02b 4.7 0.2
Diet 6 2.2% PA 1.5% Lignin 76.6 3.0 0.079 0.004a 50.5 0.5a 0.77 0.04 13.6 1.3 1.36 0.08 1.06 0.00a 4.5 0.3
Diet 9 2.2% PA 3.0% Lignin 78.3 1.2 0.078 0.001a 48.2 0.4a 0.80 0.04 11.7 0.2 1.29 0.03 1.02 0.00a 4.4 0.1
Effects
Lignin NS P<0.05 P<0.01 NS NS NS P<0.01 NS
Note: * Significant difference between pair-fed treatments and diet 8 were determined by Dunetts t tests. Difference between other diets was determined by
Tukey's HSD test (P0.05). Different letters denote significant differences; responses with no notations mean no differences among the diets. Effects of PA,
lignin and their interactions were determined by multiple linear regression (Y = b + PA + lignin + PA x lignin) and deemed significant at P<0.05.
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Table 5.5 Whole carcass nutrient deposition and retention efficiency of rainbow trout fed test diets with various levels of phytic acid
and lignin.
Whole Carcass
Lipid Energy Protein :
Protein Lipid Recovered N retention retention retention lipid
Description of treatment deposition deposition energy efficiency efficiency efficiency deposition
Phytic acid (PA) Lignin mg fish-1 d-1 mg fish-1 d-1 j fish-1 d-1 % % %
Pair-feeding
Diet 1 0.0% PA 0.0% Lignin 130.2 1.5 72.6 6.2 58.9 1.0 40.0 0.5* 47.5 4.0ab 42.5 1.1 1.8 0.2ab
Diet 2 1.1% PA 0.0% Lignin 120.2 4.2 79.7 3.7* 59.2 1.8 36.4 1.0 54.2 1.9*b 45.4 0.7* 1.5 0.0*a
Diet 4 0.0% PA 1.5% Lignin 126.2 0.8 79.7 1.8* 58.6 2.7 37.7 0.3 53.1 1.1*b 43.4 1.1 1.6 0.1ab
Diet 5 1.1% PA 1.5% Lignin 119.6 6.9 72.7 7.4 58.0 5.0 35.5 1.4 46.6 5.1ab 42.5 1.7 1.7 0.2ab
Diet 7 0.0% PA 3.0% Lignin 123.6 3.9 66.2 5.8 58.2 3.6 37.0 1.1 43.7 3.7ab 43.0 1.6 1.9 0.1ab
Diet 8 1.1% PA 3.0% Lignin 115.8 14.1 56.8 6.7 50.9 5.9 35.6 1.4 37.1 2.1a 40.4 0.9 2.0 0.1b
Effects
PA NS NS NS P<0.05 NS NS NS
Lignin NS P<0.05 NS NS P<0.01 NS P<0.05
PA x Lignin NS NS NS NS NS NS NS
Satiation feeding
Diet 3 2.2% PA 0.0% Lignin 87.1 3.1 43.2 6.3 38.3 2.2b 37.2 1.0 40.0 5.3 40.8 1.0b 2.1 0.3
Diet 6 2.2% PA 1.5% Lignin 78.9 4.4 33.7 1.5 31.3 1.3ab 37.0 1.8 35.1 1.4 39.0 0.8ab 2.4 0.0
Diet 9 2.2% PA 3.0% Lignin 77.3 1.9 26.0 7.1 27.5 2.6a 37.8 1.2 27.77.6 33.7 2.7a 3.7 1.3
Effects
Lignin NS NS P<0.01 NS NS P<0.05 NS
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Table 5.6 Nutrient deposition and retention efficiencies in dressed carcass and viscera of rainbow trout fed the experimental diets.
Dressed carcass Viscera

