VOL 9 ISSUE 2 June/July 2017

A D VA N C E S I N P R O C E S S I N G & F O R M U L AT I O N

An Aquafeed.com publication

Interview with Dr. Rick Barrows

Survey of nutrient levels in commercial shrimp feeds in India
Mycotoxins in aquafeeds and why processing does not prevent it
Taurine leaching in fish feeds
Nucleotides in Fish Nutrition
The importance of pre-extrusion process design
Mixed feed nut meal for aquafeeds
Algae as aquaculture feed ingredients
Computer-assisted image analysis to monitor whiteleg shrimp
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AQUAFEED Volume 9, Issue 2
A D V A N C E S I N P R O C E S S I N G & F O R M U L AT I O N

Contents

5
• Interview with Dr. Frederic T. (Rick) Barrows 5

• Survey of nutrient levels in commercial shrimp feeds in India 9

• How mycotoxins get into aquafeeds — and why processing does
not prevent it 15

• The importance of pre-extrusion process design 19

9
• * Taurine leaching in fish feeds 23

• The use of algae as aquaculture feed ingredients 29

• Mixed feed nut meal — natural and sustainable plant based
protein for aquaculture 33
• Nucleotides in fish nutrition — the best strategy to enhance
immunity and intestinal health 38
• Use of computer assisted image-analysis to monitor health and
nutritional status in whiteleg shrimp (Penaeus vannamei) 43
15
• Calendar of Events 47

*Cover story

33
To read previous issues in digital format or to order print copies, visit:
http://www.aquafeed.com/publications/aquafeed-magazine/
AQUAFEED
4
Volume 9, Issue 2

A D V A N C E S I N P R O C E S S I N G & F O R M U L AT I O N

Index to advertisers

Aquaculture Europe 2018 32

Aquaculture Without Frontiers (AwF) 4

Bioiberica 22
Famsun (Muyang) 8
Format Solutions 47

Hatcheryfeed 45
ILDEX Indonesia 201742 42
International Aquaculture Forum 28
Lallemand 30
Nutriad 2
Ottevanger 17

Skretting 37

Wenger Manufacturing 14

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 5

Dr. Frederic T. (Rick) Barrows is one of a very few

number of scientists with expertise in aquaculture
nutrition, feed production and fish physiology. He has
devoted his long career with the U.S. Department of
Agriculture, working at the Bozeman Fish Technology
Center, Montana, to the evaluation of novel
ingredients as alternatives to fishmeal and oil.
He retired a year ago and formed Aquatic Feed
Technologies, LLC., with the aim of assisting in the
development of a safe, sustainable, seafood supply,
working with companies with feed ingredient
development and evaluation, regulatory approval and
alternative feed development. This includes feeds
ranging from larval, weaning or starter feeds through
grow out feeds and brood feeds Rick has over 130
peer-reviewed publications through out his 28 years
of innovative feed research and has received
numerous awards for his work.

Interview
with Dr. Frederic T. (Rick) Barrows
AQUAFEED.COM You have gained a
reputation over many years for your
work on new protein and oil sources as
potential alternatives to fishmeal/oil.
Spirulina does not seem like a “new”
ingredient in aquaculture. Would you talk
about your work with this microalgae
and why you believe it to hold such
potential?
FTB Yes, you are correct spirulina has
been evaluated as a fish feed ingredient
for decades. There does seem to be an The one “new” aspect of spirulina might
interaction of spirulina and other be that the price seems to be coming
ingredients for select marine species. down to a more reasonable level as more
When spirulina was added to a fishmeal is produced overseas.
based diet for white sea bass or
There is another source of spirulina
yellowtail there was no beneficial effect.
product that could be beneficial for
When spirulina was supplemented to a
aquafeeds. Spirulina contains
fishmeal free diet, however, there was a
phycocyanin and this is now being
large increase in both growth and
extracted from the meal to produce a
survival. This suggests that spirulina is
blue pigment for foods and it is approved
supplying a nutrient that is present in
in the U.S. and Europe. The residual
fishmeal but not in the other ingredients.

biomass should be more affordable and
may contain the essential nutrients that
the unextracted spirulina contains, but
this needs to be demonstrated.
AQUAFEED.COM What are some of the
other promising ingredients you have
investigated and how do they hold up in
terms of their nutritional value?
FTB There is a lot of interest and
investment going into single cell proteins
(i.e. bacterial proteins) and insects (i.e.
black soldier fly and meal worms) and
production scale plants are being built
around the world. The products
themselves are very nutritious; the cost
per ton and the scalability are primary
questions now, but we will soon see.
I think we will be seeing the develop-
ment of regional or local ingredient
sources. One example is what AAFCO
calls mixed nut meal. This is nuts that
cannot be certified due to size, color or
broken pieces and are used for animal
feed. This material is processed to
produce a meal of approximately 62%
protein and 10% lipid. Most times the
meal is comprised of pistachios and
almonds and this meal increases feed
intake when included at 10% or greater
in the diet.
Most barley farmers want to produce
malting grade barley, but barley that is
too high in protein gets discounted and is
sold as feed grade. The feed grade
barley can then be processed to produce
ethanol and a barley protein meal. This
process is designed to protect the
protein quality of the meal and it has
been demonstrated to have high protein,
dry matter and energy digestibility. Both
barley protein concentrate and mixed
nut meal are in the pilot scale phase, but
6
the ingredients have been fed commer-
cially for several years now.
AQUAFEED.COM Are changes in animal
health and/or behavior or quality of the
finished product major issues with use of
alternative ingredients you’ve looked at?
FTB These are also important considera-
tions when evaluating new ingredients.
If a new ingredient has a negative effect
on these criteria it would be dropped
immediately until further modified.
There are certain prebiotic effects of
some ingredients, and there are several
fermented or microbially enhanced
products that show great potential
especially for the shrimp industry. One
of these fermented products was tested
on a farm scale with 8 different farms
and a significant increase in survival was
observed from approximately 40% in the
control groups to approximately 80% in
the test groups. The shrimp fed the
fermented product were also more
uniform in size.
Many consumers and fish producers are
concerned with the effect of alternative
ingredients on the flavor of the final
product. It is fairly clear that changing
protein sources does not change the
flavor of the fillet, but changing lipid
sources from fish oil to plant oils does
change the flavor. Depending on the
consumer group this can be a positive or
a negative effect but it seems a blend of
oil sources is often most effective from a
product quality and cost effectiveness
perspective.
AQUAFEED.COM Have you looked into
the physical properties and processing
parameters of any of these ingredients?
FTB Yes, as you know this is of vital
importance to feed manufactures to
know that the ingredient will not have
an adverse effect on pellet quality. This
is difficult to determine for every type of
diet due to interactions among ingredi-
ents, but a good time to evaluate the
effect of the ingredient on processing
and pellet quality is when producing
diets for a digestibility study. In these
studies there is a reference diet which is
fed to three tanks of fish and typically 9
test diets which are each fed to three
tanks of fish. The test diets contain 70%
of the reference diet and 30% of the test
ingredient. The 30% inclusion rate is
greater than I would recommend in a
practical type diet so if there is no effect
on processing or pellet quality at this
level I would expect none at lower levels.
This is also a good time to evaluate the
effect of an ingredient on the palatability
of the diet by measuring feed intake.
A good example of the effect of
ingredient on pellet quality was with
diets containing 30% Schizochytrium
algae. This is the algae that contains high
levels of lipids and high levels of DHA, the
fatty acid known for its heart and brain
healthy attributes. We processed these
product obtained from 4 different
sources and found a large difference
among the products. Since this algae can
contain over 50% lipid some of the
products felt very greasy and some were
“dry” to the touch. One of the greasy
products, however, processed well and
produces a good quality pellet. Three of
the products produced good quality
floating pellets, but the fourth product
we could not get the pellets to float no
matter how much energy we put into the
process. This inclusion level is way over
the suggested level but demonstrates

that even among related ingredients you
can have an effect on pellet quality. We
still don’t know if the difference was due
to strain of algae or how the algae was
processed.
AQUAFEED.COM Will changes in major
feed components require a change in
process technology – new equipment or
refinements to existing machines - or
just the establishment of new parame-
ters for existing processes? How will the
feed industry need to adapt?
FTB I think that the pace for change in
processing is being set by the equipment
manufacturers. As equipment refine-
ments are developed it will allow for new
type of ingredients to be used. For
example, the addition of “wet” ingredi-
ents to an extruder system could
decrease production costs for the
ingredient since drying an ingredient can
be costly only to rehydrate during feed
production. Of course this raises
questions on product stability and could
require production of the ingredient in
close proximity to the feed mill. This is
an exciting combination of innovative
ingredients, manufacturing process, and
nutrition. Progress in this area will be
interesting to watch in the future.
AQUAFEED.COM What are the
research and/or commercial application
gaps that need to be met in order to
expand industry use of promising
alternative ingredients? Is this important,
and if so why?
FTB The available nutrient content is of
course a primary concern for new
ingredients, but it is often the price or
the potential scalability of an ingredient
that is most often limiting. Increasing the
7
scale of production from the laboratory
scale (hundred of kilograms) to pilot “It is fairly clear that
scale (hundreds of tons) can be challeng-
ing, but these challenges are often on the
changing protein sources
technical side. The development of new does not change the flavor
Ingredients often get stuck moving from
pilot scale to production scale. This is of the fillet, but changing
because of the cost of building a
production scale plant is usually large lipid sources from fish oil
and the uncertainty of market
acceptance. A practical consideration
to plant oils does change
often limits the flexibility of feed mills to the flavor.
adopt new ingredients even if the
nutritional and other characteristics have Depending on the consumer
been clearly evaluated. This is the
number of ingredient bins available group this can be a positive
whether it is 6 or 12 or 20 there are
always ingredients already in them. So a or a negative effect
feed mill would have to replace a known but it seems a blend of oil
ingredient with the new ingredient. I
have often recommended that ingredient sources is often most
providers work the cost of new ingredi-
ent bins into the business plan to at least effective from a product
get started.
quality and cost
effectiveness perspective.”
AQUAFEED.COM Finally, what is the
one thing you'd like to see happen in
aquaculture before you really retire? speed innovation and commercialization
and to better communicate success’s
FTB Well, there are actually a two related
around the globe. There are high quality
things I would like to see happen for
research and development projects going
aquaculture feeds and for human health.
on everywhere, but sometimes it seems
We do have many sources of protein
that as researchers we are reinventing
ingredients, but there are no products
the wheel again. International travel is
that contain the essential fatty acids, and
expensive, but with modern technology
probably other nutrients, that is
and some organization the rate of
contained in fish oil. Fish oil is a very
development and adoption of new
nutritious product but there just isn’t
ingredients could be increased. Also
enough available to meet the needs of
many new ingredient providers often do
the growing aquaculture industry and the
not know how to determine the
growing human population even at
nutritional and economic value of their
current harvest rates from the oceans.
products and a system that allowed them
I would like to see an affordable lipid
to do this would assist the industry
product that contains an equal level of
greatly.
essential nutrients to fish oil developed
and commercialized. This would allow AFΩ
aquaculture to continue to grow and
provide nutritious products and improve
See page 33: “Mixed feed nut meal – natural
the health of the humans that consume
and sustainable plant based protein for
those products.
aquaculture” for more on this promising feed
To reach that goal and beyond I think ingredient.
there should be a system put in place to
Page 21
 9

