Feed, Nutrition, Feeding

Nutritional Biology in Farmed Fish

Nutritional Aspects Marine Fish Larvae

Feed Resources Feed Technology

Metabolism and Uptake of Carotenoids in Farmed Fish

 Olai Einen1), Henriette Alne1), Barbara Grisdale-Helland1),2), Stle J. Helland1),2), Gro-Ingunn Hemre3),
Bente Ruyter1), Stle Refstie1),2) and Rune Waagb3)

1) Akvaforsk The Institute for Aquaculture Research AS, 2) Aquaculture Protein Centre,
3) NIFES (Norwegian Institute of Fisheries and Aquaculture Research)

Nutritional Biology in Farmed Fish

Correct nutrition of farmed fish is vital to cost-effective production of healthy,

high-quality food. Feed is the greatest single cost in fish farming, and as efficiency
increases, feed costs become even more important. With the quantities produced
by the aquaculture industry today, relatively small changes in feed composition
and feeding regimens can generate substantial benefits. Many aspects of the
appetite of fish and their ability to utilise feed for growth are still unknown. The
demand for development of more cost-efficient feed means that we have to gain
a fundamental understanding of the nutritional needs and tolerance limits of
farmed fish. There are increasingly new requirements for feed and raw materials
for feed, creating a great need for research in nutritional biology. As more species
are becoming relevant for aquaculture, it is important to build specialised exper-
tise, as the needs and preferences of each species can vary greatly. For this reason
comparative nutritional studies have been done in recent years. The Norwegian
Research Councils Aquaculture programme, which has now been concluded, has
placed particular focus on salmon and cod;, this is reflected in this chapter.