Protein Lipid Recovered
Description of treatment deposition deposition energy Protein deposition Lipid deposition
Phytic acid (PA) Lignin mg fish-1 d-1 mg fish d j fish-1 d-1
-1 -1
mg fish-1 d-1 mg fish-1 d-1
Pair-feeding
Diet 1 0.0% PA 0.0% Lignin 110.7 2.8 40.4 4.1 43.5 2.3 14.8 0.4 22.8 3.1a
Diet 2 1.1% PA 0.0% Lignin 99.4 4.4 48.3 7.0 43.6 3.5 17.6 1.4 34.5 3.8*b
Diet 4 0.0% PA 1.5% Lignin 102.3 3.0 44.4 3.6 42.7 1.7 16.6 0.5 23.2 1.0a
Diet 5 1.1% PA 1.5% Lignin 107.0 9.7 55.3 6.7 49.1 5.1* 15.2 1.2 23.1 1.4a
Diet 7 0.0% PA 3.0% Lignin 100.0 3.2 38.9 4.1 39.9 1.0 14.1 0.2 28.9 5.0ab
Diet 8 1.1% PA 3.0% Lignin 88.6 11.4 34.5 4.5 35.5 4.1 16.2 1.2 22.1 1.7a
Effects
Phytic acid NS NS NS NS NS
Lignin NS NS NS NS NS
Phytic acid x Lignin NS NS NS NS P<0.05
Satiation feeding
Diet 3 2.2% PA 0.0% Lignin 62.6 5.1b 20.7 2.3 23.7 1.8b 21.8 1.8 35.1 6.8
Diet 6 2.2% PA 1.5% Lignin 35.4 1.1a 9.5 1.6 12.3 0.9a 25.3 1.6 38.3 1.8
Diet 9 2.2% PA 3.0% Lignin 39.3 3.4a 12.5 3.7 12.5 2.1a 21.3 0.6 37.8 5.0
Effects
Lignin P<0.01 NS P<0.01 NS NS
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5.4. Discussion
Effects of dietary PA and purified lignin on growth performance, physiological indices,
and nutrient deposition in rainbow trout were assessed in this study. Overall FE and growth of
fish in all treatments including those fed the control diet were low (between 0.77 and 0.87)
compared to those observed in similar trials. In this study, five EAA: His (0.9-1.0% DM), Lys
(2.4-2.5% DM), Met+Cys (1.1% DM), Thr (1.2-1.3% DM), and Trp (0.2% DM) were formulated
to be marginally adequate to the requirement of rainbow trout. It is likely that a portion of these
AA were not available resulting in lower than usual feed efficiency. Most protein sources used in
this study are animal by-products, which have subjected to various pressure and temperature
conditions during processing and manufacturing. Bureau et al. (1999) showed that manufacturing
characteristics such as duration, pressure and temperature during hydrolysis of feather meals,
during cooking of MBM or PBM, and coagulation and drying methods of blood meals, affect the
digestibility of crude proteins of these feed stuffs in rainbow trout. Proteins may react with fats
and their oxidation products, and chemicals used during processing. Amino acids involved in
these reactions are structurally altered and often, the reaction is irreversible resulting in the AA
being biologically unavailable. Lys, with its -amino group, is particularly susceptible to such
damage, and so are Met, Cys, and Trp (Batterham, 1992; Moughan and Rutherfurd, 1996).
In this study, when fish in all treatments in the first two weeks, and in the high PA
(2.25%) treatments throughout the trial were fed to satiety, feed intake appeared to be affected by
dietary PA (Figure 5.1) and lignin (Table 5.4), respectively. It is plausible that one or more of the
five marginally adequate EAA in the diets may not be completely available to fish resulting in an
imbalanced AA profiles. Despite slightly lower performances of the fish fed PA-high lignin diets
(diet 8) than the other diets, PA or lignin level did not affect TGC, FE or any of the physiological
125
indices among the pair-feeding treatments. Only FCI was affected, where fish fed PA diets
showed higher FCI (1.06-1.07) than the PA free diets (1.02). Dabrowski et al. (2007) reported
that significant differences in growth of a cichlid species (Midas, Amphilophus citrinellum) but
not in FE the fish fed a protein-bound EAA diet supplemented with free AA (FAA) and two
AA deficient diets, (-) Lys (deficient in Lys, His, Ile, Phe) and (-) Arg (deficient in Trp, and Arg,
Thr, Val, Leu, and Met). One of the earliest studies on the effect of PA on fish was conducted by
Spinelli et al. (1983) where the authors reported significant differences in weight gain between
rainbow trout fed diets with 0.5% sodium or calcium phytate (72 and 74 g, respectively) and
without PA (86 g) but observed no effects on FE. Similar observations were also reported in
salmon (Sajjadi and Carter, 2004a, b) and carps (Usmani and Jafri, 2002). In another study,
Denstadli et al. (2006) reported insignificant effect on the ADC of CP and FE (between 0.68 and
0.72) in Atlantic salmon with maximum inclusion rate of 2% PA of the diet. But feed intake was
severely affected at the dietary PA of 1% or higher (11.9-15.5 g fish-1) in the first 39 days but
after that, low feed intake were observed in the fish fed only the high PA (2%) diet (28.8 g fish-1)
compared to that of the fish fed the control diet (19.0 and 50.1 g fish-1, respectively). These
results are in agreement with the present study in that animals control their PA intake by
controlling the total feed intake reducing the effect of PA on the deposition and partitioning of
nutrients.
In this study, dietary PA levels affected only the FCI and the whole body NRE of the
fish. FCI is a measure of energy reserve where values >1 represent fish with high fat while fish
with <1 CI, represent the fish that suffered from adverse environmental or poor feeding
conditions (Rtz and Llroet, 2003). Although LD tend to decrease with the addition of lignin,
both LD and whole body LRE were not significantly affected but slightly higher in fish fed PA
126
diets than the control diets resulting in higher FCI. LD (35 mg fish-1 d-1) in viscera of fish fed
lignin free PA diets was significantly higher than the control diets (23 mg fish-1 d-1) (Table 5.6)
suggesting that much of the excess lipid were deposited as intra-peritoneal fat (IPF) rather than
as muscle fat.
Dietary PA have been reported to significantly affect CP digestibility in several fish
species, for example, Atlantic salmon Salmo salar (Sajjadi and Carter, 2004a,b), rainbow trout
Oncorhynchus mykiss (Spinelli et al., 1983; Sugiura et al., 2001), and common carp Cyprinus
carpio (Hossain and Jauncy, 1989). Recently, Laining et al. (2010) reported a decreasing ADC of
CP from 95% in the fish fed the control diet (0% PA) to 90% in those fed 1% PA diets. Selle et
al. (2000) proposed that the de novo formation of binary protein-PA complexes in the
gastrointestinal tract as the key mechanism by which PA negatively influences the AA
digestibility. Several modes of action of forming these complexes are: (1) the presence of
protein-PA complexes in feedstuffs, (2) the de novo formation of binary and ternary protein-PA
complexes in the digestive tract and (3) the inhibition of proteolytic enzymes (Kies et al., 2001;
Selle et al., 2006).
Significantly lower NRE in fish fed the PA diets observed in this study are in agreement
with several studies (Nangthu et al., 2010; Storebakken et al., 1998b; Usmani and Jafri, 2002).
Nangthu et al. (2010) reported that Lys utilization efficiency was severely affected when
formulated to be marginally adequate in a plant protein based diet resulting in a significantly
lower NRE. In an earlier study, ADC of His and Lys appeared to be negatively influcenced by
the PA intake (Chapter 3). All experimental diets in this study were marginally adequate in these
two EAA. The inclusion of PA could have further reduced their availability resulting in low NRE
in fish fed the PA diets (P<0.05).
127
None of the growth performance and physiological indices of rainbow trout was affected
by lignin in the pair-feeding treatments but when fed to satiety, lignin appeared to significantly
suppress feed intake, which likely affected the TGC and FCI of rainbow trout. No such effects on
feed intake have been demonstrated in studies with swine and broiler chickens fed purified lignin
(Schedle et al., 2008; Baurhoo et al., 2007). A synergistic interaction of lignin with PA in this
case can not be ruled out because of the very high PA concentration (2.25%) in these diets.
Schedle et al. (2008) investigated the effects of insoluble dietary fibre in the variable
lignin diets on the performance of piglets. The authors used Chinese Mason Pine pollen (29%
lignin) as the source of lignin. Higher feed intake by the piglets fed lignin diets than those fed the