Survey of nutrient levels in

commercial shrimp feeds in India
By Alexander van Halteren and Peter Coutteau, PhD., Nutriad International NV, Dendermonde, Belgium

Since the introduction of

white leg shrimp in 2009,
shrimp culture has boomed
in India to over 485,000 Mt
in 2016. During the past
years, Indian shrimp culture
has spread from one main
culture belt, Andhra
Pradesh, into two more
areas, Orissa and Gujarat.
Culture conditions vary in
salinity, water source
(borehole/seawater),
temperature and length of
the winter period in the
different culture areas.
The number of shrimp feed producers
has increased dramatically in recent
years. Initially, fish and chicken feed
producers were the first to see shrimp
feed as a potential for diversification.
They were followed by large farmers
setting up their own feed-mill operations.
Currently large seafood processors are
installing new feed-mills. It is expected
that this trend will further fragment the
feed market, challenging the existing key
feed producers.
Globally there was a decline of fishmeal
production in 2016 and also Indian
domestic fishmeal production was
affected, causing shortage of fish meal
and fish oil during the summer crop.
Contrary to earlier days when the bulk of
the marine ingredients such as fishmeal,
squid meal, and krill meal were import-
ed, today shrimp feeds are formulated
for more than 95 % with locally produced
raw materials. Thanks to the implemen-
tation of modern production technology,
most domestic producers of fishmeal/oil
in India meet international quality
standards nowadays. Although this
allows feedmills to avoid the complex
importation and stocking of marine
ingredients, the prices of these local raw
materials have increased to international
levels. The selection of raw materials will
influence the overall nutrient profile of
the feed. Reducing marine ingredients
will directly affect the level and availabil-
ity of the essential lipids such as n-3
HUFA and cholesterol, unless the
formulator compensates this by adding
specialty ingredients providing these
nutrients.
 10

Compared to other (aquatic) species, Sample collection and analysis

formulating shrimp feed is more based
on experience then on exact science. The
Since 2013, Nutriad has surveyed the … the results of this survey are
composition of commercial shrimp feeds representative for the feed
optimal nutrient profile of a shrimp feed
in India. Feeds have been analyzed on a
will depend on many factors, including
range of nutritional parameters. For the specifications during a specific
the culture density, environmental
conditions (temperature, salinity,
present study, we restricted the samples window of time during the
to one pellet size, i.e. 3P, which
oxygen, …), productivity of the pond
constitutes the main consumed volume
culture cycle of 2016.
water, the stability of the feed, feeding
of commercial shrimp feed in India. Pellet
method and frequency, … These factors
3P typically has the following specifica-
often are different depending on the hydrolysis following the Soxhlet method
tions : crude protein (35 – 36 %), crude
season, region, farm or even pond which (AOAC 996.06). Fatty acid composition
fat (4.5 – 6 %), crude ash (< 13 %) and
makes the selection of the ideal feed was determined with the gas chromato-
crude fiber (2 – 5 %).
rather a complex and often unstable graphic method following fat extraction
decision for the farmer. As a result, The selected feeds were collected from (AOAC 996.06; expressed as g/kg).
research in optimizing feed formulations the market during the second quarter of Cholesterol was determined by direct
under practical conditions continues to 2016 from farmers and feed distributors. saponification using the gas chromato-
be a major objective for feed producers. All feed samples were produced during graphic method (AOAC 994.10; ex-
Also, we can expect a wide variety in Q2 of 2016 and stored under typical lab pressed as g/kg). Phospholipids were
nutritional specifications among conditions, before sending for analysis. analysed with 31P-NMR spectroscopy
commercial shrimp feeds as composition Three different samples of each type of using the internal standard method (SAA-
may depend on the target market. feed was pooled into one representative MET002-03, expressed as % as is). The
sample. Therefore, the results of this analyses of amino acids and nitrogen
Increasing cost and fluctuating availabil-
survey are representative for the feed were performed by Evonik Degussa
ity of raw materials in combination with
specifications during a specific window of GmbH (official European method of
an increasingly competitive market is
time during the culture cycle of 2016. amino acid analysis in feed, COMMIS-
demanding a creative mind from the
SION DIRECTIVE 98/64/EC of 3 Septem-
shrimp feed formulator. The nutritional Crude protein was analyzed following the
ber 1998; official method code 994.12 of
strategy is key to maintain or gain market Kjeldahl method (Commission Directive
the AOAC International 2000, and
share. Aside from that, diseases like 93/28/EEC.OJ No L179.22.7.93). Crude
expressed % as is).
white spot, vibriosis and white gut/feces fat has been determined with acid
are an emerging risk during the produc-
tion cycle of shrimp in India and require a
good nutritional support to the animal.
The present study investigated the
different nutritional strategies in
commercial shrimp feeds during 2016,
when the number of shrimp feed
suppliers increased sharply. Feed
samples of 8 major brands were
collected in the market and analysed for
proximate composition as well as a
number of essential nutrients (amino
acids, phospholipids, cholesterol, n-3
highly unsaturated fatty acids).
 11

Crude protein and fat

Crude protein levels varied between 33.9
% and 40.7 %, with an average of 36.4 %
± 2.3 % of the analyzed samples (Fig. 1). 5
feeds out of 8 were found to have a
higher protein content than the
minimum specification on the label. On
average, the feeds were 4% above the
labelled feed specifications of 35 % crude
protein, but in one case the analysed
crude protein level was 20% higher than
the level specified in the label. The crude
fat specifications vary between different
feed producers between 4.5 – 6 % with
the majority having the fat specification
above 5 %. All the analyzed feeds have
Figure 1. Crude protein versus crude fat of 8 commercial shrimp feeds.
crude fat contents higher than 5 %, with
an average of 6.18 % ± 0.54 %. This is
approximately 25 % above the specifica-
tion. There was no clear relation
between crude protein and crude fat
content. This confirms the lack of
consensus among shrimp nutritionists on
the importance of the protein/fat ratio in
the diet. The analysis of crude fat and
protein does not reveal the origin or
quality of the fats and proteins used.

Amino acid profile

The amino acid profile gives a first idea
on the quality of the proteins used as
well as the nutrient density. This is far
from a complete picture of protein
quality which would include an assess-
ment of the ingredients regarding
processing parameters and protein
Figure 2. Amino acid profile for 8 commercial shrimp feeds (represented by different line
digestibility. Amino acid nutrition in
colors).
shrimp is complex due to the interaction
from the leaching of amino acids prior to requirements in white shrimp is still very body could give guidelines on the desired
ingestion, lack of digestibility values, and limited (NRC 2011). In the absence of profile of essential amino acids in the
the role of specific amino acids in feed scientific studies determining absolute diet (ideal protein concept). In this study
attractiveness. The current knowledge on requirements for essential amino acids the average (± standard deviation for 8
the requirements of amino acid (EAA), the composition of the whole samples) lysine content was 2.11% ±
 12

0.17% and methionine 0.63% ± 0.09%

(Fig. 2). Arginine an amino acid that could
play a role it the attractivity of the feed
has an average of 2.41% ± 0.12%.

Phospholipids, n-3 HUFA, choles-

terol
The n-3 HUFA content is an indicator for
the quantity of marine fats used in the
feed, either derived from fish oil or from
marine protein meals containing fat.
Marine shrimp do not have the ability to
biosynthesize n-3 HUFA and the dietary
requirements of Litopenaeus vannamei
Figure 3. Dietary level of n-3 HUFA versus crude fat for 8 commercial shrimp feeds.
were found to be at least 1 % (Kanazawa
et al. , 1979; Kontara et al., 1995; Shiau,
1998). Five out of the 8 feeds had n-3
HUFA values around 5 g/kg, whereas
crude fat levels varied between 5.8 and
7.1 g/kg (avg 6.18 % ± 0.54; Fig. 3). One
feed exhibited more than 7g/kg n-3
HUFA and two feeds were below 3 g/kg n
-3 HUFA.

Cholesterol requirement studies show a

wide range in cholesterol requirements
from 0.5 to 5 g/kg for L. vannamei (Chen,
1993, Duerr and Walsh, 1996; Gong et
al., 2000). Duerr and Walsh (1996)
showed that dietary cholesterol levels
below 1 g/kg limit growth in L. vannamei.
Morris et al. (2011) reported the
cholesterol requirement for L. vannamei
grow-out to be somewhere between
0.76 and 1.1 g/kg, however, a regression
analysis predicted the cholesterol
requirement for maximum growth to be
1.5 g/kg. Gong et al. (2000) estimated
that the cholesterol requirement for L.
vannamei was 3.5 g/kg in the absence of
supplemented phospholipids. At 1.5%
and 3% phospholipids, dietary cholester-
ol requirements reduced to 1.4 and 1.3
g/kg, respectively. The cholesterol levels
Figure 4. Dietary level of cholesterol versus crude fat for 8 commercial shrimp feeds.
found in the analysed feeds were all well
below 1 g/kg (Fig. 4). The average
cholesterol level was 0.64 g/kg ± 0.16,
with half of the feeds around 0.7-0.8 g/kg
and one feed sample as low as 0.34 g/kg.
Dietary phospholipid is required for
optimal growth of penaeid shrimp
including L. vannamei (Glencross et al.
1998; Paibulkichakul et al. 1998;
Thongrod and Boonyaratpalin 1998).
Addition of 1.5% of phosphatidyl choline
(PC) from either a 95% pure soybean
source, 94% pure chicken egg source, or
deoiled soybean lecithin (23% PC)
increased growth of L. vannamei relative
to a PC-deficient diet (Coutteau et al.
1996). Recommended levels of dietary
phospholipids from soybean sources
range from 1.25% to 6.5%, depending on
shrimp species, developmental stage, as
well as purity of the lecithin (Coutteau et
 13