200 Aquaculture Research: From Cage to Consumption

This chapter presents new knowledge in nutri-
tional biology relating to feed composition,
feeding regimens, absorption, metabolism and
fish health. Atlantic salmon and cod are dis-
cussed in particular, as these species have been
the focus of projects on nutrition carried out
under the Aquaculture programme during the
programme period. Results from these projects
have been included, as have results from strate-
gic institute programmes and projects funded
by the Fishery and Aquaculture Industry Re-
search Fund (FHF) for which the Aquaculture
programme has been responsible, or in which it
has taken part. Subjects such as gene-modified
raw materials, contaminants, pigmentation and
larval nutrition are discussed in separate chap-
ters in this book.
Feeding and feed intake
The growth potential of fish differs from spe-
cies to species, but is highly dependent on tem-
perature, size and genetic origin. The degree to
which this growth potential is realised is highly
dependent on feed intake and on how well the
feed has been adjusted to the nutritional needs
of the fish. Maximum growth is usually ob-
tained when the fish are fed until the point of
satiation. In earlier years, it was difficult to
accurately measure feed intake in nutritional
experiments in tanks and net pens, and it was
even more difficult in practical fish farming. In
recent years, feed intake measurement has be-
come a standard routine in many nutritional ex-
periments, and has proven to be a useful tool.
Feed intake is quantified by measuring the
amount of feed given, minus the accumulated
feed waste after the fish have been fed in surplus
(1020 per cent overfeeding). In practical fish
farming, feeding surveillance systems based on
video, sensors or collection of feed waste have
been implemented. This has given better control
over the significance of the variation parameters
in experiments and their effects on feed utilisa-
tion.
Differences between species?
Are there differences in the feed intake between
species under the same conditions? This ques-
tion was studied in an experiment that com-
pared feed intake and growth in salmon,
rainbow trout and cod; the groups had the same
starting weight (450 g) and the same environ-
mental conditions. The feed intake was highest
in the salmon and somewhat lower in the trout,
while the cod only consumed half of the aver-
age intake for salmon and trout. When compar-
ing the three species, we observed that the cod
partly compensated for its lower feed intake
with greater nutrient utilisation. However, the
level of nutrient utilisation was not high enough
to account for the differences in growth related
to the appetite of the fish. It was expected that
nutrient digestibility would be somewhat re-
duced in the cod when the feed intake was in-
creased. This was demonstrated in salmon, but
the effect on cod was marginal. A comparison
of feed utilisation results from several experi-
ments with Atlantic halibut, cod, salmon and
trout indicates that this parameter is relatively
similar in small fish (Figure 1). It appears, how-
ever, that there are differences between the spe-
cies when fish of different species, but the same
weight, are compared in the same experiment.
In the comparison of trout, salmon and cod
mentioned above, the feed utilisation was 30
40 per cent high in the cod than in the salmon
and trout (Figure1; centre ellipse). There were
no differences in feed utilisation between the
salmon and the trout in this experiment, but
other experiments indicate that salmon utilise
feed 2030 per cent better per kg growth than
trout (Figure 1; left and right ellipse).
Theme: Feed, Nutrition, Feeding 201
 1.7
1.5
Feed conversion ratio
1.3
1.1
0.9
0.7
0.5
0 200
145x100//Kap12-fig01.eps
Figure 1. Feed conversion ratio in farmed fish of different sizes; ellipses represent values from the same experiment.
How does energy content in the feed
affect the feed intake?
To compare the effect on feed intake caused by
the energy concentration in the feed, rainbow
trout, salmon and cod were given two different
feeds one feed with a medium energy level
and high protein level (22.6 MJ and 60 per cent
protein) and one feed with a high energy level
and medium protein level (24.9 MJ and 53 per
cent protein). The feed intake in the salmon and
trout was higher with high-energy feed than
with medium-energy feed, but the cod con-
sumed about the same amount of each of the
two feeds. It seems the salmon and the trout
regulated the feed intake so that the protein in-
take per kg fish for the two feeds was the same.
The cod, however, did not show any obvious re-
sponse to the energy or protein levels in the
feeds, consuming about the same amount of
both feeds.
202 Aquaculture Research: From Cage to Consumption
Trout
Salmon
Halibut
Cod
400 600 800 1000 1200
Mean weight in experiment, g
Vegetable protein ingredients contain a high
percentage of indigestible carbohydrates (30
50 per cent) in the form of oligosaccharides and
different fibre fractions, giving a lower concen-
tration of digestible energy in the feed. In experi-
ments with rainbow trout and cod it has been
shown that the fish can compensate for the
lower energy level in the feed by increasing the
feed intake. In some experiments with cod, the
proportion of gastrointestinal tract to body
weight has increased as a consequence of long-
term use of soybean meal in the feed, whereas
other experiments have not revealed the same
tendency. It has also been shown that cod digest
vegetable protein better than salmon. The
growth rate in both cod and salmon is sustained
because the fish compensate for poorer diges-
tion with higher feed intake. This leads to re-
duced feed utilisation when the percentage of
vegetable protein in raw materials increases, i.e.
a higher feed conversion ratio and lower reten-
tion of protein and energy. Cod seem to be more
flexible than salmon and trout in adjusting feed
intake and sustaining good digestion of low-
cost vegetable raw materials in the feed.
How often should the cod be fed?
Feeding regimens are important in fish farming
in order to optimise the feed intake of the fish in
relation to feed utilisation and fish growth.
There has been much discussion on how often
cod should be fed, and whether feeding fre-
quency affects feed intake and fish growth. In
an experiment where young cod were held at a
water temperature of 8C, no significant differ-
ences in growth were found between groups
that were fed to satiation every day and groups
that were fed until satiation every other day.
Moreover, the feed conversion ratio and liver
index were not affected by the various feeding
regimens used in this study.
Feed composition
In recent years, there has been increased aware-
ness of the scarcity of marine raw materials
such as fish meal and fish oil. There is no longer
a feed quota regime in Norway for salmon
farming, which gives greater opportunities for
using new raw materials in feed. This, in turn,
gives rise to a new demands for knowledge
about the nutritional needs of fish and their util-
isation and conversion of each nutrient. Good
feed utilisation is a prerequisite for good
growth, and is also related to a high retention of
protein and fat in salmon. It is important to note
that feed manufacturing companies have carried
out a considerable amount of unpublished re-
search on feed composition in order to develop
new feeds and feed concepts; this is not dis-
cussed here.
Do high energy feeds give the same
results in salmon and cod?
As mentioned above, differences in feed intake
were found in salmon, rainbow trout and cod
that were given feeds with a medium or high
energy content. In this comparison of the three
species, no difference was found in the salmon
and the trouts retention of amino acids (per-
centage of the intake of amino acids) when
given a medium energy and a high energy feed.
However, the cod, the fish fed a high energy/
medium protein diet retained 45 per cent more
essential amino acids than the fish fed a me-
dium energy/high protein diet with. In this ex-
periment, the salmon retained 17 per cent more
of the digested energy than the trout, while the
retention of digestible energy in the cod was no
different than in the other two species. The
comparison between the three species showed
that the cod retained on average 14 per cent
more of the digested protein than the salmon,
and 34 per cent more than the trout.
What is the optimal feed composition for
cod?
An experiment with Atlantic cod (from 200 to
approx. 850 g) showed that growth was nega-
tively affected by low protein and high starch
concentrations in the feed. The fat content,
however, had no significant effect on growth.
Feed utilisation was improved by increasing the
amount of fat and protein in the feed, while it
was reduced by increasing the concentration of
starch in the feed. One challenge with farmed
cod is unwanted deposits of fat in the liver,
which raises the liver index (liver weight to
body weight ratio). The liver index rose with a
higher content of fat in the feed, and fell with a
higher content of protein. The level of starch in
the feed, however, had an insignificant effect on
the liver index. The conclusion is that in order
to obtain good growth and protein retention,
Theme: Feed, Nutrition, Feeding 203
 avoid a high liver index and achieve low feed
conversion ratios in young cod, the feed should
contain according to the results in this experi-
ment 500600 g raw protein/kg feed, 130200
g fat/kg feed and <150 g starch/kg feed.
In another experiment in which cod were al-
lowed to grow from 125 to 250 g, growth was
not greatly affected by feed composition. Four-
teen different feeds, containing 4560 per cent
protein, 1027 per cent fat and 521 per cent
starch, were compared. Feed utilisation was, as
in the experiment above, reduced when the con-
centration of starch in the feed was increased.
Feed utilisation was improved when the amount
of protein in the feed was increased; however,
the results indicated that this improvement only
continued until a certain level of protein in the
feed was reached. The liver index rose from 7 to
13 per cent when protein was replaced by fat in
the feed. The liver index fell when there was a
higher content of ash in the feed. The conclu-
sion in this experiment was somewhat different
from the experiment above; in order to obtain
good growth and feed utilisation in young cod,
the feed can, without problem, contain >200 g
fat/kg feed and <120 g starch/ kg feed. There
were no advantages gained by increasing the
protein level to over 510 g/kg feed.
Should the feed be adjusted to the
season?
Large seawater salmon have a seasonally deter-
mined rhythm of energy use from feed in rela-
tion to their own energy depots. These natural
seasonal rhythms are also found in farmed
salmon which are fed year-round according to
appetite with a well-balanced feed, even when
continuously exposed to simulated daylight.
The salmon become leaner in spring and grow
extensively in the summer and autumn, accu-
mulating large lipid deposits. Due to the annual
variation in salmon body energy stores, we
aimed to discover if, by manipulating the
energy density of the feed contrary to the
salmons natural annual rhythm (i.e. by feeding
the fish a lipid-rich spring feed and a lean
autumn feed), we could achieve more uni-
form fillet quality year-round.. If such feed
could guarantee invariable fillet quality, it