lignin-free diet resulted in a significantly higher weight gain. However, apparent crude protein
digestibility was significantly lower only in piglets fed high lignin (1.7%) diets than those fed the
other diets. In another study, Wang et al. (2009b) fed feedlot lambs diets containing 1.5% and
3% purified lignin, and reported no differences in the weight gain between lambs fed diets with
and without lignin.
In this study, lignin did not affect the whole carcass PD and NRE but the LD (P<0.05),
LRE (P<0.01), and PD:LD (P<0.05) in the pair-feeding treatments, where as in the satiation-
feeding treatments, only whole carcass RE (P<0.01) and ERE (P<0.05) appeared to be
significantly affected. Glencross (2009) reported a comparable effect of lignin where not the
ADC of N but those of GE and DM were significantly reduced from 92% and 84%, respectively
in rainbow trout fed the control diet to 68% and 77%, respectively in those fed the high lignin
(3%) diets. In this study, unlike the whole carcass, NRE (P<0.05) and ERE (P<0.01) in dressed
carcass were significantly affected by lignin in the pair-feeding treatments. Although not
128
significant, LD (P=0.061) and RE (P=0.096) in the dressed carcass of fish pair-fed PA-high
lignin treatment was lower than those in the other treatments.
The lack of notable interactions between PA and lignin in this study suggests that their
mechanisms and regions of actions are different. In nature, hydrophobic lignin molecules co-
exist with the hydrophilic cellulose, and together, they act as nutrient transporter in various plant
tissues. It can be speculated that a portion of dietary lipid might have bound by the lignin
molecules affecting digestibility of lipids. This is the first detailed study elucidating effects of
lignin and its interaction or not with PA on performance, physiological indices and nutrient
deposition of rainbow trout. It can be concluded that fish, like any other animal, controls intake
of the feed when fed diets with imbalanced AA profile or deficient in one or more EAA, or when
one or more ANF are present in order to maintain their homeostasis. Neuro-gastric mechanisms
to control feed intake in such situations is not well-understood and should be explored further to
improve our understanding on anti-nutritional components of plant ingredients and their effects
on nutrient dynamics in farmed animals.
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CHAPTER 6
ASSESSING THE EFFECTS OF LIGNIN ON PHYSICAL PROPERTIES OF STEAM-
PELLETED EXPERIMENTAL TROUT FEED
Abstract
A series of trials were conducted to evaluate the effects of lignin on the physical properties
of a dietary-mash and a steam-pelleted fish feed. Oil and water absorption of dietary mash and
physical properties of the pellets were assessed with graded levels of purified lignin. Absorption
of both vegetable oil (VO) and fish oil (FO) decreased significantly from 91% to 80% and from
84% to 76% in the mash with 0% and 3% lignin, respectively, but did not change thereafter.
Water absorption capacity was not affected (P>0.05) with increasing levels of lignin. Bulk
density, sinking velocity, hardness and water stability (i.e. dry matter, nitrogen and lipid loss) of
the steam-pelleted feed were examined at 0%, 1.5% and 3% lignin levels. Bulk density increased
significantly with increasing levels of lignin but hardness was not affected with inclusion of up
to 1.5% (75 and 73 Newton for 0% and 1.5% lignin pellets, respectively), that reduced
significantly (P<0.01) at 3% inclusion (51 Newton). Water stability of the pellets was
significantly reduced by increasing duration in water and smaller size but not affected by lignin
(P<0.05). Loss of nutrients increased linearly with increasing time where 2-mm pellets lose
nutrients faster than the 3.35-mm pellets. Overall, lipid loss was higher (P<0.05) than dry matter
and nitrogen loss for all treatments. This study suggests that at level up to 1.5%, lignin had no
effect on the physical quality of the steam-pelleted aquaculture feed.
130
6.1. Introduction
Physical properties are a measure of the effect an ingredient has on the characteristics of
a feed (Aas et al., 2011). These properties include fluid absorption capacity of mash and pellets,
and bulk density, hardness, sinking velocity, viscosity and water stability of the pellets. Water
stability of a pellet is usually determined by the time it takes to disintegrate and may include the
determination of the dry matter loss. Rapidly disintegrating feeds cause significant
environmental and economical losses and are highly undesirable in commercial farming
practices (Cho and Bureau, 2001; Obaldo et al., 2002). Water stability can be improved by
adding starch and soluble (e.g., guar gum, Brinker, 2007; Brinker and Reiter, 2011; Storebakken,
1985) or insoluble (e.g., cellulose, Bromley and Adkins, 1984; Hansen and Storebakken, 2007;
Lupatsch et al., 2001) fibres as binders in fish feed. Level and composition of the fibre
components in plant protein ingredients alter the physical properties of mash and pellets during
processing often requiring modifications of the manufacturing parameters. Until today, few
studies have assessed physical properties of aquaculture feed affected by the fibre components of
an ingredient (e.g., Baeverfjord et al., 2006; Glencross et al., 2007b; Hansen and Storebakken,
2007; Kraugerud et al., 2011), and the criteria for evaluation of these properties are not well-
established (e.g., Glencross et al., 2007b; 2010).
The insoluble lignin, a highly polymerized phenolic plant polymer responsible for
providing structural support and the flow of nutrients, is the second most abundant compound in
plant kingdom after cellulose (Jung and Fahey, 1983). Lignosulfonates and Kraft lignins,
products of pulping, are primarily used as binders to improve durability of the animal feed pellets
(Gargulak and Lebo, 2000). These lignin derivatives are composed mostly of high-molecular-
weight fragments and have shown some toxicity in rainbow trout (Pessala et al., 2010; Roald,
131
1977). Purified lignin, in contrast, consists mostly of low-molecular-weight phenolic fragments
with putative anti-microbial and prebiotic effects (Davidson and Brenen, 1981; Kleinert and
Barth, 2008; Lora et al., 1993; Roller and Seedhar, 2002; Ultee et al., 2000). Prebiotic effects of
purified lignin are reported recently in broiler chickens (Baurhoo et al., 2007a), and in feedlot
lambs (Wang et al., 2009b) at low inclusion level. Baurhoo et al. (2008) hypothesized that lignin,
in its purified form, possesses functional properties not available in other lignin compounds. In
addition, apparent digestibility of nutrient, gut morphology, growth and feed intake in poultry
(Baurhoo et al., 2007b) and rabbits (Chiou et al., 1994) remain unaffected when fed diets with
<4% of lignin suggesting the possibility of using purified lignin in limited quantity in
aquaculture feed both as binder and as pre-biotics.
In this study, an attempt was made to standardize the assessment criteria of some physical
properties of compounded aquaculture feed with inclusion of purified lignin, as a model
ingredient. The physical properties assessed are the oil and water absorption in the mash and
bulk density, hardness, sinking velocity, and water stability of the steam-pressed pellets
containing various levels of lignin.
6.2. Methods
Purified lignin, extracted from pine (Pinus sylvestris L.) bark using a proprietary process,
was obtained from the Pure Lignin Environmental Technology, Kelowna, BC, Canada. A dietary
mash consisting of fish meal, animal by-products (meat and bone meal, feather meal, blood meal
and gelatin), and corn protein concentrate as protein source was prepared (Table 6.1). Dextrin,
vitamin and mineral premixes, L-lysine, DL-methionine, di-calcium phosphate, and NaCl were
also added to reflect the composition of a typical trout feed (Table 6.1). The basal mash,
132
produced by mixing the ingredients in a Marion mixer (Marion, IA), was used to determine oil