al., 1997). The average levels of

phospholipids in the current study were
1.74 % ± 0.43, with the lowest level being
1.13% and the highest 2.5% (Fig. 5).
Comparing different studies on phospho-
lipid requirements can be troubled by
the differences in analytical methods
used. The present study used NMR
spectroscopy to quantify total phospho-
lipid content in the shrimp feeds. This
method is more accurate than traditional
methods based on HPLC or iatroscan as
the NMR quantification is independent
from fatty acid composition and
phospholipid profile of the phospholip- Figure 5. Dietary level of phospholipids versus crude fat for 8 commercial shrimp feeds.
ids.
36.4% in 2016. Average crude fat levels Reducing the level of essential lipids like
The level of the essential fat nutrients,
are similar in both surveys, ie around cholesterol, phospholipids and n‐3 HUFA
cholesterol, n-3 HUFA and phospholipids,
6.2%. n-3 HUFA and cholesterol levels significantly affected growth, feed
were not correlated at all with the total
have dropped with 16% and 24%, conversion and protein efficiency in
level of dietary fat (Fig. 3, 4, 5 : non-
respectively, , whereas the average white shrimp in a controlled feeding trial
significant correlations). This indicates
inclusion of phospholipids increased with in clear water (van Halteren et al., 2016).
that the dietary fat originates from
38 % between 2014 and 2016. The The above trends in the feed industry
blending fats from marine as well as
changes in the lipid profiles reflect promote the application of digestibility
vegetable origin.
possibly the effects of the trend to enhancing additives which improve the
replace fish meal and fish oil, particularly absorption efficiency of the increasingly
in 2016 when the domestic supply of limited levels of cholesterol and n-3
Trends revealed from comparing
these raw materials was insufficient. HUFA. Digestive enhancers like bile salts
surveys 2014 and 2016 Average levels indicate that overall and phospholipids are natural emulsifiers
Comparing the present survey for 2016 reduced levels of cholesterol and n-3 capable of enhancing the digestive
with a similar survey in 2014 (based on 4 HUFA, likely due to increased replace- capacity for lipids in the digestive system
commercial feeds), we see some ment of marine by vegetable ingredients, of shrimp by improving the lipid
significant trends. Overall the average were compensated with increased levels emulsification and micelle formation,
crude protein increased from 35.8% to of phospholipids and crude protein. resulting in a faster absorption of lipids in
the hepatopancreas. Furthermore, bile
salts constitute an alternative source for
Table 1. Variation in fat composition in commercial shrimp feeds between 2014 versus 2016
the steroid ring which shrimp cannot
(data represent average and stdev).
synthesize, which is at the basis of their
2014 2016 requirement for dietary cholesterol.
Average ± s.d. (n=4) Average ± s.d. (n=8) Adding bile salts to the diet lower in
Crude fat (%) 6.28 ± 0.86 6.18 ± 0.54 essential lipids restored the performance
Crude protein (%) 35.82 ± 0.20 36.44 ± 2.25 of the shrimp to the same level as the
n-3 HUFA (g/kg) 5.23 ± 2.48 4.42 ± 1.74 control diet with elevated levels of
Cholesterol (g/kg) 0.84 ± 0.16 0.64 ± 0.16 essential lipids (van Halteren et al.,
Phospholipids (%) 1.26 ± 0.29 1.74 ± 0.43 2016). By improving the utilization

efficiency of dietary lipids, shrimp
formulations can be made more cost-
effective by reducing the formulated
values for phospholipids, cholesterol and
n-3 highly unsaturated fatty acids
(HUFAs) without affecting the perfor-
mance (Coutteau et al., 2011).
Conclusions
The present study collected samples
from 8 commercial shrimp feed brands
(pellet size P3) in India during 2016 for
the analysis of selected nutrients. The
shrimp feed samples exhibited a wide
range of levels for the nutrients
analysed. The shrimp feeds contained on
average (± s.d. for the eight samples)
36.4 ± 2.3% crude protein, 6.28 ± 0.86 %
14
crude fat, 2.11 ± 0.17% lysine, 0.63 ± and phospholipid levels were increased
0.09% methionine, 2.41 ± 0.12% over the same period, likely due to the
arginine, 4.42 g/kg ± 1.74 g/kg n-3 HUFA, increasing replacement of marine
0.64 ± 0.16 g/kg cholesterol, 1.74 ± ingredients by vegetable raw materials.
0.43% phospholipids. Furthermore, the
survey showed an overall trend in the
AFΩ
industry between 2014 and 2016 to offer
feeds with lower levels of cholesterol
and n-3 HUFA, whereas crude protein
More information
Alexander van Halteren
Business development manager Aquaculture
Nutrition, Nutriad International, Belgium.
E: a.vanhalteren@nutriad.com
Peter Coutteau, PhD
Business Unit Director Aquaculture,
Nutriad International, Belgium.
E: p.coutteau@nutriad.com
 15

How mycotoxins get into aquafeeds

and why processing does not prevent it
Rui Gonçalves, Aquaculture Scientist, Biomin

The topic of mycotoxin has started to draw increasing attention from the aqua industry in
recent years. There is, however, nothing new about fungal contamination. Fungi have
always held biological, ecological and economic importance when it comes to plants,
aquafeeds and aquaculture. The role fungi play ranges from positive to neutral to negative,
depending on the species and circumstance.

The Good, The Bad and The Friend

The importance of fungi to the eco-
system is immeasurable. Fungi are
present in all terrestrial habitats from
Antarctica to the hot deserts of Namibia,
and in most aquatic environments. Being
opportunistic heterotrophs, they have
specialized in penetrating solid sub-
strates (rock, bark, dead branches, bare
soil or grains), nutritionally exploiting
almost any food substrate. Fungi are
essential to recycle nutrients. Most of
the known taxa are specialized in
decomposing complex plant and animal
debris.

The Good
Several species of fungi, namely from
genus Penicillium, play an important role
in industry. For example penicillin,
produced by P. chrysogenum (formerly P.
notatum), discovered by Alexander
Fleming in 1929, was probably the most
important discovery of the last century
Fusarium verticillioides (major producer of FUM) colonies morphology on petri dish.
and changed the course of medicine.

Aspergillus-flavus (major producer of AF) colonies morphology on petri dish.
Fungi also play a central role in the food
industry (cheese and various meat
products) and are becoming increasingly
important in the biotechnology field,
especially in the production of enzymes
(e.g. gluconic, citric, and tartaric acids,
several pectinases, lipase, amylases,
cellulases, and proteases).
The friend
Fungi are vital to agriculture and forestry
through their global involvement in
mycorrhizae (fungus roots). They also
established mutualistic symbioses with a
wide range of organisms like cyano-
bacteria (blue-green ‘algae’) and
chlorophycota (green algae) in lichens.
The bad
Minor fungi taxa can have a parasitic
behavior, and in certain cases can be
16
pathogenic. The ability to penetrate
almost any surface can be used to invade
host organisms. Fungi attack almost all
known taxa of plants and animals,
including shrimp (e.g. Fusarium sp in
penaeids) and fish (e.g. Saprolegniasis).
Focusing on fungi as plant pathogens,
they attack all parts and all stages of crop
plants (from root hairs to apical buds,
grains or fruits). The fungal infections
may be restricted to small leaf spots, or
may be systemic—killing their host very
quickly or remain invisible until it is time
to appropriate crucial energy resources,
such as those concentrated by the host in
anthers, bulbs or seeds.
Toxic secondary metabolites
Taking into account the available nutrient
content, plant stuffs are desirable targets
for fungus. Fungi successfully colonize all
plant parts, at all stages of crop/storage
and utilize substrates efficiently by
growing over their surfaces and
penetrating into their matrices. Fungal
metabolic processes originate an
apparently endless diversity of organic
compounds which are not obviously
required for normal growth and
metabolism: these are called secondary
metabolites. Not all secondary metabo-
lites are mycotoxins. Simplistically, we
could split them in 3 broad groups, being
1) toxic to bacteria (antibiotics), 2) toxic
to plants (phytotoxins) and 3) toxic to
animals (mycotoxins).
Mycotoxin contamination in aquatic
species is often associated with poor
growth and low feed efficiency. The lack
of obvious pathological signs make it
difficult to identify the source of the
problem.
Mycotoxin producing fungi
Aflatoxins (AF), ochratoxin A (OTA),
deoxynivalenol (DON), zearalenone
(ZEN), fumonisins (FUM) and ergot
alkaloids are within the most common
mycotoxins found in agriculture
commodities and responsible for millions
of dollars annually in losses worldwide.
These toxins are produced by just a few
species from the common genera
Aspergillus, Penicillium, Fusarium, and
Claviceps. All Aspergillus and Penicillium
species either are commensals, growing
in crops without obvious signs of
pathogenicity, or invade crops after
harvest and produce toxins during drying
and storage. The most important
Aspergillus species, occurring in warmer
climates, are A. flavus and A. parasiticus,
which produce aflatoxins in maize,
groundnuts, tree nuts, and, less

frequently, other commodities.
Penicillium verrucosum also produces
ochratoxin A, but occurs only in cool
temperate climates, where it infects
small grains.
In contrast, the important Fusarium and
Claviceps species infect crops before
harvest. F. verticillioides is ubiquitous in
maize, with an endophytic nature, and
produces fumonisins, which are generally
more prevalent when crops are under
drought stress or suffer excessive insect
damage. It has recently been shown that
Aspergillus niger also produces
fumonisins, and several commodities
may be affected. F. graminearum, which
is the major producer of deoxynivalenol
and zearalenone, is pathogenic on maize,
wheat, and barley, and produces these
toxins whenever it infects these grains
before harvest.
17
How mycotoxins reach aquafeeds
Despite efforts to control fungal
contamination both on the field and in
storage, extensive mycotoxin contamina-
tion has been reported in both plants
and finished feeds. The type and
prevalence of mycotoxin contamination
will depend on the type of substrate
(plant meal type; finished feed character-
istics) as well as geographical area,
seasonal and local weather conditions
during critical plant growing stages or
storage.
Factors contributing to the presence or
production of mycotoxins include:
environmental (temperature, humidity)
and ecological conditions (insect attacks,
physical plant damage and general
stress)—though these are often times
beyond human control.
Incredibly durable, even in
processing
The mycotoxins commonly occurring in
plant stuffs are not destroyed during
most processing operations. On the
contrary, processing affects mycotoxins
distribution and concentrates mycotoxins
into fractions that are commonly used as
animal feed (plant by-products; e.g. corn
gluten meal, DDGS, etc). The fate of
mycotoxins during cereal processing,
such as sorting, cleaning, milling and
thermal processes has been studied by
several authors. However, their level in
feedstuffs is variable and affected by
several factors such as the type of
mycotoxins, the level and extent of
fungal contamination, and the complexi-
ty of the cereal processing technology.