Growth
9000 Feed Conversion Ratio (FCR)
1,4
1.4
8000
1,2
1.2
7000
1,0
1.0
6000
0,8
0.8
5000
0,6
0.6
4000
0,4
0.4
3000
0,2
0.2
2000
Direct
rstid
Kontroll
Control 0
1000 Sesong
Season Spring
Vr Sommer
Summer Hst
Autumn Vinter
Winter
start spring
vr sommer
summer hst
autumn vinter
winter

145x100//Kap12-fig02.eps

Figure 2. The Direct group was given feed that varies with the marine raw materials protein-energy composition (P/E).
(The raw material was filleting waste from herring.) The control group was given feed with a constant P/E ratio. The
Season group was given an energy-dense spring feed and a lean autumn feed. The figure to the left shows growth
development with samples taken in March (start), May (spring), August (summer), November (autumn) and February
(winter). The Season group was almost half a kilo heavier than the Direct group after one year in the sea. The figure to the
right shows FCR values (g dry feed eaten/g growth) for the three feeding groups: Direct, Control and Season, measured in
the period March-May (spring) June-August (summer), September-November (autumn) and December-February (winter).
Red=Direct, yellow=Control, blue=Season.

204 Aquaculture Research: From Cage to Consumption

would have a competitive advantage on the
market. This group was given the acronym Sea-
son A second group of triplicate sea cages were
fed a diet that was the opposite of the Season
diet. This diet was designed to be lean in spring
and lipid rich in autumn, following the natural
variation in energy deposits in herring catches.
This group was given the acronym Direct. Her-
ring trimmings were used as a major source of
lipids and protein in the diets. The control diet
did not vary in composition throughout the four
seasons, corresponding to a high-energy salmon
feed with a composition similar to that of a
commercial feed. The control diet was included
in order to compare data from the experimental
situations against data from a normal situation.
The fish were fed according to appetite, two
meals a day until satiation. There were slight
differences in growth in the course of a year;
however, the fish fed the Season diet were 0.5
kg larger on average compared to the fish fed
the Direct diet. Feed utilisation also varied
throughout the year (Figure 2). The results illus-
trate that when salmon are fed according to ap-
petite, they adjust their intake of energy and
protein themselves. For fish farmers, the mea-
surable differences related to production data
first and foremost concern the amount of feed
necessary to achieve the same growth. It is pos-
sible to use less feed by giving fish energy-
dense feeds in the spring and summer, when the
diet richest in energy gives the best feed utilisa-
tion compared to the two other diets. In spring
the feed conversion ratio (FCR) varied from
0.58 to 1.18, and in summer it varied from 0.83
to 1.12. The control diet was utilised better than
the Season diet during spring, but worse during
summer. The salmon showed the highest
growth rate in the fall; this was also the season
during which dietary energy levels did not af-
fect feed utilisation, within a variation in fat
levels of 2744 per cent. The highest protein
retention was achieved in summer by feeding a
lipid-rich (44 per cent) feed with a medium
amount of protein (44 per cent). There were
also differences in growth rate between dietary
treatments during the various seasons. Total
growth throughout one year of feeding was
from 1.7 to 7.6 kg for the fish in group Season.
Digestion and absorption
Knowledge about how the digestive system
works and how different nutrients behave in the
gastrointestinal tract is critical to optimising the
utilisation of new raw materials in feed. There
are also significant differences between species;
for example, Atlantic salmon and rainbow trout
have short gastrointestinal tracts, whereas cod
have a considerably longer one. In recent years,
several studies have generated new knowledge
in this area, and are a result of, among other
things, newly developed methods which makes
such studies possible.
Digestion of proteins
In fish the majority of amino acids are absorbed
from digested protein as di- and tri-peptides ac-
tively transported through the membranes of in-
testinal cells. Oligo-peptides are furthermore
substrates for enzymes aminopeptidases in
the intestinal mucosa. It appears that young fish
can grow almost normally when amino acids
are provided in the form of dipeptides, contrary
to when they are provided as free amino acids.
Hence, the need for amino acids in young fish
can be determined by using feeds in which lim-
ited amino acids are provided in the form of di-
and tripeptides as substrate for specific peptide
transporters. This also has consequences for op-
timisation of the amino acid profile in feed for
fish larvae with incompletely developed diges-
tion channels (see separate chapter).
Theme: Feed, Nutrition, Feeding 205
 Figure 3. Methodology for studies of uptake of fat in the intestine and liver. In recent years, a method for quantifying fat
in the intestine and liver has been established. Thin cuts of intestine and liver tissue are stained with toluidine blue and
alcaline fuchsine. Afterwards the drops of fat appear with a green colour against cytosol, which appears violet or pink
under light microscopy. The fat can thus be quantified by digital image analysis. The method has proven to be useful for
studying how water temperature and fat content in feed affect deposition of fat in the intestine and liver. (Photo: A.
Vegusdal/Akvaforsk)
145x100//Kap12-fig03.eps

Anti-nutrients from vegetable protein raw mate-
rials present challenges with regard to distur-
bances in the digestive processes. In plants
these substances are a heterogeneous group of
structural components and/or chemical de-
fences against herbivores. Anti-nutrients are
therefore often found in low-refined vegetable
ingredients, for example soybean meal, an inex-
pensive and easily accessible protein source.
Soybeans and other legumes contain proteins
which bind themselves covalently to the active
sites in pancreas proteases such as trypsine and
chymotrypsine. In vitro experiments have
shown that phytic acid (myo-inositol-6-phos-
phate) also inhibits the activity of both cationic
and anionic salmon trypsine. Moreover, these
anti-nutrients inhibit the proteolytic activity in
the intestinal contents, which in turn leads to in-
creased loss of proteases through the faeces.
This shows that high levels of soybean meal in
the feed leads to depletion of enzymes contain-
ing granules in exocrine pancreas cells in rain-
bow trout. Soybean meal also contains
unidentified anti-nutrients that cause inflamma-
tion in the mucous membrane of the hindgut of
Atlantic salmon. At the same time, the activity
of enzymes in the intestinal mucous membranes
of the brush border is reduced, depending on the
amount of soybean meal in the feed. Active
transport of amino acids into the enterocytes de-
creases, and the activity of cytosolic enzymes in
the intestinal mucous membrane is reduced,
which indicates reduced super nuclear forma-
tion of vacuoles and therefore reduced absorp-
tion of intact macro molecules (proteins) by
endocytosis. All in all, this will reduce the fishs
ability to digest and absorb proteins. This seems
to be due to an increased exchange of cells in
the intestinal mucous membranes surface as a
response to the intestinal inflammation. The in-
testinal mucous membrane of the fish is strong-
ly folded, but has no villi.
With soya in the feed, the number of proliferat-
ing cells on the surface of the folds increases.
These immature cells have reduced function be-
cause of a lower number of functional proteins
(enzymes, transporters etc.) in the apical cell