and water absorption with five levels of lignin (0%, 1.5%, 3%, 6% and 12%). Fish oil (16%), and
appropriate amount of kaolin (an inert product with low shrink-swell capacity) and lignin were
added to the mash to prepare three diets of 0%, 1.5% and 3% lignin. The diets were thoroughly
mixed (using a Hobart mixer Hobart Ltd., Don Mills, Ontario), pelleted (using a laboratory
steam pellet mill California Pellet Mill Co., San Francisco, California), and dried in forced air
at ambient temperature for 24 h. Pellets of 2.00-mm and 3.35-mm diameter were produced to test
the size effects on bulk density and pellet water stability (loss of dry matter (DM), nitrogen (N)
and lipid). Another set of pellets of 3.35-mm diameter were produced by mixing mash diet with
0%, 1.5% and 3% lignin using a laboratory built pellet gun for assessing the effects of lignin on
sinking velocity and hardness of the pellets.
133
Table 6.1 Ingredient composition of the basal mix prepared for assessing oil and water
absorption capacity with the addition of lignin. 16.0% fish oil was added before mixing with
kaolin and lignin to prepare three diets of 0%, 1.5% and 3% lignin.
Ingredient (% in diet)
Herring fish meal, 64% CP 6.8
Meat-bone meal, porcine, 47% CP 10.7
Poultry by-products meal, 66% CP 4.9
Feather meal, 85% CP 2.0
Spray dried blood meal, porcine, 93% CP 2.0
Corn protein concentrate, 77% CP 15.6
Gelatin 9.8
Gelatinized starch 20.1
Wheat middlings 2.0
Vitamin premix1 1.0
Lysine (Bio-lys) 1.3
DL-Met 0.1
Mineral premix2 1.0
Ca(H2PO4)2 0.5
NaCl 0.5
Total (% diet) 78.0
1
acetate (vitamin E), 75 IU; menadione sodium bisulphite (vitamin K), 1.5 mg; ascorbic acid polyphosphate (Stay
CTM 25% ascorbic acid), 75 mg; choline chloride, 1500 mg; cyanocobalamine (vitamin B12), 0.03 mg; biotin, 0.21
mg; inositol, 450 mg; folic acid, 1.5 mg; niacin, 12 mg; Pantothenic acid, 30 mg; pyridoxineHCl, 7.5 mg;
riboflavin, 9.0 mg; thiaminHCl, 1.5 mg.
2
Provides per kg of diet: sodium chloride (NaCl, 39% Na, 61% Cl), 3077 mg; ferrous sulphate (FeSO 47H2O, 20%
Fe), 65 mg; potassium iodide (KI, 24%K), 11 mg; manganese sulphate (MnSO4, 36% Mn), 89 mg; zinc sulphate
10 mg.
134
6.2.1. Oil and water absorption of the mash
Absorption of FO, VO, and water was determined according to a method slightly
modified from Sathivel et al. (2005). 500-mg of sample mash with appropriate amount of lignin
was placed into a 50-ml centrifuge tube, 10-ml of oil or de-ionized water was addred, then
thoroughly mixed using a steel spatula for 2 minutes, and kept for 30 minutes at ambient
temperature mixing intermittently in every 10 minutes. The mixture was centrifuged at 2560 x g
for 25 minutes using an Eppendorf centrifuge model 5810R (Eppendorf AG, Hamburg,
Germany). After decanting the free oil or water, absorption was determined from the weight
difference and expressed as percent of the sample weight.
6.2.2. Physical properties of the pellets
Bulk density, sinking velocity, hardness and water stability of the pellets with 0%, 1.5%
and 3% lignin were assessed. For bulk density, pellets of each size were filled in a 50-ml
measuring cylinder up to the mark, and weighed in g. The bulk density of the pellets was then
calculated as the weight of this volume and expressed as g L-1. Sinking velocity was determined
by placing a pellet 1-cm below the surface in a 500-mL measuring cylinder filled with water.
Time taken by a pellet to travel to the bottom (26.7 cm) was measured using a digital stop-watch
and expressed as cm s-1. Ten randomly selected pellets were used to determine average sinking
velocity for each treatment.
Hardness was determined as the force to crush a pellet across diameter using a texture
analyzer, Instron Universal Testing Machine, Model 1122 (Instron, Norwood, MA) equipped
with a load-cell and a flat disk. Ten pellets of each treatment were randomly selected for the
analysis. The analyzer was set with a test speed of 0.17 mm s-1. The probe was set to pass a
135
maximum distance of 3 mm and trigger at a contact pressure of 5 g. Crush strengths were defined
as the peak force at breaking of the pellet and expressed in Newton (N).
A horizontal shaking method, slightly modified from Obaldo et al. (2002), was employed
using a shaking water bath (Thermo Scientific Haake SWB25, Thermo Fisher Scientific, MA)
for assessing water stability of pellets measured as the amount of nutrients leached over time.
Representative feed samples of 10 g for each of the 2-mm and 3.35-mm pellets were taken into
250-ml Erlenmeyer flasks containing 100-ml de-ionized water, and were subjected to horizontal
shaking (at 100 rpm) for 1, 10, and 20 min at 20oC. Remaining solids were then separated using
a 1-mm mesh sieve, carefully poured into pre-weighed aluminum pans with minimal disturbance,
and dried at 105oC for 24 hr for dry matter loss (AOAC, 1995). Nitrogen (N) and lipid-contents
of the dried samples were determined using a LECO Analyzer (LECO Corporation, St. Joseph,
MI) and by petroleum ether extraction at 90oC and 150 PSI for 60 min using ANKOM XT-20
analyzer (ANKOM Technology, Macedon, NY), respectively.
All data were subjected to GLM procedure using SAS 9.2, SAS Institute, Cary, NC,
USA. Effects of lignin, pellet size, time and their interactions were assessed for pellet water
stability. For the bulk density, effects of lignin level, pellet size and their interactions were
assessed while for sinking velocity, hardness, and water and oil absorption, a simple linear
regression (a + bx) model was used. A parameter was deemed significant at P<0.5. Differences
among the mash or pellets with various levels of lignin were assessed by Tukeys-b test and
considered significant at P<0.05.
136
6.3. Results
6.3.1. Oil and water absorption
The absorption of VO (average 82%) was significantly higher (P<0.01) than the FO
(average 76%). Absorption of both VO and FO decreased significantly (P<0.05) from the mash
containing 0% lignin to those with 3% lignin (from 84% to 76% for FO and from 91% to 80%
for VO, respectively), but did not change at higher concentration (Table 6.2). Water absorption
was not affected by lignin (P>0.05).
6.3.2. Physical properties
Sinking velocity of the pellets decreased significantly with increasing levels of lignin
(Table 6.3), from 5.1 cm s-1 to 4.5 cm s-1 to 3.2 cm s-1 with the 0%, 1.5% and 3% lignin,
respectively. Hardness of the pellets did not differ (P>0.05) between the pellets without (75 N)
and those with 1.5% lignin (73 N), but decreased significantly (P<0.05) with 3% lignin (47 N,
Table 6.3). Lignin levels (P<0.05), pellet size (P<0.01), and their interactions (P<0.05), all
affected the bulk density (Table 6.4). Among the water stability parameters, loss of DM from the
pellets ranged between 7% and 13%, respectively within the first minute in the water and
increased significantly for both 2-mm (between 45% and 63%) and 3.35-mm (between 23% and
28%) pellets at the 10th minute in water (Figure 6.1). Duration in water affected nutrient loss the
most (P<0.01) followed by size x time interactions (affecting only the DM and N loss, P<0.05),
where as lignin and pellet size did not affect the loss of any nutrient (P>0.05, Table 6.4).
137
Table 6.2 Oil and water absorption capacity (in %) of the diet mash with various levels of
lignin (Mean SE). The difference in the absorption capacity of oil and water among different
levels of lignin was assessed by Tukeys b test and considered significant at P<0.05.
Absorption capacity of mash (%)
Lignin (% in mash) Fish oil, herring Mixed vegetable oil Water
(%) (%) (%)
0% 84 1b 90 4b 134 3
1.5% 77 1ab 86 2ab 134 2
3% 75 3a 80 1a 140 3
6% 75 0a 78 0a 139 2
12% 74 2a 78 0a 134 2
138
Table 6.3 Sinking velocity (cm s-1) at water temp. 22OC, hardness (Newton) and bulk density
of the diet pellets with various levels of lignin (mean SE). The difference among the pellets of
three lignin levels (0%, 1.5% and 3%) was assessed by Tukeys-b test and considered significant
at P<0.05.
Sinking Hardness Bulk density