The mycotoxins commonly
occurring in plant stuffs
are not destroyed during
most processing operations.
On the contrary, processing
affects mycotoxins
distribution and concentrates
mycotoxins into fractions
that are commonly used as
animal feed (plant
by-products; e.g. corn gluten
meal, DDGS, etc).
18
Penicillium-verrucosum (major producer of OTA) colonies morphology on petri dish.
Mitigation methods
Not all molds produce mycotoxins and
even the ones that have that capacity
may be present without producing any
toxin. Thus, the confirmation of mold
contamination is not the same thing as
the demonstration of mycotoxin
contamination. As a result, the use of
mold inhibitors does not guarantee that
feed is free of mycotoxins, as they are
also produced in crops and not destroyed
during processing. It is recommended More information
that aquafeed and aquaculture produc-
ers regularly monitor raw commodity
feed ingredients and finished feeds for
mycotoxin contamination—either
through on-site rapid testing or through
an external laboratory that may be
equipped with more powerful detection
equipment. In cases where feed quality
has been compromised by mycotoxins,
the use of a mycotoxin deactivator is
Rui Gonçalves
advised. Aquaculture Scientist, Biomin
E: rui.goncalves@biomin.net
AFΩ
 19

The importance of pre-extrusion

process design
By Hennie Pieterse

Extrusion is the heart-beat

of an aquatic feed
processing plant. Still it
largely depends on a well
designed pre-extrusion and
post-extrusion process to
ensure the desired
nutritional and physical
outcome of the extruded
final product is achieved.
Knowledge and
understanding of the raw
materials, both dry and wet
(or fresh) to be used in
formulations are of utmost
importance in designing and
specifying a process that will
Some critical pre-extrusion Dry ingredient intake, cleaning and
optimize extrusion storage.
processing steps
performance. Pre-extrusion The range of ingredients we use to
It is difficult to accentuate only a few of
process design plays a key manufacture modern-day aquatic feed
the processing steps that make up a
are becoming increasingly more
role in producing extruded complete pre-extrusion process. The next
expensive. We should therefore take
topics cover some of the more critical
aquatic feeds that are feed ones:
care as to how we acquire, accept,
handle and store these ingredients on
safe and environmentally
site. The following design criteria should
friendly. be taken into consideration:

1. Intake sampling and quality control
2. Flexibility in intake & storage design
allows for utilizing a wide range of raw
ingredients
3. Good aspiration and dust removal
from the intake pits.
4. Cleaning of the raw ingredients and
removing foreign plants and other
materials including metals, sand and
stones
5. Safe storage of raw ingredients in bulk
storage silos, flat storage and dosing silos
Dry ingredient batching
Accurately combining ingredients
together to create a proper formulation
to achieve species health, species
performance and production costs. All
ingredients whether dry, liquid or fresh
go through an accurate batching process
ensuring the final extruded product
Figure 1. This flow shows the steps we have to take before extrusion. The numbers show: 1. First Stage grinding, 2. Fine grinding , 3. Vitamin &
mineral additions, 4. Fresh additions.
20
meets the species nutritional and
lifecycle requirements. Process control
and communicating formulation details
between least cost formulation software
and the field devices controlling the
process play a key role in a successful
feed mill design.
Size reduction
1. Size reduction is the first processing
step where the physical and potentially
nutritional characteristics of ingredients
are changed. Physical ingredient size is
reduced to an average particle size. Heat
damage to the nutritional value of amino
acids and some micro ingredients may
take place when the size reduction
system is not well designed or specified.
The traditional size reduction rule during
extrusion is to have no particle larger
than one third of the die openings in the
extruder die plate. This is easily
achievable using a single grinding step
when producing larger extruded pellet
sizes. It becomes a challenge when
producing smaller pellets for shrimp and
micro feed for hatcheries and ornamen-
tal fish.
Traditional hammer mill designs were in
most cases inadequate for fine grinding
in a single step. It was and still is
common to use a course grinding
hammer mill followed by a fine grinding
hammer mill to achieve the required fine
grinding specifications. For ultra-fine
grinding, to achieve an average particle
size below 300 micron, a course grinding
hammer mill is used in conjunction with a
pulverizer. This is a common setup for
producing shrimp feed and micro feed
for hatcheries or ornamental fish.
A range of high efficiency hammer mills
are now on the market, capable of
producing narrow particle size

distribution curves with average particle
size down to the 300-350 micron mark.
The design of these hammer mills allow
for shorter grinding chamber retention
times and therefore lower grinding
temperatures. Coupled with a variable
frequency drive and special features in
terms of breaker plate design and sieve
types, these hammer mills can mill a
large range of cereals and other aquatic
feed ingredients at lower cost compared
to traditional systems. Figure 2 shows
typical application range for size
reduction technology in relation to
average ingredient size and feed type
requirements.
In addition an attempt in design should
be made to mill only the ingredients in
the formulation that requires size
Figure 2. Size reduction technology should be specified with final product requirements and ingredient characteristics in mind.
21
reduction. Funneling unnecessary
ingredients through the size reduction
system increases operational costs
through wear and could also increase
capital cost through a higher capacity
requirement.
More critical pre-extrusion
processes
Other pre-extrusion processing steps of
critical importance are:
1. Mixing: a range of single shaft ribbon
mixers, single and twin shaft paddle
mixers are on the market. The ultimate
goal is to deliver a homogeneous mix to
the extruder surge bin, representing the
specific formulation to be extruded. A
well-designed mixing environment makes
provision for additional liquids and dry
ingredients as well.
2. Vitamins & minerals addition: it is
common for large feed mills to have
dedicated micro ingredient dosing
sections. Such capability provides more
control and could limit costs when
compared to pre-packed vitamins and
minerals.
3. Fresh ingredient intake & preparation:
the demand to include fresh meat, fish
and other wet by-products into the
formulations of aquatic species is
increasing. Great care should be taken to
ensure ingredient streams do not cause
cross-contamination hazards. Keeping
dry, fresh and final product separated.
Fresh products could be pre-processed
 22

into slurries, hydrolysates, concentrates cross contamination and segregation of

and other forms prior to inclusion into mixed formulations
the feedstock stream. 5. Feed plant zoning in line with
functional and feed-safety requirements
Ultimately the pre-extrusion process
Critical process design aspects
should supply meal to the extruder that More information
A range of operations make up the pre- will allow the extrusion operation to be Hennie Pieterse is
extrusion process. The discussion in this consistent, constant and easily controlla- a graduated
article covered some aspects of the more ble. And all of this in a cost efficient and agricultural
important operations. Ultimately the reliable way. engineer and has 25
process has to be engineered into a three years experience
dimensional space that will meet serving the
operational requirements and other feed AFΩ extrusion industry.
milling aspects that are frequently He represents
Ottevanger Milling
overlooked:
Engineers in Australia and New Zealand
1. Sound insulation through hp dezign Pty Ltd. He is also
2. Dust and odor control full-time on-board Ottevanger Milling
Engineers as part of the Aquatic Feed
3. A safe environment for workers Technology team .
4. Layout design and transitional E: h.pieterse@ottevanger.com
equipment sections that will minimize

Taurine leaching in fish feeds
By Guillaume Salze1, T. Gibson Gaylord2, D. Allen Davis1
1
School of Fisheries Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849
2
US Fish and Wildlife Service, Bozeman Fish Technology Center, Bozeman, MT 59715
Leaching of water soluble
compounds is an issue for fish
and shrimp feeds often discussed
among nutritionists. Indeed, such
compounds include critical
nutrients and supplements such
as vitamins and free amino acids.
Yet, this issue has received
limited attention in terms of
dedicated, quantitative studies.
Taurine has been the subject of
an intensive research effort, and
is now widely recognized as an
essential nutrient in many fish
species, such as European
seabass, California yellowtail, or
Japanese flounder. Traditionally
supplied through animal meals
such as fishmeal, taurine must be
supplemented in formulations
containing high levels of taurine-
poor ingredients such as plant-
based protein sources. As of
January 2017 taurine is listed as
an approved feed additive for fish
feeds in the United States; thus it
is only appropriate to look at this
important nutrient and its
retention in fish feeds.
23
Descriptions of symptoms of taurine
deficiencies in fish have mostly been
limited to depressed growth, reductions
in immune response and occurrence of
green liver syndrome in juveniles of some
species, and development retardation in
larvae. In mammals taurine is attributed
many functions spanning from bile-salt
formation, osmoregulation, and
inhibition of oxidative stress (Huxtable,
1992). In yellowtail taurine deficiency
plays a role in osmoregulation (Takagi et
al., 2006), and taurine-deficient red
seabream exhibit a decrease in taurine
conjugated bile-salts (Matsunari et al.,
2008). In Florida pompano, taurine
deficiency caused significant changes in
several biomarkers of liver function,
including a decrease in alkaline phospha-
Photo: Guillaume Salze, Ph.D
tase activity, accumulation of glycogen
and lipid with marked vacuolation, and
reduced mitochondrial function in the
liver. Taken together, these findings
indicate severe abnormalities in liver
metabolism in fish fed taurine-deficient
diets, and provide an explanation for the
reduced growth, feed efficiency and liver
issues commonly observed with taurine
deficiency (Salze et al., 2016).
Taurine is a polar molecule, and since it is
a beta-amino acid and cannot form a
peptide bond to form a protein, it is
almost always found in free form.
Taurine is therefore prone to leaching,
and it is important to quantify leaching
rates from practical feeds. Indeed, there
are a number of factors that will affect
 24

the rates of nutrient loss due to leaching Table 1: Experimental setup

such as time of exposure, pellet size, and
extrusion technology. The time of water
Pellet size Extrusion temperature Time points
exposure can vary dramatically with slow
-eating species, as well as when feeding 2.0 mm Cold 1, 5, 20, 40 min
response is reduced with medicated 2.0 mm Hot 1, 5, 20, 40 min
feeds or during winter months. Both
4.0 mm Cold 1, 5, 20, 40 min
time in water and pelleting technology
will affect the physical integrity of the 4.0 mm Hot 1, 5, 20, 40 min
pellet at the time of ingestion. Pellet size
will change with fish species and across Table 2: Formulation of the diet mash
fish life stages. Quantitative knowledge
of factors driving leaching rates is critical Ingredient Inclusion Diet composition
to adapt formulations accordingly and (% as-is) (% dry matter)
Menhaden fishmeal 12.00 Crude protein 45.07 ± 0.36
ensure that taurine intake in susceptible
Corn protein concentrate 10.00 Crude lipid 14.61 ± 0.78
species are sufficient to meet the dietary
Soybean meal 10.00 Taurine 1.17 ± 0.03
requirement and avoid the consequences
of a deficiency as described above. Poultry by-product meal 10.00
Therefore, the goal of this study was to Wheat gluten meal 4.00
quantify the effects of major drivers of Blood meal, spray dried 2.50
taurine leaching and evaluate possible Wheat flour, durum 30.03
interactions between duration of pellet Menhaden fish oil 10.01
immersion, size of the pellet, along with Other (premixes, supplements, etc.) 10.36
the effects of diet manufacturing Taurine 1.00
process. Yttrium oxide 0.10

Experimental design
To shed some light on factors influencing
leaching, a series of four diets varying in
size and extrusion temperature (cold- or
hot-extruded) were produced and then
immersed in water for 1, 5, 20, and
40min. Table 1 summarizes the general
experimental setup.
Diets were manufactured using commer-
cial methods starting with ingredient
weighing of the control grind mixture.
Ingredients minus fish oil were mixed in a
paddle mixer in a 100-kg batch followed
by grinding to a particle size of <200 µm
using an air-swept pulverizer.
The 2mm and 4mm pellets were
manufactured with a twin-screw
extruder at the U.S. Fish and Wildlife
Service, Fish Technology Center,
Bozeman, Montana. The processing
parameters were set to either cooking
extrude or cold process the pellets.
Cooking extrusion is defined when
temperatures above 110oC are used to
gelatinized the starch, and cold is defined
as when the starch is not gelatinized and
ingredients are primarily pressed into a
form.
Hot-extruded diets were cooked using
conventional settings on the extruder.
Diet mash was exposed to an average of
110oC for approximately 14 seconds in
five barrel sections and the last section
was maintained at 62oC. Pressure at the
die head was approximately 50 bar, and
screw speed was maintained at 423rpm.
The 4.0mm floating pellets were
manufactured through a 3mm die while
the 2 mm floating pellets were manufac-
tured through a 1.5 mm die. The diets
were dried in a pulse bed drier until
moisture readings were below 6%. Drier
conditions were maintained with
incoming air temperature at approxi-
mately 107°C with an upper limit outflow
air temperature of approximately 88°C.
The diets were then cooled at ambient
air temperatures for final moisture levels
of <10%. The remaining fish oil was
applied using a vacuum infusion coater
after the pellets were cooled.
The cold-extruded diets had all the oil
mixed in the mash prior to pelleting. No
additional heat was added and barrel
tempering units were set at 15oC, which