206 Aquaculture Research: From Cage to Consumption

membrane and/or other glucosylation profile in
membrane-bound receptors.
Corresponding intestinal changes were not
found in cod in an experiment in which the fish
were given feed with up to 44 per cent soya and
maize gluten. Plant protein mixtures up to 44
per cent did not affect the capacity for enzym-
atic hydrolysis of protein and absorption of
amino acids in any part of the cod intestine, but
did reduce the digestibility of fat and led to in-
creased microbial activity and changes in
microflora in the intestine. Reduced digestibili-
ty of fat with vegetable proteins in the feed was
also observed in salmon.
Digestion, intake and transport of fat
Despite the fact that todays commercial high-
energy feeds can contain up to 40 percent fat,
little is known about how the various fatty acids
from different sources affect the absorption pro-
cess along the digestive tract. Furthermore,
little is known about how different fatty acids
are converted in intestinal cells (enterocytes)
and thereafter transported in lipoproteins to dif-
ferent tissues, to be used for combustion, meta-
bolic conversion and/or storage. Increased
knowledge in this area is especially important
because new dietary fat sources will be used to
a greater extent in commercial feeds.
There is little knowledge about how lipids are
transported from the fishs intestine to the tissues
where they are to be utilised. Therefore, one ob-
jective has been to obtain a fundamental under-
standing of absorption of fat in different parts of
the intestine and further transport of fatty acids
with different chain lengths to different tissues in
salmon. By feeding salmon with isotope-marked
fatty acids of short (C10) and long (18:1) chain
lengths, it has been possible to follow the fatty
acids absorption into various parts of the intes-
tine and transport to different tissues. The pyloric
caeca area appeared to be the most important
area for absorption of both short and long
chained fatty acids, but longer fatty acids were
absorbed in more parts of the intestine, including
large parts of the middle intestine. Both short and
long chained fatty acids were principally drained
from the intestine via portal blood, and trans-
ported directly to the liver. In the liver, the fatty
acids were incorporated into lipoproteins before
further transport. Significant amounts of the long
chained fatty acids from the feed were directly
deposited in visceral fat depots.
When partly digested fat is incorporated into
the enterocytes, it has to be reassembled into
triglycerides before it can be transported to the
organs. A concrete question studied during the
programme period was whether fish have a high
activity of monoacylglycerol acyltrasferase
(MGAT), the pathway of re-synthesis of triglyc-
erides after the fat has been digested in the in-
testine and absorbed. Initially, it was assumed
that the fish could have two mechanisms for
synthesis: the MGAT pathway and the glycerol
3-phosphate (GPAT) pathway. The MGAT
pathway is very fast, demands little energy and
is dominant among mammals. The alternative
GPAT pathway is energy-intensive and rela-
tively slow. The problem with the MGAT path-
way is that it requires a partly digested
triglyceride, 2-monoacylglycerol (2-MAG), as
the first building block. Earlier studies have
shown that fish possibly do not have the diges-
tive enzyme that creates 2-MAG, but probably
have a less specific enzyme which creates all
kinds of products. Thus, it was also considered
a possibility that the MGAT pathway was not
active in fish. A partial characterisation of the
enzyme MGAT (+DGAT) from salmon micro-
somes did, however, show that the enzyme was
very active in the intestine. A further compari-
Theme: Feed, Nutrition, Feeding 207
 son of the two alternative pathways of synthe-
sis, using entire intestinal segments, also
indicated that the MGAT pathway is far more
active than the alternative GPAT pathway. It is
also possible that these two pathways of synthe-
sis have different functions in the cells. Further-
more, it was shown that the fat from the diet can
be used directly for creating phospholipids in
the enterocytes (CPT), probably for direct use
in the lipoprotein synthesis, but also for direct
incorporation into membranes. Therefore, this
makes the enterocytes very vulnerable to
changes in the composition of the diet, and
symptoms of badly balanced fat mixtures will
often first manifest themselves here. There was,
however, little that indicated that the composi-
tion of the diet had any effect on the activity of
these enzymes (MGAT, DGAT, CPT).
It is known that the fat from the liver is princi-
pally transported in lipoproteins in the blood of
the fish, but there is not much knowledge about
synthesis, secretion, transport, reception and re-
moval of the different lipoproteins. To provide
improved analytical tools for identification and
quantification of lipoproteins at the apo-lipo-
protein and lipoprotein levels, NIFES has de-
veloped polyclonal antibodies against HDL and
VLDL from salmon. A method for quantifica-
tion of vitellogenine in salmon plasma has also
been developed.
Intestinal flora
Like other animals, fish have bacterial flora in
the intestinal channel. This micro biota consists
of aerobe, facultative anaerobe and obligate
anaerobe bacteria, but the composition varies
with age, nutrition and water environment. The
amount of bacteria increases along the intestine
and most is found in the distal part of the intes-
tine. Using electron microscopy this has been
shown with both transient bacteria in the intes-
208 Aquaculture Research: From Cage to Consumption
tinal lumen and fixed bacteria associated with
the intestinal epithelium in a number of farmed
species, among them salmon and cod. The fixed
bacteria are associated with the viscous car-
pet of hydrated mucines which are excreted
from the intestinal epithelium to protect the sur-
face of the enterocytes against the intestinal
lumen. For the time being, it is unknown to
what degree intestinal flora contribute to micro-
bial digestion in fish.
The intestinal flora in fish are sensitive to stress,
antibiotics and changes in feed composition. It
has been shown that different types of oils and
protein meals affect and change the intestinal
flora in fish. Low stability in the microflora
might have consequences for the fishs resis-
tance to disease, as it can result in the growth of
pathogen bacteria which damage the intestinal
epithelium at the expense of neutral and/or
favourable bacteria (for example, lactic acid
bacteria). Pathogens may also be absorbed
through the intestinal mucosal membrane by
endocytosis, as shown in salmon with electron
microscopy. This indicates that the intestine is
one of the main routes of infection in fish.