Lignin level velocity
(%)
cm s-1 Newton g L-1
2.0 mm 3.35 mm
c b
0% 5.1 0.3 753 594 12b 522 5a
1.5% 4.50.2b 733b 581 5a 537 13b
3% 3.20.1a 513a 580 2a 540 12b
139
Figure 6.1 Effect of lignin inclusion rate (0%, 1.5% and 3%) on dry matter loss and nitrogen
leaching from pellets of two sizes (2-mm and 3-mm) following immersion in shaking water bath
at 100 rpm min-1 for >1-min, 10-min and 20-min of durations at 22oC.
Pellet size (2.00 mm) Pellet size (3.35 mm)
80 80
Dry matter loss (%)
Dry matter loss (%)

60 60
40 40
20 20
0 0
0 10 20 0 10 20
Time (min) Time (min)
60 60
50 50
N loss (%)
40 40
N loss (%)
30 30
20 20
10 10
0 0
0 10 20 0 10 20
80 80
60
Lpid loss (%)
Lpid loss (%)
60
40 40
0% Lignin 0% Lignin
20 1.5% Lignin 20 1.5% Lignin
3% Lignin 3% Lignin
0 0
0 10 20 0 10 20
140
Table 6.4 Parameter estimates (b) of the effect of lignin levels (0%, 1.5% and 3%), pellet size, duration in water (time) and their
interactions on pellet water stability (%), parameter estimates (b) of the effect of the three lignin levels on sinking velocity and
hardness of the pellets, and oil and water absorption of the mash (lignin levels 0%, 1.5%, 3%, 6%, and 12%). Pellet water stability was
measured as dry matter (DM), nitrogen (N), and lipid loss.
Pellet quality Diet mash