Photo: Ryan Hagerty/USFWS
resulted in an average barrel tempera-
ture mid-way through the production of
only 23.2oC. Solids feed rate was half of
the cooking extruded pellets which
resulted in a longer retention time in the
barrels (28 seconds), but only 13 bar of
pressure at the end plate. The 2mm and
Figure 1: Typical leaching response observed with increasing time spent in water (A) and on logarithmic scale (B). The logarithmic trajectory indi-
cates fast leaching rates immediately after immersion, and slower rates thereafter.
25
4mm pellets were manufactured with 2
and 4 mm dies, respectively. The
resulting extrudate did not expand and
resulted in sinking pellets. Drier
conditions and diet storage were as
described for the cooking extruded
pellets. These temperatures and
pressures are typically observed in what
is described as a cold extrusion process.
The leaching trials were conducted
following methods described by Watson
et al. (2015) with modifications. At 1, 5,
20, and 40min after immersion, two
flasks were removed from the water bath
and the pellets were dewatered as
quickly as possible, dried, and frozen. All
leached pellets were analyzed for amino
acids and yttrium oxide, in duplicates.
Taurine leaching was expressed as the
percentage lost relative to the initial diet
content. Effects of duration of immer-
sion, pellet size, and extrusion tempera-
ture were evaluated in a linear model
along with interactions. The duration of
immersion was linearized by log-
transformation prior to statistical
analysis. The model was run using
Program R (version 3.3.2, www.r-
project.org) and predicative equations
developed to determine estimates of
taurine leaching.
Results and interpretation
As expected, immersion duration, pellet
size, and extrusion temperature all
significantly affected leaching of taurine:
higher losses were seen with longer
immersion time, smaller pellets, and
lower extrusion temperature. Regardless
of the conditions, taurine leached out of
the pellet in a logarithmic fashion:
quickly during the first few minutes after
immersion, after which the leaching rate
slowed down progressively (Figure 1A).
Therefore this relationship can be plotted
on log-scaled x-axis to observe a straight
line, facilitating interpretation (Figure
1B). Taurine losses after 40min immer-
sion ranged from 44.5% to 93.6% across
diets and conditions.

There were significant interactions
between the factors we tested, meaning
that data must be interpreted according
Figure 2: Interaction between immersion duration and pellet size. The 2mm pellets leached 35%
faster than the 4mm pellets.
Figure 3: Interaction between pellet size and extrusion temperature. The cold-extruded 2mm pellets leached 54% faster than the 4mm pellets,
whereas this difference was reduced to 20% in hot-extruded pellets.
26
to these four pairs of factors. Figure 2
illustrates the relationship between
pellet size and immersion duration.
Regardless of extrusion temperature, the
2mm pellets leached faster than the
4mm pellets. While differences in taurine
loss are barely noticeable after 1min in
water, the slope of the regression lines
separates the two groups by 5min of
immersion. The ratio of the slopes
indicates that the 2mm pellets leached
about 35% faster than the 4mm pellets.
This is directly attributable to the
increased area of contact between the
pellet and surrounding water in a smaller
pellet: the larger the surface area, the
more and faster the leaching.
The cold-pelleted diets had a higher
leaching rate than the hot-extruded
pellets (31% overall), which is most likely
explained by a lower physical integrity of
the cold-extruded pellets vs. hot-
extruded pellets. Indeed, the hot-
extrusion process increased carbohy-
drate gelatinization, which traps soluble
compounds in the matrix more effective-
ly than a cold-extrusion where carbohy-
drates gelatinization is limited. However,
there is an interaction between pellet

Photo: Ryan Hagerty/USFWS
size and extrusion technology (Figure 3),
where that difference was only noticea-
ble in a 2mm pellet, whereas leaching
rates were similar in a 4mm cold- or hot-
extruded pellet. As a result, a 2mm cold-
extruded pellet leached 54% faster than
its 4mm counterpart, whereas hot-
extrusion reduced this difference to 20%
of the 4mm pellet.
Conclusion
Beyond confirming the importance of
pellet size, extrusion technology and
immersion duration, these results
quantify these effects. More importantly,
they reveal and quantified interaction
effects between these factors. This
information can be used toward
adjusting the inclusion of crystalline
taurine in dietary formulations.
For example, Salze et al. (2014)
estimated the taurine requirement of
Florida pompano at 0.54% of the diet,
using cold-pressed pellets. Assuming that
the pellets were immersed for about 15
seconds before being ingested by the
fish (a conservative estimate allowed by
an aquarium-based research system),
27
leaching can be estimated at approxi-
mately 5%, i.e. the fish would have eaten
a pellet containing 0.51% taurine. If
formulation were to be transferred from
a 2mm cold-extruded to a 4mm hot-
extruded for commercial purposes
where immersion time are expected to
be up to 5min (i.e. leaching rates of
about 25%), then such pellets should be
formulated with 0.64% taurine to
account for leaching and achieve the
same ingested dose. Accounting for
leaching rates as illustrated allows
nutritionists to reduce the risks of either
under-formulating (leading to deficien-
cies and reduced performances) or over-
formulating (reduced economic
performances as well as potential water
quality issues from sub-optimal nutrient
utilization).
More information
Guillaume Salze, Ph.D., Research Associate III,
School of Fisheries,
Aquaculture, and Aquatic Sciences,
Auburn University, Auburn, AL 36849, USA.
E: gzs0010@auburn.edu
References
Huxtable, R.J., 1992. Physiological
actions of taurine. Physiological Reviews
72 (1), 101-163.
Matsunari, H., Yamamoto, T., Kim, S.-K.,
Goto, T., Takeuchi, T., 2008. Optimum
dietary taurine level in casein-based diet
for juvenile red sea bream Pagrus major.
Fisheries Science 74 (2), 347-353.
Salze, G.P., Davis, A.D., Rhodes, M.A.,
2014. Quantitative requirement of
dietary taurine in Florida pompano
Trachinotus calorinus. Aquaculture
America 2014, Seattle, WA, Feb 11th.pp.
Salze, G.P., Spangler, E., Cobine, P.A.,
Rhodes, M., Davis, D.A., 2016. Investiga-
tion of biomarkers of early taurine
deficiency in Florida pompano Trachino-
tus carolinus. Aquaculture 451, 254-265.
Takagi, S., Murata, H., Goto, T., Hayashi,
M., Hatate, H., Endo, M., Yamashita, H.,
Ukawa, M., 2006. Hemolytic suppression
roles of taurine in yellowtail Seriola
quinqueradiata fed non-fishmeal diet
based on soybean protein. Fisheries
Science 72 (3), 546-555.
Watson, A.M., Barrows, F.T., Place, A.R.,
2015. Leaching of taurine from commer-
cial type aquaculture feeds. Aquaculture
Research 46 (6), 1510-1517.
AFΩ
 29

The use of
ALGAE
as aquaculture
feed ingredients

High-quality microalgae (Reed Mariculture Shellfish Diet 1800®)

By Eric Henry, Ph.D., Research Scientist, Reed Mariculture Inc.

Interest in using algae as fish feed
ingredients
In recent years there has been great
interest in the potential of algae as
ingredients in aquaculture feeds. Even a
cursory Internet search of the topic will
find numerous web sites describing
studies of the nutritional value of algae,
or touting the potential of algae as a feed
ingredient, or even announcing the
launch of new aquaculture feeds made
with algae as ingredients, although
technical information about the products
may be conspicuously absent. All this
attention is largely driven by the need to
find replacements for fish meal and fish
oils, as awareness grows of the unsus-
tainability of the practice of feeding wild-
capture fish to support a rapidly growing
farmed fish industry. Demand for
aquaculture feeds that provide the high
nutritional value found in fish meal and
oils is also spurred by the expansion of
aquaculture of high-value fish such as
Sea Bass, Sea Bream, Red Drum, Seriola,
Grouper, etc. These carnivores require
high-quality protein and omega-3 fatty
acids in their diets, nutrients that are
difficult or impossible to supply from
conventional plant-based feeds.
Why Algae?
Algae, including both macroalgae
(“seaweeds”) and microalgae (e.g.
phytoplankton), are the base of the
aquatic food chains that generate the
nutritional resources that fish are
adapted to consume. Certain algae are
already recognized as premium aquacul-
ture feeds, for both direct feeding and to
produce zooplankton (e.g. rotifers,
copepods, Artemia) for fish and shrimp
larviculture, and for bivalve larviculture.
So it is not surprising that many algae are
nutritionally superior to the land plants
used in formulated aquaculture feeds.
Which Algae?
It is often not understood that the term
“algae” commonly refers to what is really
an arbitrary grouping of organisms that
encompass a bewildering variety of
forms, and an even more bewildering
biochemical diversity. Algae may vary in
the properties of their cell walls, which
can impede digestion or extraction of
nutritional components, presence of
toxins or anti-nutritional factors, as well
as desirable nutritional components. It is
therefore impossible to make meaningful
generalizations about the nutritional

value of this extremely diverse group of
organisms, so it is always necessary to
consider the particular qualities of
specific algae.
Nutritional value of Algae
Protein
Fish meal is so widely used in feeds
largely thanks to its substantial content
of high-quality proteins, containing all
the essential amino acids. A critical
shortcoming of the crop plant proteins
commonly used in fish feeds is that they
are deficient in certain amino acids such
as lysine, methionine, threonine, and
tryptophan (Li et al. 2009). By contrast,
analyses of the amino acid content of
numerous macro- and micro-algae have
found that they generally contain all the
essential amino acids (Brown et al. 1997,
30
Dawczynski et al. 2007, Ortiz et al. 2006,
Rosell & Srivastava 1985, Wong & Peter
2000).
Lipids
Certain fish oil lipids, called
“PUFAs” (polyunsaturated omega-3 and
omega-6 fatty acids), have become highly
prized for their contribution to good
cardiovascular health in humans. But it is
not always appreciated that these “fish
oil” fatty acids are in fact originated by
algae at the base of the aquatic food
chain. These desirable algal fatty acids
are passed up the food chain to fish, and
they are indeed essential nutrients for
many fish.
Algae have been recognized as an
obvious alternative source of these “fish
oil” fatty acids for use in fish feeds (Miller
et al. 2008), especially eicosapentaenoic
acid (EPA), docosahexaenoic acid (DHA),
and arachidonic acid (ARA). There is a
substantial literature devoted to analysis
of the PUFA content of microalgae,
particularly those used in aquaculture,
because they have long been recognized
as the best source of these nutrients that
are essential for production of the
nutritious zooplankton necessary for the
first feeding of larval fish (Holt 2011), as
well as filter-feeding shellfish.
Tests of Algae in formulated fish
feeds
Various species of macroalgae and
microalgae have been incorporated into
fish feed formulations to assess their
nutritional value, and many have been
shown to be beneficial for fish such as
Tilapia (Tartiel et al. 2008), Korean
Rockfish (Bai et al. 2001), Sea Bream
(Yone et al. 1986, Mustafa & Nakagawa