The use of molecular methods for characterisa-
tion of the micro biota in fish and how it is af-
fected by feed and water environment will
therefore be important. This will pave the way
for a more correct use of raw materials in feed,
as well as for promoting the growth of favour-
able microflora through feed or water (probiot-
ics) as a prophylactic measure.
From intestine to muscle and fat
tissue: conversion of nutrients
When the feed is digested and absorbed, the nu-
trients are distributed via the blood to the liver,
muscles and other tissues. Here the nutrients are
deposited, combusted or metabolised into other
substances, which in turn can be excreted
through the gills or urine, or transported back to
the intestine. The way in which and the efficacy
with which the different nutrients are converted
are important to feed utilisation, growth, health
and the quality of the final product. The access to
nutrients often controls the expression of genes
which are essential to the metabolism of nutri-
ents, such as enzymes, growth factors and factors
that are important for the fish immune system.
Protein metabolism
In the body of fish, synthesis and degradation of
protein is ongoing. Most studies show that pro-
tein synthesis increases together with increases
in protein intake, up to a maximum level (de-
pendent on genetic, environmental and nutri-
tional conditions). The linear proportion which
is found between protein intake and synthesis is
explained by the fact that the feeds amino acids
stimulate protein synthesis to different degrees.
By measuring feed intake and protein synthesis,
it has been found that approximately 7080 per
cent of a measured growth variation can be ex-
plained by variation in feed intake. The degra-
dation rate of protein will also strongly affect
the growth rate; it is uncertain whether this is
dependent on feed intake. Some individuals
grow rapidly, while others grow slowly with the
same amount and type of feed. Thus, in the
aquaculture industry there is the potential for
selective breeding of individuals with both a
high growth rate and good feed utilisation. Rap-
idly growing individuals seem to store a larger
share of the synthesised protein than individuals
with a lower growth rate. When protein meta-
bolism is measured in order to investigate the
quality of a protein source, it is necessary to
give standardised amounts of feed and energy
because both these factors affect the synthesis
and degradation rates. Fish that are given a
high-quality protein source grow more rapidly
and show both increased synthesis and in-
creased degradation. In other words, they have
a higher total metabolism of protein than slower
growing individuals. Synthesis of protein costs
the same for the two types of fish, but the fish
that retain more of the synthesised protein have
a net gain which is higher than the fish that has
a higher decomposition rate.
Previously, there has been little research done on
how the amino acid balance affects the growth
process in fish. Protein metabolism, and with it
growth, is modulated by a series of nutrients
which behave as individuals or through interac-
tions with other nutrients or hormones. Interac-
tions occur when one nutrient directly affects the
utilisation of another nutrient, either on an ab-
sorptive level or on a cellular level. This can have
a positive or negative influence on fish growth.
Results from experiments have shown a correla-
tion between the concentration of polyamines in
muscle and growth. The polyamines spermidine
and spermine are growth-regulating compounds
that are related to cell division. These are syn-
thesised from the amino acid arginine, but the
process is also dependent on a form of the
amino acid methionine. The amino acid lysine
can give reduced availability of arginine. An
imbalance in content and availability of argin-
ine, lysine and methionine in the feed can there-
by affect the growth process in fish. Inclusion of
more than 16 g lysine per kg feed in an experi-
mental feed based on wheat gluten gave re-
duced growth, most likely because of the
lysines effect on arginine metabolism. Taurine
in salmon feed has to some extent resulted in
improved growth and feed utilisation as long as
the methionine level in the feed is low. The
branched amino acids leucine, iso-leucine and
valine have appeared to have a modulating
Theme: Feed, Nutrition, Feeding 209
 effect on protein metabolism in fish. An imbal-
anced intake may possibly affect the meta-
bolism of branched amino acids as well as the
protein synthesis capacity in muscle tissue,
which in turn can affect the growth process. In
an experiment with salmon, it was shown that
these three branched amino acids had an inhib-
itive effect on each others reception in the in-
testine, measured in vitro.
There is little known about the need for arginine
and other amino acids in the feed in the early
life stages of fish larvae and fry. Contrary to
adult fish, the enzymes in the urea cycle of lar-
vae and fry are often measurable. This cycle
can, in theory, take part in producing the essen-
tial amino acid arginine. It is therefore possible
that the need for arginine in fry can be covered
by endogene synthesis. In an experiment with
trout under initial feeding, feed in which the
protein had been replaced with di-peptides (two
amino acids chained together) was used. One
feed contained di-peptide arginine, while an-
other contained no arginine. The results were
clear: trout do need arginine in the feed during
initial feeding, and any synthesis of arginine in
the fry themselves is not sufficient to cover the
need for arginine for maintenance and growth.
This finding also strengthens the hypothesis
that the urea cycle is expressed in fish larvae
and fry, and is significant in order to remove
ammonia, which is a toxic end-product created
through the degradation of amino acids. The ex-
periment shows the importance of developing
experimental dry feeds for larvae in order to de-
termine the quantitative need for arginine and
other amino acids in the feed. Such experiments
can contribute to reduced mortality and in-
creased growth in larvae and fry in culture.
Degradation of nucleic acids is also associated
with urea production in fish. In humans, overly
high levels of nucleic acids in the diet can lead
to gout. BioProtein (based on bacteria cultiv-
ated on natural gas) is a product with a high
content of nucleic acids (approx. 10 per cent),
and it was therefore of interest to investigate
how salmon handles such relatively large
amounts. A central enzyme in the metabolism
of these substances is urate oxidase; mRNA for
this enzyme was first isolated and sequenced in
several fish species. The sequence was then
used to make a probe in order to measure the
regulation of this enzyme. These results, to-