Pellet water stability Oil & water absorption
N- Bulk Sinking Vegetable
DM loss loss Lipid loss density velocity Hardness Fish oil Oil Water
% % % g L-1 cm s-1 Newton % % %
Intercept 18.8 13.4 40.7** 423.7** 5.2** 78.2** 79.8** 86.7* 135.4**
Lignin 5.0 3.5 5.6 21.6** -0.6** -7.9** -0.6* -0.9* 0.5
Pellet size -2.3 -1.2 -5.7 50.3** - - - - -
Time 3.8** 3.6** 3.0** - - - - - -
Lignin x size -1.8 -1.8 -2.0 -7.9* - - - - -
Lignin x time -0.0 0.1 -0.0 - - - - - -
Pellet size x time -0.7* -0.7* -0.5 - - - - - -
Lignin x size x time 0.1 0.0 0.1 - - - - - -
2
Adj. R 0.88 0.88 0.92 0.90 0.61 0.49 0.40 0.52 0.33
**P < 0.01; * P < 0.05
141
6.4. Discussion
Effects of lignin on oil and water absorption by a dietary mash and on some physical
properties of steam-pressed pellets of an experimental salmonid feed were investigated in this
study. The focus of this study was to develop protocols for assessing the quality of steam-pressed
pellets. Type of feedstuffs (Olsen et al., 2006; Refstie et al., 2006), feedstuff components
(Hansen and Storebakken, 2007; Hilton et al., 1981), and processing and manufacturing
conditions (Sorensen et al., 2005; Sorensen et al., 2009) have substantial effects on the stability
of a feed requiring to adjust processing and manufacturing parameters including physical,
chemical and thermal treatments (e.g., moisture, temperature, pressure, flow, torque, screw
speed) (Draganovic et al., 2011; Thomas, 1996).
Modern farming practices involve storage and transportation of large volume of feed that
are mechanically or manually distributed to the rearing vessels (e.g., ponds, tanks, pens, cages
etc.) demanding highly durable and water-stable feeds. The feeds also need to be stable enough
to avoid rapid disintegration after ingestion. Besides increasing the waste-outputs (Cho and
Bureau, 2001), rapid disintegration of a feed after ingestion appeared to cause oil-belching in
salmonids fed high-fat diets often coinciding with gastric dilation air sacculitis (GDAS)
(Baevefjord et al., 2006; Forgan and Forster, 2007; Lumsden et al., 2010).
6.4.1. Oil and water absorption
Sathivel et al. (2005) defined absorption of any fluid as the ability of a mash to absorb
and retain water and oil against a centrifugal force. Glencross et al. (2009) adopted a similar
method to assess water absorption by dry mash but for oil absorption, the authors proposed a
vacuum infused oil uptake method where oven-dry pellets placed in a mixer with excess oil,
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thoroughly mixed for 1-min, transferred to a beaker, then placed in the vacuum chamber of a
freeze drier. After reaching the vacuum, the chamber was re-equilibrated to the atmospheric
pressure and oil infused to the pellets was then weighed by difference. This method deemed
suitable for extruded pellets where pellets are expanded during production and have air pockets
but may not be feasible for steam-pressed hard pellets.
In this study, oil absorption decreased significantly with lignin levels of up to 3%.
Significant differences were observed between the mashes with 0% and 3% or more lignin for
both FO and VO (Table 6.2). Approximately 7% more VO was absorbed than the FO
irrespective of the lignin levels. FO used in this study is North American herring oil composed of
26% SFA, 57% MUFA and 17%PUFA (Bureau et al., 2008). Significantly higher density of the
VO (specific gravity, 0.874 0.01) than that of the FO (specific gravity, 0.846 0.02) used in this
study may explain the differences in the absorption of these oils.
6.4.2. Pellet quality
The bulk density, sinking velocity, and hardness of the pellets were significantly affected
by lignin in this study. Bulk density were affected by both lignin (P<0.05) and pellet size
(P<0.01) (Table 6.4). Larger steam-pressed pellets are usually denser than the smaller pellets
resulting in higher bulk density. Other factors that may affect the density of pellets are: condition
temperature (C), screw speed (rpm), die temperature (C), pressure (bar), torque (N m), and
specific mechanical energy SME (Wh kg-1), and the composition of non-starch polysaccharides
and level of starch gelatinization (Kraugerud et al., 2011; Sorensen et al., 2009).
The common method to measure sinking velocity is to drop a pellet into a measuring
cylinder filled with water and recording the time to reach the bottom (Glencross et al., 2011;
143
Kannadhason et al., 2009), where some pellets tend to float because of the surface tension or oils
around the pellets or air-bubbles, producing erroneous results (e.g., Glencross et al., 2011). To
avoid this, in this study, individual pellets were dipped into 1-cm below the surface for 2 seconds
using a forcep and then released. This has eliminated the risk of floatation, and should be
preferred to determine sinking velocity.
Hardness of the pellets was reduced significantly from 75 and 72 N (0% and 1.5% lignin,
respectively) to 51 N (3% lignin), suggesting that up to 1.5% of purified lignin can easily be
added without affecting the durability of the pellets. Durability is usually measured as hardness
(the force to disintegrate) and as water stability (Refstie and sgrd, 2009). Obaldo et al. (2002)
defined water stability as the retention of physical integrity of the pellets with minimal
disintegration and nutrient leaching. The authors tested static water method, horizontal and
vertical shaking methods to test water stability of shrimp feed, and concluded that both shaking
methods are suitable for routine laboratory analysis as they allow pellet and water movement like
normal farming conditions.
The leaching of nutrients is generally determined as dry matter loss assuming that protein
ingredients and oils are uniformly distributed in the pellets and therefore, estimating the dry
matter loss could represent the protein and lipid loss from the pellets (e.g., Aas et al., 2011;
Baeverfjord et al., 2006; Lim and Dominy, 1990; Obaldo et al., 2002). Findings from this study
suggested otherwise where the dry matter loss differed significantly from the lipid loss, and did
not represent the loss of individual nutrients. Not the levels of lignin but the duration in water
and pellet size significantly affected the nutrient loss where smaller pellets lost some of the
nutrients at faster rate than the larger pellets. While DM loss closely corresponded to the N-loss
144
(e.g., 46% and 42%, respectively, after 10-min in water for 2-mm pellets), loss of lipid was
significantly higher for all treatments (e.g., 58% after 10-min in water for 2-mm pellets).
6.4.3. Conclusion
This study provided general guidelines and methods for assessing quality of mash and
steam-pressed pellets of an experimental aquaculture feed. Significant changes in oil absorption
of dietary mash and in bulk density, sinking velocity, and hardness of steam-pressed pellets
occurred with increasing level of purified lignin. Dispersing, binding and emulsifying properties
of lignin are different than other binders such as cellulose or guar gum contributing to the
changes in the physical properties of mash and pellets. Pellet water stability is highly vulnerable
to the changes in ingredient composition of the diet, physical and chemical properties of the
ingredients, and processing and manufacturing conditions. While highly durable and water stable
pellets that are able to withstand shear handling and distribution stresses, and disturbances in the
water are desirable to the farmers, they may not be suitable for digestion and absorption of
nutrients (Hilton et al., 1981). Careful attention to modification of processing and manufacturing
conditions is therefore needed to maintain the delicate balance of producing highly stable pellet.
145
CHAPTER 7
GENERAL DISCUSSION
7.1. Evaluation of A Novel Plant Protein Ingredient: Why is It Important?
Modern compounded aquaculture feeds include protein sources ranging from fish meal to
variety of animal and plant products. In the past few decades, several animal by-products such as
poultry meal and blood meal have been tested and effectively utilized in salmonid diets (Bureau
et al., 1999; Gaylord and Barrow, 2008). However, world production of the animal by-products
(13 MMT) is limited and much less than the production of plant proteins (Table 2.1). The advent
of novel processing techniques and avaialability of high quality products with excellent amino
acid profiles comparable to fish meal have caused plant protein sources to become more
prominent ingredients in aquatic animal diets (Gaylord et al., 2010). These novel techniques have
also allowed manufacturers to remove some of the inherent anti-nutrients that limited usage of
plant protein sources. Many high-quality plant ingredients are a complex mixtures of proteins,
fibre and starch components that require thorough evaluation in order to determine their
nutritional value and appropriate use levels in prospective diets (Glencross 2007b). Gatlin et al.
(2007) recently reviewed the use of sustainable plant products in aquafeeds and highlighted the
need for better characterization of these ingredients. Glencross et al. (2007b) also reviewed
strategies generally employed in ingredient evaluation of aquaculture feeds and suggested
several general but key components of the ingredient evaluation process, starting with
characterization of macro- and micro-nutrients (Figure 7.1), digestibility and utilization of
nutrients, effects of anti-nutritional components on nutrient utilization and health condition of the
target animals, and finally, functional properties.
146
An important evalution criterion for any protein source has traditionally been to
determine the digestibility of crude protein. Fish feeds are increasingly formulated digestible
amino acid basis and the paucity of information on available amino acid content in ingredients
forced feed formulators to use broader safety margins when formulating feeds (Bureau, 2006).
Accurate information on the available amino acid contents in traditional and novel ingredients is,
therefore, crucial for cost-effective formulation of modern aquaculture feeds.
7.2. Digestibility of Amino Acids from Plant Protein Sources
In fish, digestibility of nutrients is usually assessed as total G-I tract digestibility, using
indirect methods by adding indigestible markers to the diet, and expressed as apparent
digestibility coefficients (ADC). When evaluating a protein ingredient, typically a portion of a
standard reference diet is partially replaced with the test ingredient, and then the ADC of
nutrients in the test ingredient is calculated using the indirect method from ADC values obtained
for the reference diet and the test diets (Bureau and Hua, 2006; Bureau et al, 1999; Cho et al.,
1982; Forster, 1999). Several in-vitro methods, such as enzymatic- or acid-digestion methods or
a combination of both have also been developed (Baasompierre et al., 1998; Carter et al., 1999;
Dimes and Haard, 1994). Various methods of assessing digestibility of nutrients in fish are
described in the Chapter 2.
Several factors may affect the digestibility values in replacement studies and they are: the
amount of test ingredient included in a test diet (20-40%), the types of indigestible markers,
faecal collection methods, and composition of fibre components and anti-nutritional factors.
Fibre components and anti-nutritional factors constitute a significant portion of the plant
proteins, composition of which vary considerably based on species, genetics, source, processing
147
techniques and conditions. These components in plant proteins may substantially interfere with
nutrient utilization, growth and intestinal morphology of salmonids (Bureau et al., 1998; Francis
et al., 2001; Kaushik et al., 1995; Krogdahl et al., 2010; Wang et al., 2009a). Fractionation of
different components to produce high quality proteins helps to isolate some fibre components but
may increase the concentration of some ANFs. Some of the ANF can be removed by further
mechanical, chemical or biological treatments.
The key assumptions when using indirect methods are that there are no interactions
among ingredients that differentially affect digestibility, and altering the level of an ingredient in
a diet does not change the digestibility values of that ingredient. It has been shown that ADC of
nutrients of some ingredients, particularly ingredients with high carbohydrate content, varies
considerably (Stone, 2003). A critical review of various assumptions in a typical digestibility
study, and their implications for derived ADC values can be found in Glencross et al. (2007b).
In this study, ADC of crude protein and amino acids of two novel plant protein