1995), European Sea Bass (Valente et al.
2006), Striped Mullet (Wassef et al.
2001), Gilthead Sea Bream (Wassef et al.
2005), Atlantic Cod (Walker et al. 2009,
2010), and Salmon (Norambuena et a.
2015). But unfortunately, it is often
impossible to determine the particular
nutritional factors responsible for these
beneficial effects, either because no
attempt was made to do so, or due to
the poor design of the studies.
In recent years there has been great
interest in the potential of algae as a
biofuel feedstock, and it has often been
proposed that the protein portion
remaining after lipid extraction could be
a useful input for animal feeds (e.g. Chen
et al. 2010). However, the algae chosen
for biofuel production may not be
optimal for use as a feed input, and the
economic pressure for the lowest-cost
methods of fuel production can result in
protein residues with contamination that
makes them unfit for use as feed (e.g.
Hussein et al. 2012).
Choosing the right Algae
Just as it would be senseless to arbitrarily
substitute one conventional crop plant
for another (e.g. potatoes for soybeans)
when formulating a feed, the particular
attributes of each alga must be carefully
considered. In addition to the protein/
amino acid profile, lipid/PUFA/sterol
profile, and pigment content, there are
important additional considerations. The
type and quantity of extracellular
polysaccharides, which are very
abundant in certain algae, can interfere
with nutrient absorption, or conversely
be useful binding agents in forming feed
pellets. The thick cell walls of microalgae
such as Chlorella can prevent absorption
31
of the nutritional value of the cell
contents. Inhibitory compounds such as
the phenolics produced by some kelps,
and brominated compounds produced by
red algae, can render an alga with an
excellent nutritional analysis unsuitable
for use in a feed. Depending on growth
and processing conditions, algae can
contain high concentrations of trace
elements that may be detrimental.
Just as it would be senseless
Economic constraints
to arbitrarily substitute one
Despite the high nutritional value
provided by some algae, their adoption
conventional crop plant for
as ingredients for aquaculture feeds another (e.g. potatoes for
remains constrained by the high cost of
soybeans) when formulating a
production and processing. Recent
economic analyses (Beal et al. 2015, feed, the particular attributes
Maisashvili et al. 2015, Voort et al. 2015) of each alga must be carefully
provide useful insights toward under-
standing how technological progress and
considered.
market forces will determine whether
algae will soon become significant inputs
into the aquaculture feed market. Such
factors as the current moderation in
fishmeal prices (Byrne 2017) will
continue to have a strong influence on
how rapidly such changes in feed
formulation are adopted by the
aquaculture industry.
AFΩ
More information
Eric Henry, Ph.D., Research Scientist, Reed Mariculture Inc.
E: techsupport@reedmariculture.com
References available by request.
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Mixed feed nut meal – natural and
sustainable plant based protein for
aquaculture
By John Bowman, General Manager, Adaptive Bio-Resources
Aquaculture feed formulations have
undergone significant change in
recent years as researchers sought
plant based alternatives to supple-
ment fish meal and fish oil. The staff
at Adaptive Bio-Resources, LLC has
worked with esteemed aquaculture
nutrition researchers for over 10
years to identify viable alternative
protein sources, and recently
collaborated with Dr. Frederic
Barrows and his team at the USDA -
ARS Fish Technology Center in
Bozeman, Montana, in researching
several potential sources of plant
based proteins. The key criteria used
to determine the commercial
potential of a newly developed feed
ingredient include:
• Nutrient profile
• Fish performance (growth and
health)
• Availability
• Economics (1)
33
Most of the plant based feeds had
significant challenges to overcome,
including vastly different nutrient and
anti-nutrient profiles from fish meal, high
cost, limited availability, poor perfor-
mance, and/or lack of sustainability.
Dietary inclusion levels were often
limited due to the presence of anti-
nutrients or an imbalanced nutrient
profile that resulted in reduced feed
performance (growth and health). The
collaborative research project culminat-
ed with the identification, testing,
development of an exciting new source
of protein, Mixed Feed Nut Meal, an all
natural material that has a nutrient
profile similar to fishmeal with no
discernible anti-nutrients. The study
found Mixed Feed Nut Meal produced
comparable feed performance (growth
and health), at virtually the same cost as
fishmeal. The following summarizes the
technical results of the research and
testing against the key criteria.
The Nutrient Profile of nut meal typically
includes 50-55% protein, 8-11% lipids,
and little fiber or ash. The specific
nutrient profiles for materials tested in
the study are shown in Table 1. Amino
acid profiles showed similarities in the
ingredients, making nut meal a strong
candidate for further testing. Several
different forms of almond and pistachio
nut material were tested in a series of
trials to discern the feed performance
versus alternative diets. Other nut meals
such as walnut were evaluated but did
not perform as well and were discarded
from consideration.
The Performance of the diets was tested
over the life stages of rainbow trout from
fry to juvenile. The first trial was a six
week fry screening study, with nut meals,
spirulina, Algae, and mussel meal tested
against the baseline fishmeal diet. The
positive performance results from this
initial trial suggested that Nut Meal
(pistachio and/or almond) is palatable,
digestible and does not contain
detrimental anti-nutrients (Table 2). The
trout fed a diet containing the nut meals
had survival and growth that was similar
to trout fed a diet with 55% fish meal.
Both pistachio and almond meals had
strong apparent digestibility coefficients
for crude protein, lipid, and amino acids.
 34
Table 1. Nutrient compositions of nut meals, soy protein concentrate and whey for macro-nutrients and minerals.
g/100g g/100g
Dry Dry
weight Weight
Test Ingredient DM Lipid Protein Energy P K S Zn
Almond Meal, first cut, Adaptive Bio-Resources 92.4 8.5 53.4 4986 1.09 0.00 0.18 6.23
Almond Meal, second cut, Adaptive Bio-Resources 93.0 10.8 51.2 5053 1.09 0.01 0.18 6.28
Blanched Almonds, Adaptive Bio-Resources 93.6 9.1 61.5 5032 1.77 1.42 0.29 5.71
Almond meal, Adaptive Bio-Resources 93.7 8.6 54.4 4986 1.53 1.43 0.28 7.69
Pistachio meal, Adaptive Bio-Resources 94.2 10.7 58.8 5267 1.90 1.67 0.49 8.89
Whey Protein Isolate 94.1 1.8 79.7 5645 0.67 0.61 1.09 0.75
Soy Protein Concentrate, Profine VF, Solae 94.6 1.5 69.9 4803 1.43 NA 0.55 2.98
Fish meal, Menhaden, Special Select™, Omega
94.3 8.2 69.0 4705 5.08 1.31 0.81 10.68
Prot.
Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an
invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014)

Table 2. The effect of diet on growth and survival of first feeding rainbow trout
3 weeks 6 weeks
Ingredient g/fish Gain, % Survival g/fish Gain, % Survival
Fish meal control 2.7 394 100 7.6 1285 99.3
Almond 1st cut 2.6 365 99 7.7 1297 96.7
Almond 2nd cut 2.4 331 99 7.1 1195 98.0
Almond blanched 2.3 324 100 6.8 1145 96.0
Almond meal 2.5 360 100 7.8 1331 97.7
Pistachio meal 2.6 372 100 7.4 1244 99.3
Spirulina 1.9 244 99.7 5.8 948 97.7
Algae strain 1 1.9 239 99 4.9 795 94.3
Algae strain 2 1.8 220 100 4.1 658 96.3
Mussel meal 2.9 427 99 4.1 636 39.0
Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an
invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014)

The second trial compared digestibility,
and it confirmed that Nut Meal had high
levels of digestible protein, energy and
amino acids consistent with, and in some
cases exceeding digestibility of the
reference diet (Table 3, Table 4). The
third trial was a juvenile growth study in
which trout with an average initial weight
of 20 g/f were fed one of ten experi-
mental diets for 12 weeks. This trial
confirmed the results of the previous fry
and digestibility trials (Table 5), with
comparable to superior weight gain and
survival rates. Trout gained between
600% and 900% of their initial weight.
There were two series of diets, one
where protein from each of the nut
meals replaced fishmeal at a 50% or
100% rate, and the second where the
protein from soy protein concentrate
(SPC) was replaced with the nut meals
 35
Table 3. Apparent digestibility co-efficient of nut meals, soy protein concentrate and whey for amino acids.

Sum
Test Ingredient Arg His Ile Leu Lys Met Phe Thr Tyr Val
AA
Almond meal, Adaptive Bio-Resources 97 88 97 95 94 85 96 92 98 95 95
Pistachio meal, Adaptive Bio-Resources 96 68 97 94 90 85 96 89 85 96 93
Whey Protein Isolate 99 95 99 99 94 100 99 94 100 99 98
Soy Protein Concentrate, Profine VF, Solae 99 94 98 98 97 97 99 93 100 96 96
Fish meal, Menhaden, Special Select™, Omega Prot. 94 91 95 94 95 93 93 91 96 93 90
Reference diet #3 97 93 97 97 96 97 97 93 99 96 95
Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an
invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014)

Table 4. Apparent Digestibility Co-efficient of nut meals, soy protein concentrate and whey for macro-nutrients and minerals.

Dry Crude
Test Ingredient Fat Energy P K S Zn
Matter Protein
Almond Meal, first cut, Adaptive Bio-Resources 59 100 90 70 4 NA 55 31
Almond Meal, second cut, Adaptive Bio-Resources 61 98 87 69 27 NA 36 79
Blanched Almond meal, Adaptive Bio-Resources 59 90 92 76 NA 88 50 16
Almond meal, Adaptive Bio-Resources 57 92 92 72 NA 90 44 24
Pistachio meal, Adaptive Bio-Resources 64 96 86 75 27 91 47 50
Whey Protein Isolate 98 95 96 98 91 97 70 100
Soy Protein Concentrate, Profine VF, Solae 72 75 94 81 NA NA 58 35
Fish meal, Menhaden, Special Select™, Omega Prot. 71 92 83 88 40 94 70 74
Reference diet #3 80 87 91 89 62 61 66 70
Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an
invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014)

(Table 6). Both pistachio and almond
meals could serve as a complete
alternative to fish meal and Soy Protein
Concentrate with similar or improved
performance. These trials confirmed
that almond and pistachio meals have
good levels of available nutrients,
support a high level of fish performance,
are highly palatable, digestible, and
support high levels of growth whether
replacing fish meal or soy protein
concentrate.
The trials confirmed the first two criteria,
nutrient profile and performance, but the
last two criteria must also pass the test
of commercial viability, adequate
availability and cost.
Availability: The U.S. tree nut industry is
based in California with almond and
pistachio production estimated at 2.9
billion pounds (1.45 million tons) in 2016
(USDA, California Pistachio Growers).
Most of this high value crop is consumed
directly by humans, with only a small
portion of broken, chipped, or off color
material available for feeds and oil
production. ABR estimates that with
conservative 30% diet inclusion rates,
about 2.5% of raw nut material has the
potential to provide for the diet needs of
current U.S. trout and west coast salmon
aquaculture. Check criteria #3.
Economics/ Cost: ABR has developed
proprietary production methods to safely
 36

Table 5. Effect of replacing fish meal or soy protein concentrate with almond or pistachio meals at two levels on growth of juvenile rainbow trout.