Figure 4. A method for cultivating fat cells from fish in culture has been established to improve the understanding of
development of fat tissue and deposition of fat in fish. The method makes it possible to follow the development of fat
tissue step by step, from pre-adipocytes, which are the precursors of the fat cells, to mature fat cells (adipocytes). The
method is used in studies to see how high-energy feeds affect the deposition of fat in Atlantic salmon. Using this method,
the presence of PPAR (left), C/EBP (centre), leptin and cPLA2 is shown. The accumulated amount of intracellular lipids is a
good indicator of the degree of cell differentiation: oil red-O colours drops of lipid red (right). Results achieved using the
method indicate that the development of fat tissue in salmon is regulated in the same way as it is in mammals.
(Photo: A. Vegusdal/Akvaforsk)
145x100//Kap12-fig04.eps

210 Aquaculture Research: From Cage to Consumption

gether with traditional enzyme measurements
and measurement of several metabolites, gave
no indication that the high content of nucleic
acids was problematic for Atlantic salmon. The
salmon responded well to the BioProtein feed,
growing better than on fish- meal-based control
feeds. It is possible that the nucleic acids had a
positive effect on the salmons growth.
Metabolism of fat
In recent years, substantial research has been
carried out on the consequences of and possibil-
ities for using an increased amount of vegetable
fat and protein sources in fish feed. The fishs
ability to metabolise and, in turn, deposit fat de-
pends on the complex interaction of many fac-
tors, among them life span, water temperature,
and the types of fat and protein sources used in
the feed. In most cases, fat is a less expensive
energy source than protein, and the goal is to
optimise fat metabolism in fish, so that protein
can be saved for muscle growth. An unwanted
effect of a high fat level in the feed is increased
deposition of fat. Until now, little has been
known about the function of fat tissue in fish,
even though it was proven relatively recently
that fat tissue has great significance for the reg-
ulation of energy metabolism in mammals. Fat
tissue produces several important regulatory
factors such as leptine, PPARy, adiponection,
etc. To increase the understanding of the devel-
opment of fat tissue in fish and how it is affected
by different fatty acids in the feed, Akvaforsk
has established a method for cultivating fat cells
from fish in culture (Figure 4).
Several experiments on the addition of both nat-
ural and synthesised bioactive fatty acids to
salmon feed have been done to see whether
these additives will lead to increased oxidation
of fatty acids, thus the desired effect of salmon
with a lower fat content. The addition of tetra-
decyl-thio-acetic acid (TTA) in the salmon feed
resulted in increased mitochondrial oxidation of
fatty acids in both the liver and red muscle as
well as a moderate reduction of fat in the mus-
cle. By replacing up to 70 per cent of the fish
(capelin) oil with rapeseed oil in a salmon feed,
the -oxidation capacity in white muscle was
increased compared to fish whose only oil
source was fish oil.
It has been proven that water temperature can af-
fect both the fat deposition level and the fatty
acid -oxidation capacity in fish. In an experi-
ment in which the oil source was either fish oil or
soybean oil, it was found, among other things,
that low water temperature resulted in increased
deposition of fat in both the intestine and the liv-
er. Low water temperature also seems to induce
the peroxysomal metabolism of fat in fish.
Composition of fatty acids in the fish,
and product quality
The composition of fatty acids in the fish large-
ly mirrors the composition of fatty acids in the
feed, which is significant for the evaluation of
product quality. When fish oil is replaced with
vegetable oil in salmon, the level of long-chain,
healthy, favourable n-3 fatty acids is reduced in
the fish muscle. To avoid too high a loss of these
healthy fatty acids, much research has been
done on stimulating the capacity of Atlantic
salmon to produce the healthy n-3fatty acids,
EPA and DHA from 18:3n-3 from plant oils.
Among other things, it has been shown that the
addition of both soybean oil and rapeseed oil to
salmon feed gives increased 5-desaturase activ-
ity, which in turn leads to increased formation
of DHA. Even though it is possible to stimulate
the capacity of Atlantic salmon to produce EPA
and DHA by adding plant oils to the feed, the
increase is not sufficient to maintain the level of
healthy n-3 fatty acids in the fish muscle. Partial
Theme: Feed, Nutrition, Feeding 211
 reestablishment of EPA and DHA levels in the
fish muscle can be achieved by implementing a
washout period before slaughter, during which
plant oil in the feed is replaced by an EPA/
DHA-rich fish oil. Like the addition of fatty
acids to the feed, water temperature may also
affect the capacity of Atlantic salmon to meta-
bolise fatty acids.
It has been tested whether the flesh of Atlantic
salmon that had been fed a diet rich in vegetable
fatty acids had less liquid-binding properties
than the flesh of Atlantic salmon fed a diet rich
in fish oil. In studies with both small (approx. 1
kg) and large (approx. 3 kg) Atlantic salmon, no
substantial loss of liquid from the muscle after
ice storage or smoking was found. However,
feeding fish with vegetable oil did seem to af-
fect fillet colour and lipid oxidation. Fish fed
with fish oil have shown somewhat better
colouration than fish fed with vegetable oil. On
the other hand, feeds containing vegetable oil
did seem to have a positive effect on lipid
oxidation, which can occur during frozen stor-
age, because the fish fed with vegetable oil have
a reduced content of n-3 fatty acids that easily
oxidise during storage. Quality parameters such
as fillet gaping, texture and consumer prefer-
ence were not affected.
Nutrition and health in fish
In the growing aquaculture industry there are al-
ways new challenges concerning fish health and
fish diseases. In Norway, effective vaccines
have been developed for many diseases affect-
ing Atlantic salmon, thus limiting the spread of
these diseases. Nevertheless, disease and health
problems lead to substantial losses every year.
Aquaculture has been undergoing rapid devel-
opment, and fish growth has increased signifi-
cantly in the past decade. This gives rise to new
212 Aquaculture Research: From Cage to Consumption
questions regarding the health and welfare of
the fish. Several diseases and production-
related disorders have been proven to be pre-
ventable by correct nutrition.
Alternative feed ingredients How do
they affect health?
A large number of experiments have shown that
a relatively high intake of vegetable oil under
normal production conditions does not affect
the growth and feed utilisation of salmon to any
notable degree. However, much research re-
mains to be done on the amounts and length of
time fish can be fed various types of vegetable
oil without negative consequences on fish
health, especially under suboptimal conditions
such as fluctuating farming conditions, stress
and poor health. Experiments with Atlantic
salmon have shown that substantial changes in
the composition of fatty acids can affect the im-
mune system by affecting phagocytotic capa-
city of macrophages and production of signal
substances (eicosanoids). These effects were
especially clear at low water temperatures
(5 C). It has also been proven that rapidly
growing salmon that were only given vegetable