ingredients, Indian mustard protein concentrate and Indian mustard protein meal, were compared
to those of a commonly used soy protein concentrate in rainbow trout and Atlantic salmon using
the indirect method, where 30% of a high quality reference diet was replaced with the three test
ingredients (Chapter 3). High ADC values of most essential amino acids (>90%) of these novel
ingredients are comparable to those of other commonly used protein sources indicating the
possibility of their utilization as excellent protein sources in salmonid diets.
Among the essential amino acids, ADC of lysine, isoleucine and phenylalanine were
significantly lower in fish fed Indian mustard protein concentrate than in those fed the Indian
mustard protein meal and soyprotein concentrate. It is hypothesized that low ADC of crude
protein and some amino acids in IMC for rainbow trout could be related to its high PA content
148
(4.2%) compared to IMM (2.8%) and SPC (1.8%). PA intake by rainbow trout fed 30%-IMC
diet was significantly higher (37 mg fish-1 d-1) than those fed 30%-IMM (19 mg fish-1 d-1) and
30%-SPC (25 mg fish-1 d-1) diets. A relationship may exist between the ADCs of crude protein
observed in this study and the PA intake of the animals. High dietary PA intake appears to have
more significant impact on the ADC of the lowest pKa (dissociation constant) His than the
higher pKa AA, Lys and Arg (Figure 3.1). Histidine, lysine, and arginine are highly susceptible
to chelation in both stomach and intestine as they become polar and positively charged at pH
below their pKas (6.1, 10.5 and 12.5, respectively; Cosgrove, 1966). Broken-line regression
analysis of ADC of Phe and PA intake showed that ADC of the AA was not affected below the
PA intake of 17 mg-1 fish d-1 (Figure 3.2). The results suggested that the level of phytate intake
may also influence the AA utilization in rainbow trout besides the formation of indigestible PA-
AA binary or PA-mineral-AA ternary complexes.
7.3. Bio-availability of Amino Acid and Slope-ratio Assay
In Chapter 4, bioavailability of arginine for fish from two novel plant proteins, Indian
mustard protein concentrate and Indian mustard protein meal, and a commercially available soy
protein concentrate relative to those from a crystalline L-Arg source was explored. Both TGC
and protein deposition were significantly linear in relation to both Arg level and intake, and used
as response variables for the assay.
The findings indicated a significantly higher Arg availability from novel Indian mustard
protein concentrate and protein meals than that from the crystalline L-Arg. The responses tended
to plateau in regression analysis with dietary Arg level as predictor resulting in reduced
bioavailability of Arg with increasing dietary Arg level as determined by two (~1.23 and ~1.35%
149
Arg) or three (~1.23, ~1.35% and ~1.5% Arg)point assays. In contrast, responses were
significantly linear with Arg intake and did not affect the slopes in either assay. A similar
quadratic effect was also observed by El-Haroun and Bureau (2007) in three-point analyses to
determine bioavailability of Lys from various sources in rainbow trout, where slope-ratios were
determined with Lys level (%) as predictor variable.
Several important inferences can be made from this study for a statistically valid slope-
ratio assay. The foremost criterion for a valid slope-ratio assay should be an appropriate
experimental design relevant to the chosen statistical model. For example, a linear regression
model can be performed with two levels of the target AA from the test sources but care should be
taken to maintain the AA intake below 80% of the requirement to ensure a linear response. In
such scenario, feed intake need to be controlled and equalized for all treatments. On the other
hand, there should be more than two levels of target AA for a non-linear or broken line approach,
where at least two levels must be below the requirement of the animal including the diet highly
deficient in the target amino acid.
Variations in the basal diet composition among experiments may also result in significant
variations in RBV of an AA from the same test ingredient. Practical basal diets in bioavailability
studies in fish have been formulated with either corn gluten meal or wheat gluten meal as the
major protein source (Li et al., 2008, 2009; Nangthu et al., 2007; Poppi et al., 2011). Nangthu et
al. (2007) compared lysine utilization efficiency in rainbow trout fed CGM and WG based diets
and reported that the imbalanced amino acid profile in CGM induced by excess leucine and high
Arg-Lys ratio, may have affected the feed intake, suggesting WG to be better source of AA than
the CGM. The difference in the lysine utilization efficiencies observed by Nangthu et al. (2007)
could also be an effect of the variations in digesta viscosity and passage rate because of variable
150
composition of NSPs in these two ingredients. Besides the variable compositions of other
nutrients or anti-nutrients in the test sources, variations in ingredient compositions of the basal
diets may also hinder the meaningful comparison of the results among the slope-ratio studies
illustrating the need for a standardized basal diet for bioavailability studies along with the
standardized experimental designs and feeding protocols.
7.4. Effects of Phytic Acid and Lignin on Nutrient Utilization (except minerals)
Effects of dietary phytic acid (PA) and purified lignin on growth performance,
physiological indices, and nutrient deposition in rainbow trout are described in chapter 5. Overall
feed efficiency and growth rate of fish in all treatments including those fed the control diet were
low compare to those observed in similar trials. It is likely that a portion the amino acids
formulated to be marginally adequate, were not completely available resulting in lower than
usual growth rate and feed efficiency.
Several studies suggested that reduced availability of one or more dietary AA negatively
influences feed intake in animals including fish (Aja et al., 1999; Koehnle et al., 2004;
Yamamoto et al., 2001; 2004). Feed intake in fish as in other animals is regulated by
neuropeptides that vary by origin, mode and regions of actions depending on external
environment, life-stage and composition of the diets. Several neuropeptides such as ghrelin,
neuropeptide-Y family of peptides (NPY-Y), galanin, orexins (hypocretin-1 and hypocretin-2)
and agouti-related proteins have been identified in fish as appetite stimulators (Nieminen et al.,
2004; Silverstein et al., 1998; Silverstein and Plisetskaya, 2000; Volkoff and Peter, 2001a;
Volkoff et al., 2005).
151
Among the pair-feeding treatments, lipid deposition in fish fed PA diets was slightly
higher than those fed PA-free diets. Imbalanced profile of available AA originated from the
reduced digestibility of some AA as reported in the chapter 3 may have resulted in more AA
available for gluconeogenesis resulting in higher lipid deposition by increasing fatty acid
synthetase (FAS) activity. Wang et al (2009a) also reported higher lipase activity in rainbow
trout fed untreated soybean meal based diets compared to those fed the phytase-treated diets.
FA and TAG synthesis is a highly regulated cellular process crucial to the metabolic
homeostasis of organisms. Figueiredo-Silva et al. (2010) reported significantly higher FAS
activity in the liver of blackspot seabream fed plant protein based diets (30.0 mIU mg-1 protein)
than those fed fishmeal based diets (20.5 mIU mg-1 protein) resulting in high total lipid
deposition. Lipid retention exceeded the intake in both treatments but deposited at much higher
amount in the fish fed the plant protein based diets than those fed the fish meal based diets
(188% and 127% of the intake, respectively). In contrast, activity of the enzyme lipoprotein
lipase (LPL) in adipose tissue were lower (P>0.05) in fish fed the plant protein diets (1.3 mIU
mg-1 protein) than in those fed fish meal based diets. Similar effects were also observed in the
adipose tissue of pigs fed high protein diets, where significantly lower FAS activity was
observed when fed a high protein diet than those fed low protein diets (Zhao et al., 2010). Dias et
al. (2005) suggested that AA imbalance, particularly a deficiency of exclusively ketogenic AA
such as lysine, leads to increase catabolism of other AA, leaving more carbon chains and
glucogenic substrates available for lipogenesis.
None of the growth performance and physiological indices was affected by lignin in the
pair-feeding treatments but when fed to satiety, lignin appeared to suppress the feed intake
contributing to the differences in growth and Fultons condition index. No such effects on feed
152
intake have been demonstrated in studies with pigs and broiler chickens fed purified lignin
(Schedle et al., 2008; Baurhoo et al., 2007). Diets of the satiation feeding treatment are very high
in PA contents, and a possible synergistic interaction of lignin with PA may have further reduced
the feed intake.
7.5. Beyond Tradition
Protein ingredients for aquaculture feed are usually evaluated in digestibility trials by
assessing the ADC of crude protein. Diets are increasingly formulated on a digestible amino acid
basis that requires accurate information on digestible amino acid values of the protein
ingredients. A significant amount of efforts have recently been invested to determine digestible
amino acid values of protein ingredients of plant origin (Gatlin et al., 2007; Hardy 2010).
However, digestibility is a measure of disappearance of nutrients that doesnt reflect the
utilization of nutrients by the animal for deposition and metabolism. A more practical approach
is to determine the bioavailability of amino acids using growth trials, and protein deposition as
an indicator variable.
Bioavailability assays are attractive because the response criteria are similar to those
under practical conditions. A potential advantage of these assays is that metabolic costs to the
animal that are induced by feeding a particular ingredient and that are associated with digestion
and absorption are reflected in this measure of availability. On the other hand, these assays are
tedious and costly, and can only determine the availability of one amino acid at a time. These are
the main reasons that there are so few studies on the bioavailability of amino acids in various fish
species. In addition, most of these studies are not properly designed for a statistically valid slope-
ratio response. There is a serious need for standardizing the experimental design, research
153
protocols and ingredient composition of the basal diets for valid determination of the
bioavailability of amino acids that is comparable among laboratories and the research groups.
Plant protein ingredients also contain antinutritional compounds and fibrous components
that usually interfere with nutrient utilization in animals. In the last two decades, studies on these
components have mainly focussed on nutrient digestibility, feed intake, growth, nutrient
deposition and retention efficiencies providing some very useful information on the effects of
these anti-nutrients on various economically important fish species including salmonids. There
has been increasing number of studies on various hormones and their effects on the regulation of
enzymes affecting feed intake, digestibility, and nutrient utilization and partitioning dynamics in
various fish species in recent years. Plant proteins now represent the majority of the dietary
proteins in fish feed and therefore, there is a serious need to improve our understanding on how
anti-nutrients and various fibre components affect regulation of these hormones and enzymatic
activities in fish species.
A framework for evaluation of plant protein ingredients is provided in Figure 7.2. It can
be concluded that a comprehensive evaluation of a plant protein ingredient for their inclusion in
compound aquaculture feed should include series of studies encompassing digestibility and bio-
availability of amino acids, effects of anti-nutrients and fibre components, effects of these
ingredients on physical quality of the pellets (chapter 6), and probably, how inclusion of these
ingredients and their various antinutrients and fibre components affect the nutrient utilization
dynamics and welfare of the fishes.
154
Figure 7.1 A flow-diagram for characterization of the components of a plant protein ingredient.
155
Figure 7.2 A frame-work for evaluation of a plant protein ingredient.
156
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M A Kabir Chowdhury, January, 2012