Gain/fish, Survival, Feed Intake,

Diet n Gain, % of initial FCR HSI
grams % % bw/day
Fish Meal Control 3 131.2 b 753 ab 93.3 0.92 0.78 1.33 ab
Fish, Almond, mid 3 104.5 d 614 d 100.0 0.95 0.79 1.10 cd
Fish, Almond, high 3 124.5 bc 711 bc 98.3 0.88 0.74 1.04 cd
Fish, Pistachio, mid 3 131.0 b 752 ab 98.3 0.91 0.76 1.16 cd
F-Pistachio, high 3 138.7 ab 790 ab 90.0 0.92 0.77 1.00 d
Soy Protein Control 3 111.8 cd 648 cd 96.7 0.99 0.83 1.39 a
SPC, Almond, mid 3 126.8 bc 728 bc 90.0 0.97 0.82 1.33 ab
SPC, Almond, high 3 111.0 cd 650 cd 95.0 0.97 0.81 1.16 cd
SPC, Pistachio, mid 3 123.1 bc 718 ab 90.0 0.97 0.82 1.09 cd
SPC, Pistachio, high 3 149.3 a 831 a 88.3 0.95 0.8 1.17 bc
a
Menhaden Special Select, Omega Proteins Corp, 610 g/kg crude protein b Adaptive Bio-Resources, 490 g/kg protein c Adaptive
Bio-Resources, 546 g/kg protein d IDF Inc., 832 g/kg protein e Solae, Pro-Fine VF, 693 g/kg crude protein f Cargill, Empyreal 75, 756
g/kg crude protein g Manildra Milling, 120 g/kg protein h Omega Proteins Inc., Virginia Prime menhaden oil iARS 702; contributed,
per kg diet; vitamin A 9650 IU; vitamin D 6600 IU; vitamin E 132 IU; vitamin K3 1.1 gm: thiamin mononitrate 9.1 mg; riboflavin 9.6
mg; pyridoxine hydrochloride 13.7 mg; pantothenate DL-calcium 46.5 mg; cyancobalamin 0.03 mg; nicotinic acid 21.8 mg; biotin
0.34 mg; folic acid 2.5 mg; inostitol 600 mg. j Contributed in mg/kg of diet; manganese 13; iodine 5; copper 9; zinc 40. k Stay-C,
35%, DSM Nutritional Products l Carophyll Pink 10, DSM Nutritional Products

Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an
invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014)

Table 6. Effect of replacing fish meal or soy protein concentrate with almond or pistachio meals at two levels in the diet of juvenile rainbow trout
on growth performance; factorial analysis.

Diet n Gain/fish, grams Gain, % of initial Survival,% Feed Intake, % bw/day

Fishmeal control 3 131.2 753 93.3 0.92
Fish, Almond, mid 3 104.5 614 100.0 0.95
Fish, Almond, high 3 124.5 711 98.3 0.88
Fish, Pistachio, mid 3 131 752 98.3 0.91
F-Pistachio, high 3 138.7 790 90.0 0.92
Soy Protein Control 3 111.8 648 96.7 0.99
SPC, Almond, mid 3 126.8 728 90.0 0.97
SPC, Almond, high 3 111 650 95.0 0.97
SPC, Pistachio, mid 3 123.1 718 90.0 0.97
SPC, Pistachio, high 3 149.3 831 88.3 0.95
Barrows, Frederic T., Frost, Jason B., Evaluation of the nutritional quality of co-products from the nut industry, algae and an
invertebrate meal for rainbow trout, Oncorhynchus mykiss, Aquaculture (2014)
 37

modify the nutritional profile of nut market opportunities. Check criteria #4.
material to meet the specifications for
We at ABR are committed to maintain
aquaculture feeds without degrading the
and consistently supply high quality
basic proteins. This results in the
commercial ingredients needed to
conversion of a low value raw material
support this growing premium market at
into a high value product with very little
competitive costs. A truly sustainable,
waste. ABR began producing commercial
viable, and high quality aquaculture
volumes of Mixed Feed Nut Meal at its
industry is not a far off dream dependent
central California facility in 2014, with
on new technology. The all natural More information
ample room for expansion. Our
alternative is here today for those ready
customers have shown strong interest in
to embrace the opportunity.
building a sustainable aquaculture
industry that produces the highest
quality farmed fish. Some might expect References:
there to be an economic penalty for early
(1) Barrows, Frederic T., Frost, Jason B.,
adopters of plant based feeds, but that is
Evaluation of the nutritional quality of co
clearly not the case. Mixed Feed Nut
-products from the nut industry, algae John Bowman,
Meal is priced at virtually the same level
and an invertebrate meal for rainbow General Manager,
as fishmeal. In addition there is another
trout, Oncorhynchus mykiss, Aquaculture Adaptive Bio-Resources
substantial benefit as the taste, texture,
(2014) E: abrjbowman@gmail.com
and overall quality of the sustainably
farmed fish has opened up premium AFΩ
 38
Nucleotides in Fish Nutrition:
The best strategy to enhance
immunity and intestinal health
By Oriol Roigé (Bioiberica), Barcelona, Spain
Nucleotides and aquaculture: a review
The aquaculture feed industry, as well as other animal feed
industries, is constantly screening the market and the latest
research looking for new ingredients that can give an added
value to their formulations in terms of quality and functionali-
ty, having a direct impact on the production parameters and
performance of the animals.
In such a growing industry that is aquaculture, where fish is
being cultured more and more intensively and production of
new species is being researched, the addition of new function-
al ingredients to the diet plays a very important role and will
become even more important in the future.
One of these new ingredients are nucleotides. Nucleotides are
low-molecular-weight intracellular compounds that play key
roles in literally every biochemical and cellular process of all
living beings. This is because nucleotides are the building key
parts of DNA, the molecule of life that contains all the
information about everything that happens and defines the
living being. Dietary nucleotides have been researched and
studied in animal and fish nutrition for some years, although in
a small extent. They are formed by three different units: a
pentose (sugar), a nitrogenous base and a phosphate group.
The pentose is either a ribose or a deoxyribose (forming RNA
and DNA respectively), the nitrogenous base can be a purine or
a pyrimidine depending on the molecular structure, and the
phosphate group can contain from one to three phosphates.
Nucleotides can be acquired by the animals from two different
sources: an endogenous route through de novo synthesis, and
an exogenous route through diet. Because nucleotides can be
synthetized by living organisms, they are not considered
essential nutrients. However, in specific life periods with
stress, diseases, fast growth or limited nutritional support,
nucleotide de novo synthesis can be limited and might not
cover the needs of the animal. This is because endogenous
production of nucleotides is a highly energy-demanding
process for the body, and during these periods energy can be
limited. For this reason, nucleotides are considered semi-
essential nutrients, because their supplementation through
the diet is very important during some specific and crucial
stages of the development of the animals, such is early stages
of life, vaccination, disease...
Nucleotides, as building blocks of DNA, are involved mainly in
processes where cell replication is important and necessary.
Cell replication is related to global growth and tissue develop-
ment and reparation. For this reason, nucleotides are very
useful in early stages of life, where the animals are growing
quickly and tissues need to replicate fast. More specifically,
some key systems benefit directly from this property of
nucleotides: the immune system and the digestive system.
Both systems are of huge importance for animal production in
aquaculture: the immune system protects the body from
diseases and is responsible for survival and healthy develop-
ment, while the digestive system is responsible of the correct
development and health of the intestines, absorption of
nutrients and overall growth and production. Thus, the more
developed these systems are, the better their survival,
performance and productivity will be.

Dietary nucleotides in fish and
immunity
There are many reasons why dietary
nucleotides are necessary in aquaculture.
Economic losses of fish producers caused
by pathological problems are becoming
more and more common in the industry.
These diseases have multiple causes:
unbalanced nutritional conditions, wrong
production management
(superpopulation) or poor water quality.
All this factors affect the fish by causing Figure 1. Weight gain was higher in the nucleotide group compared to the control group. Data
stress, which has a direct impact on the was collected every 15 days.
immune system of the animal lowering
their resistance to infections and diseases and limiting their
feed intake. This, of course, means a decrease on survival and
production.
A trial carried out by Bioiberica, supplementing nucleotides
(Nucleoforce Fish, Bioiberica) to Tilapia, showed a positive
impact on performance and health of the animals. A supple-
mentation of 500 ppm in the feed during 135 days was enough
to increase survival and significantly improve productivity
parameters such as body weight and FCR (Figure 1 & Table 1).
This trial proved that nucleotides are very helpful as they are
immunomodulators: they get the immune system ready
sooner and better, as they help immune cells to replicate and
grow faster. At the end, it is translated into higher survival,
body weight and improved FCR, which means better produc-
tion in a more economical way.
In another trial in Chile, nucleotides were tested against
Pisciricketsia salmonis infection, one of the main pathogens
affecting aquaculture of salmons. The experiment was
designed with four experimental groups of salmons: (1) a
group with placebo vaccination fed with a control diet, (2) a
group with SRS (Salmon Rickettsial Septicaemia) vaccination
Table 1. Productive parameters were significantly higher in the nucleotide group. Survival rate mortality. Furthermore, when productivity
also increased when compared to the control group.
Treatment Inital weight (g) Final Weight (g)
Control 1.86 130.14
Nucleoforce
1.43 182.5
FISH
39
fed with control diet, (3) a group with placebo vaccination fed
with an experimental diet containing 300ppm of nucleotides
(Nucleoforce Salmondis, Bioiberica), (4) and a group with SRS
vaccination fed with an experimental diet containing 300ppm
of nucleotides (Nucleoforce Salmondis, Bioiberica). Salmons
had an average weight of 10g at the start of the trial, were
vaccinated at 21d, challenged at 71d and results were
collected to 103d. As nucleotides are immunomodulators, the
idea was to see whether nucleotides had any effect as vaccine
enhancers, considering that vaccines require the activation
and development of the immune system. Results are shown in
Figure 2.
Surprisingly, it was found that the group of salmons fed with
experimental diet containing nucleotides but with placebo
vaccination (3) improved their survival rate even more than
the group fed with a control diet but with SRS vaccination (2).
This is a clear indicator of how nucleotides are useful in the
early maturation of the immune system, which allowed the
salmons to be strong enough to overcome the infection even
better than the SRS vaccinated salmons. However, the group
that showed the best results was the one combining SRS
vaccination and a diet supplemented with nucleotides (4). This
combination showed the best reduction on
parameters were analysed before the
challenge (at day 70), it was observed that
FCR Survival (%)
the groups supplemented with nucleotides
1.582 85 showed a higher body weight than the
salmons fed with the control diet (Figure
1.264 91 3).