oil in the feed (with fish meal as the protein
source) had a higher frequency of cataract.
A high fat level in the blood has been proven to
be a serious risk factor for the development of
lifestyle-related diseases in humans, for exam-
ple cardiovascular diseases and Type 2 diabetes.
It is uncertain whether this is a risk factor in fish
as well. Experiments have shown that the lipo-
protein secretion from liver cells is significantly
higher in salmon fed with vegetable oil than in
salmon fed with fish oil. Little is known about
whether the composition of fatty acids in the
feed can affect the development of cardiovascu-
lar diseases in salmon. One experiment investi-
gated the short-term effects of replacing marine
oils with vegetable oils on atherosclerosis in At-
lantic salmon. Looking at the results of this ex-
periment, the possibility that changes in the
composition of fatty acids in fish feed can affect
the development of such disorders cannot be
ruled out, but more research is needed before
any conclusions can be drawn. It is still impor-
tant to keep this danger in mind when replacing
increasing amounts of the fat in salmon feed
with vegetable oil, which have a lower content
of the healthy n-3 fatty acids.
Bone deformities
Deformities are a problem in aquaculture, fin-
ancially and ethically. Nutrition, especially vi-
tamins and minerals, seems to have significance
for the development of deformities. Results
from experiments indicate that a deficiency of
phosphorus and zinc in periods of rapid growth
can affect bone mineralisation in Atlantic salm-
on, and thus give a higher frequency of deformi-
ties. If this deficiency is present already at start-
feeding, and becomes chronic, deformities will
occur even if there is only a moderate defi-
ciency of these minerals. Once needs for phos-
phorus and zinc are met, the deformities will be
mineralised and become permanent. Vitamin A
is probably a contributing factor to the develop-
ment of spinal deformities in several species.
However, in an experiment with salmon eggs no
relationship was found between vitamin A sta-
tus in eggs and the development of deformities
in the growing embryo. Too much vitamin A in
the feed has, in other experiments, proven to
lead to abnormal development of the vertebrae
in several species, among them salmon. Vita-
min C deficiency in salmon fry gives classic
vertebrae deformities in the form of scoliosis
and lordosis. Vitamin C deficiency over a short-
er period of time in larger fish may also lead to
deformities; this has been suggested as the
cause of deformities in the jaw area.
Cataract
Cataract is an eye disorder which appears peri-
odically in and has a high impact on Atlantic
salmon. Several nutritional factors seem to af-
fect the development of cataract. Both high fat
levels in the feed and high concentrations of
pro-oxidants such as iron, zinc and manganese
have been proven to increase the frequency of
irreversible cataract in salmon. Meanwhile,
antioxidants (astaxanthin and vitamin C) have
been proven to reduce the frequency. This indi-
cates that a balanced diet with pro- and anti-
oxidants will have a preventive effect against
cataract. The content of fat in the feed also af-
fects the growth rate, and it is possible that the
effect of fat content on frequency of cataract
may be related to fast growth. There is also an
increased potential for oxidation in high-fat
feeds because of a higher content of polyunsat-
urated fatty acids.
A deficiency of the amino acids methionine and
tryptophan as a factor in the development of cata-
ract has long been a topic of discussion in aquac-
ulture research. However, in recent years,
particular focus has been placed on histidine.
Several studies have shown that the optimal level
of histidine in the feed is often far higher than the
assumed requirement in order to prevent cataract.
In an experiment, Atlantic salmon fed a com-
mercial feed to which histidine, iron and zinc
had been added showed a lower frequency of
cataract than Atlantic salmon fed a commercial
control feed. It has since been proven that histi-
dine alone can reduce the development of cata-
ract, and very high histidine concentrations in
the lens of the fish have been identified, which
might explain the relationship between dietary
histidine and the development of cataract. The
experiments that have concluded that iron can
increase the frequency of cataract were most
Theme: Feed, Nutrition, Feeding 213
 likely done with feed that contained a signi-
ficantly higher amount of added iron. Blood
meal in the feed has been proven to reduce the
frequency of cataract for pre-smolt, smolt and
adult fish in the sea. This is related to the natu-
rally high content of histidine in the blood meal.
IPN
Infectious pancreatic necrosis (IPN) is a conta-
gious viral disease that attacks farmed fish and
can result in serious economic losses for fish
farmers. Experiments have been done to identi-
fy additives in the feed that can strengthen the
immune system. Among other things, it has
been found that feed with added nucleotide re-
duced IPN mortality in rainbow trout; however,
there were only a small number of fish used in
the experiment. Several experiments indicate
that increased access to energy for smolt in the
weeks after transfer to seawater, when the need
for energy is high, results in increased survival
in the case of natural IPN outbreaks after sea-
water transfer.
Future challenges
To maintain the growth of the aquaculture in-
dustry in Norway, it is important to continually
generate new knowledge that can be utilised to
formulate optimal, cost-effective fish feeds that
will promote normal growth, good health and
high quality. There is still a great lack of know-
ledge in nutritional biology regarding salmon,
trout, cod and Atlantic halibut. As the aquacul-
ture industry develops, feeds will have to be for-
mulated much more specifically in accordance
with the environment, life cycles of the various
species and critical life stages. A continued fo-
cus on food safety, quality and fish health is also
vital to future economic growth and to main-
taining the good reputation of the Norwegian
aquaculture industry. Molecular biology and
214 Aquaculture Research: From Cage to Consumption
gene technology offer the potential for enhanc-
ing the fundamental understanding of fish nutri-
tion, the interaction between nutrients, and the
interaction between nutrients and various con-
ditions in aquaculture. These new methodolo-
gies also offer significant possibilities for
developing practical, easy-to-use tests for both
raw materials and fish. To ensure that the
knowledge developed in nutritional biology is
utilised on a broad basis, it is necessary to
strengthen the ties between the Research Coun-
cils research initiatives and research being car-
ried out in the aquaculture industry, both
nationally and internationally.
Acknowledgements
The authors would like to thank Sjur E. Svik
for translating this chapter from Norwegian to
English.
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Atlantic salmon Hepatocytes. Lipids 40, 477486.
Waagb, R., Hamre, K., Bjerks, E., Berge, R., Wathne,
E., Lie, . and Torstensen, B., 2003. Cataract for-
mation in Atlantic salmon, Salmo salar L., smolt
relative to dietary pro- and antioxidants and lipid
level. Journal of Fish Diseases 26, 213229.
rnsrud, R., Graff, I.E., Hie, S., Totland, G.K. and
Hemre, G.-I., 2002. Hypervitaminosis A in first-
feeding fry of the Atlantic salmon, Salmo salar L.
Aquaculture Nutrition 8, 713.
Kristin Hamre1), Ivar Rnnestad2), Jos Rainuzzo3), Yoav Barr4) and Torstein Harboe5)