ASSESSING QUALITY OF NOVEL PLANT PROTEINS FOR SALMONIDS

M A Kabir Chowdhury Advisor:

of plant protein ingredients for use in aquaculture feeds.

nothing but utmost respect for you. Thank you, Dom.

made a stressful day more rewarding.

helping each other out at difficult times.

Table of Contents.................................................................................................................... vii

List of Tables........................................................................................................................... xiii

List of Figures......................................................................................................................... xvii

Chapter 1 General Introduction.......................................................................................... 1

Chapter 2 Review of Literature........................................................................................... 7

2.2. Review of Aquaculture Feed Production and Aquaculture Nutrition........................ 9

2.2.1. Evolution of salmonid feed formulation............................................................. 9

2.2.2. Evolution of fish meal usage.......................................................... 15

2.2.3. Desired characteristics of a potential feed ingredient......................................... 15

2.3. Overview of World Feed Ingredients......................................................................... 16

2.3.1. Production of oilseed meals............................................ 18

2.3.2. Production of protein concentrates and isolates............................................. 18

2.3.3. Further improvement of the protein quality............................................ 19

2.3.4. Production approaches................................................ 20

2.3.2. Rapeseed / canola / mustard (Brassicacae)........................................................ 22

2.3.2.1. Indian mustard (Brassica juncea)............................................................... 22

2.3.2.2. Nutritive value of mustard products: issues and challenges....................... 23

2.4. Nutritive Value of Protein Ingredients to Fish........................................................... 24

2.4.1. Overview of methodological approaches........................................................... 25

2.4.2.1.. Digestive process related to protein and amino acids... 26

2.4.2.2. Assessing digestibility in fish: techniques, issues and limitations......... 30

2.4.2.2.1. Fecal collection methods.................................................................. 30

2.4.2.2.2. Indigestible markers.......................................................................... 31

2.4.2.2.3. In-vitro assessment of digestibility................................................... 32

2.4.2.2.4. Limitations of digestibility of proteins in feed ingredients.............. 33

2.4.3. Bioavailability of amino acids............................................................................ 33

2.4.3.1. Methodological approaches........................................................................ 35

2.4.3.2. Limitations of slope-ratio approaches......................................................... 36

2.4.4.1. General overview........................................................................................ 37

2.4.4.2. Phytic acid................................................................................................... 41

2.4.4.2.1. Interactions with minerals................................................................... 41

2.4.4.2.2. Interactions with protein and amino acids.......................................... 45

2.4.4.2.3. Interactions with proteolytic enzymes................................................. 46

2.4.4.2.4. Effect of PA on nutrient utilization................................................. 46

2.4.4.3. Fibre components and their effects............................................................. 47

2.4.4.4.1. Lignin biosynthesis............................................................................. 48

2.4.4.4.2. Effects of purified lignin..................................................................... 50

2.4.4.4.3. Effects of lignin on minerals and amino acid availability................... 50

2.4.5. Physical characteristics of aquaculture feed...................................................... 51

2.4.5.2. Physical properties of pellets and their importance.................................... 54

2.4.5.3. Different factors affecting physical properties........................................... 56

2.4.5.4. Components of plant proteins influencing pellet characteristics................ 56

Chapter 3 Digestibility of Amino Acids in Two Novel Ingredients, Indian Mustard

Protein Concentrate and Indian Mustard Meal Compared to That of A

Soy Protein Concentrate in Rainbow Trout and Atlantic Salmon............... 59

3.2.1. Dietary design..................................................................................................... 63

3.2.2. Experimental conditions..................................................................................... 67

3.2.3. Chemical analyses.............................................................................................. 68

3.2.5. Statistical analysis.............................................................................................. 69

3.3.1. Apparent digestibility of energy and nutrients................................................... 70