Figure 2. Accumulated mortality of the different experimental groups
with different combinations of diet and vaccination.
gr P<0,05
Figure 3. At day 70 (before challenge) salmons with control diets and
experimental diets (with nucleotides) were compared. Salmons that
had been taking nucleotides showed a significantly higher body weight.
Dietary nucleotides and intestinal health:
compensating the negative impact of vegetable
proteins.
It is a growing trend to include proteins from vegetable origin
40
Figure 4. Comparison of the body weight of fish with control diets and
experimental diets (supplemented with nucleotides).
in fish diets (among other species), and the inclusion rate of
these proteins is also increasing in feed formulations. It is
known, however, that plant-based proteins have a negative
impact on the intestinal health of fish, as sources like soy
contain antinutritional factors that damage the intestine
causing inflammation and limiting its correct development,
which has a direct relation with a decrease in productivity.
Considering this, it is logic to think that supplementation with
nucleotides could be a good solution to compensate the
negative impact of vegetable proteins: Nucleotides help
replication of tissues and thus tissue repairing; intestinal
mucosa is damaged when using vegetable proteins, and has a
limited capacity of de novo synthesis of nucleotides. In
Bioiberica, several trials have been done to prove this thesis.
In a first trial in Portugal, supplementation with 1000 ppm of
nucleotides (Nucleoforce Fish, Bioiberica) was tested in
juvenile meagre with initial average weight of 37,2g over a 60-
day period. Nucleotide group and control group had diets with
high vegetable protein content (75%) (Figure 4)
Results showed a significant improvement on body weight
after 60 days in the nucleotide group and a significant increase
in leucine aminopeptidase activity (data not shown), sug-
gesting improved enterocyte status and increased digestion
capacity. Histological sections also showed a better developed
intestine (Figure 5).
 41

over a 134-day period. Both nucleotide

and control group received 100% plant-
based fishmeal-free diets. Results are
shown in Figure 6 and Figure 7.

Fish supplemented with 250 ppm of

nucleotides performed significantly
better than fish fed with control diet.
After 134 days, there were significant
A B improvements in body weight, growth
rate and FCR when compared to the
Figure 5. Histological sections of the intestine at 60 days. Figure 5A is from fish with
control group.
supplementation of nucleotides, 5B is from fish with control diets.
Both trials suggest that nucleotides are
an excellent strategy to significantly reduce the bad impact of
vegetable proteins in fish feed. In other words, supplementing
fish diets with nucleotides is very useful to increase the
vegetable protein content and reducing fishmeal, as they
reduce significantly the negative impacts of these proteins.

Figure 6. Differences in weight gain of gilt-head sea bream fed with a
control diet and with a diet supplemented with nucleotides.
Conclusions
Nucleotides are semi-essential nutrients that have key roles in
the body of fish and animals. Nucleotides are very useful in
some specific stages of development, especially in those
where cell and tissue replication is very important: early stages
of life, infection and diseases, vaccinations, stress…
Nucleotides help the immune system to develop faster,
making it ready to any challenge. Furthermore, they contrib-
ute to the healthy development of intestines, improving
productivity and overall performance.

AFΩ

More information
Oriol Roigé
Figure 7. Differences in Individual Growth Rate (IGR) and Feed Product Manager Animal Nutrition
Conversion Rate (FCR) of gilt-head sea bream fed with a control diet Bioiberica
Barcelona, Spain.
and with a diet supplemented with nucleotides.
E: oroige@bioiberica.com
In a second trial in Valenica (Spain), supplementation with 250 References and bibliography are available by
ppm of nucleotides (Nucleoforce Fish, Bioiberica) was tested in request.
juvenile gilt-head sea bream with initial average weight of 11g

Use of computer-assisted image
analysis to monitor health and
nutrition status in whiteleg shrimp
(Penaeus vannamei)
By Fabio Cervellione, Charles McGurk, Skretting ARC and Wim Van den Broeck, Ghent University
The aquaculture of penaeid shrimp has
grown from its experimental beginnings
three decades ago into a major industry.
Almost from the start, diseases and
adverse environmental conditions were
recognized as threats to the shrimp
industry, causing serious economic
losses. Feeding strategies form an
important part of modern farm manage-
ment. Feed manufacturers invest in R&D
to develop and offer new feeds and
feeding solutions aimed at helping
shrimp farming to grow, and to support
against emerging health issues and
adverse environmental conditions.
Current methods to assess health and
nutritional status in shrimp mainly rely
on clinical examination followed by
laboratory investigations and evaluation
of growth performance. One of the key
indicators of health in shrimp is the
perigastric organ (formerly known as the
hepatopancreas), making it one of the
most useful to examine.
The perigastric organ is the site of
synthesis and secretion of digestive
43
enzymes, digestion and nutrients
absorption, reserve storage and
detoxification. The perigastric organ is bi-
lobed and composed of many blindly
ending tubules, which wrap over the
dorsal and lateral sides of the posterior
part of the stomach and the anterior part
of the midgut. It is the principal organ of
the digestive tract and vulnerable to
pathophysiological changes.
Feeding regime affects the perigastric
organ. Under starvation and refeeding
conditions, beneficial lipid droplets are
rapidly depleted and haemocytes
(comparable to the white blood cells in
mammals) are recruited to mitigate
degenerative processes and inflamma-
tion occurring in the organ. Haemocyte
infiltration in the intertubular spaces of
the perigastric organ is also observed in
instances of tissue damage (traumatic or
due to infection), toxicity and environ-
mental stress.
In current practice, histological analysis
of the perigastric organ is based on
qualitative features interpreted by
pathologists. Pathologists’ quantification
is generally time-consuming and poorly
objective, with significant discrepancies
in scoring results reported between
observers. This has motivated the
development of computer-assisted
image analysis (CAIA) methods for
producing unbiased, objective, reproduc-
ible, and reliable data. The basic principle
of automated image analysis for
histology is the use of a series of
mathematical algorithms to process
images, enabling the segmentation of
picture elements into regions of interest
based on their colour, texture and/or
context. CAIA is currently used by
Skretting Aquaculture Research Centre
(ARC) to investigate a number of organs
(gills, gut and skin) across various fish
species.
The success of these methods in fish
recently led to the development of a
semi-quantitative histological method to
monitor health and the nutritional status
in whiteleg shrimp using CAIA on
microscopic sections of the perigastric
organ. After the optimization of fixation
 44

methods (both for paraffin and frozen

sections) and staining protocols for the
perigastric organ, customized software
was used to develop and validate
protocols for the quantification of
following morphological parameters of
the organ: areas of haemocyte infiltra-
tion (inflammation), F-cells (production
of digestive enzymes), B-cell vacuoles
(site of intracellular digestion), lipid
droplets (reserve energy stored in the
R-cells) and their ratios to total tissue
area and total lumen area. Figure 1
shows the development of one protocol
for CAIA.

The effect of feed deprivation and

refeeding was studied using the
developed method. Shrimp of 2 ± 1 g
were divided into three groups (40
shrimp each), receiving three treatments
(one treatment per group) for 15 days:
FE (fed), ST (starved), and REF (starved
for 5 d and then re-fed for 10 d). Shrimp
were sampled at day 0, 5, 10, and 15 and
selected for the C-intermolt stage. Figure 1. Development of computer assisted image analysis protocol for the detection of
Software was used to quantify the haemocytes and B-cell vacuoles in the hepatopancreas (HP) of whiteleg shrimp (Penaeus
following morphological parameters, vannamei). Paraffin section (5 µm) of the HP stained with immunohistochemistry using
monoclonal antibodies WSH8 counterstained with Mayer´s haematoxylin (a). Protocol workflow
indicating different parameters:
(b,c,d): pre-processing (b), classification (c), post-processing (d). In d: total tissue area (pink),
inflammatory index (HIA:TTA), nutritional
lumen area (grey), B-cell vacuoles (light blue), haemocytes (blue). lu = lumen; tb =
index (LDA:TTA), and other parameters
hepatopancreatic tubule; vB= B-cell vacuole; black arrows = haemocytes. Scale bar = 100 µm
indicating the enzymatic and digestive

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 45

activity of the organ. Starvation led to

significant morphological changes in the
HP. Significant changes were measured
for the inflammatory index (Figure 2) and
nutritional index (Figure 3) during
starvation (increase of inflammation
associated with decreased of lipid
reserves) and starvation followed by
refeeding (decrease of inflammation
associated with increase of lipid
reserves). Refeeding did not result in a
complete recovery of the perigastric
organ.

The main findings of this three-year PhD

study can be considered as a starting
base to implement image analysis in the
shrimp histology labs for diagnosis
purposes and to start collecting
reference values of the developed
morphological parameters to be used for
screening health and nutritional status in
crustaceans. The development of specific
indices (inflammatory and nutritional)
will be used in the future to collect
reference values for monitoring health,
assess the impact of different diets and
feeding regimes, facilitate early diagnosis Fig 2. Effect of starvation and refeeding on the inflammatory index of the perigastric organ in
of diseases and study the pathophysiolo- whiteleg shrimp (Penaeus vannamei). Note: HIA:TTA = ratio of Haemocytic Infiltration Area to
Total Tissue Area (as %). FE = fed; ST = starved; REF = refed; R = start of refeeding after 5 days of
gy of the perigastric organ.
starvation.
The PhD has been funded by Skretting
ARC and the Research Council of Norway.

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References:
Cervellione, F., McGurk, C., Silva, P.,
Owen, MAG & Van den Broeck, W.
(2016) Optimization of fixation methods
for image analysis of the hepatopancreas
in whiteleg shrimp, Penaeus vannamei
(Boone). Journal of Fish Diseases,
doi:10.1111/jfd.12531.

Cervellione, F., McGurk, C., Berger

Eriksen, T. & Van den Broeck, W. (2016)
Use of computer-assisted image analysis
for semi-quantitative histology of the
hepatopancreas in whiteleg shrimp
Penaeus vannamei (Boone). Journal of
Fish Diseases, doi:10.1111/jfd.12599.

Cervellione, F., McGurk, C. & Van den

Broeck, W. (2017) Effect of starvation
and refeeding on the hepatopancreas of
whiteleg shrimp Penaeus vannamei
(Boone) using computer-assisted image
analysis. Journal of Fish Diseases,
doi:10.1111/jfd.12599.

Cervellione, F., McGurk, C. & Van den

Broeck, W. (2017) “Perigastric organ”: a
replacement name for the
“hepatopancreas” of Decapoda. Journal
of Crustacean Biology, doi: 10.1093/
jcbiol/rux020.
Fig 3. Effect of starvation and refeeding on the nutritional index of the perigastric organ in
whiteleg shrimp (Penaeus vannamei). Note: LDA:TTA = ratio of Lipid Droplet Area to Total Tissue
Area (as %). FE = fed; ST = starved; REF = refed; R = start of refeeding after 5 days of starvation.

AFΩ

More information
Fabio Cervellione, DVM, MSc, PhD, Research
Pathologist, Skretting ARC, Norway
E: fabio.cervellione@skretting.com

Charles McGurk, DVM, MSc, PhD, Fish & Shrimp

Health Manager, Skretting ARC, Norway
E: charles.mcgurk@skretting.com

Wim Van den Broeck, DVM, MSc, PhD, Professor of

Histology and Cell Biology, Ghent University,
Belgium
E: wim.vandenbroeck@ugent.be
 47

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