1) NIFES (National Institute of Nutrition and Seafood Research), 2) University of Bergen,

3) The SINTEF Group, 4) Akvaforsk The Institute of Aquaculture Research, 5) Institute of Marine Research

Nutritional Aspects Marine Fish Larvae

In the course of the most recent programme period we have determined the nu-
trient content of the live feed organisms, rotifers and Artemia, which are used in
the intensive aquaculture of marine fish larvae. These were subsequently com-
pared with the nutrient content of copepods, which are the natural prey of these
larvae. Rotifers and Artemia may be particularly low in omega-3 fatty acids and
have low levels of specific vitamins and minerals. This may partly explain why
fish larvae in intensive farming grow more slowly and suffer a higher proportion
of developmental anomalies than larvae that are kept under natural conditions
and feed on copepods. We can enrich the prey species with a number of the nu-
trients that they possess in insufficient amounts, but it is still difficult to obtain a
satisfactory combination of fatty acids in Artemia. One of the goals of nutritional
research on marine fish larvae is to generate knowledge that will enable us to re-
place live feed organisms with formulated feed as early as possible in the larval
stage. However, such a development is held back by a number of factors, such as
the fact that the tiny feed particles have an extremely high rate of leaching of
water-soluble nutrients. Fat and protein, in the form in which they are normally
given to larger fish, are difficult for the larvae to digest, and thus have poor avail-
ability to their absorption processes, since their digestive tract is not fully deve-
loped. The mapping of larval development, for example the capacity for
digestion and absorption of nutrients, should take place in parallel with the
development of feeds and feedstuff raw materials with good technical character-
istics and high nutrient availability.

Thematic area: Feed, Nutrition, Feeding 217

 Cod and halibut are the main marine fish spe-
cies with altricial larvae (i.e. larvae that lack a
stomach) that are currently being farmed in
Norway. Halibut have been farmed continu-
ously since the mid-1980s, while the most re-
cent efforts using cod began around 2000. Work
on larval nutrition has been divided between
studies of live feed aimed at characterising
optimal nutritional content, the development of
formulated feeds, and research on larval biolo-
gy, nutritional physiology and nutritional re-
quirements. Developments in raising larvae
have led to techniques of stocking in semi-
enclosed bays being abandoned and being
replaced by the adoption of more intensive
methods that employ indoor tanks and feeding
with rotifers and Artemia. This has brought a
number of challenges with respect to lower
growth rates and higher incidences of develop-
mental anomalies and deformities in juveniles,
conditions that appear to be partly due to subop-
timal nutrition.
Cod larvae raised in intensive aquaculture grow
much more slowly than cod produced in semi-
enclosed bays. (Ten per cent per day compared
with up to 30 per cent per day.) There have also
been problems of deformities in cod juveniles
produced under intensive farming conditions;
such problems are extremely rare in cod pro-
145x100//Kap13-bilde01.eps
Rotifers. Photo: The Institute of Marine Research
218 Aquaculture Research: From Cage to Consumption
duced in semi-enclosed bays. The percentage of
deformities is currently declining, but there is
still room for improvement. There are no reli-
able figures regarding different rates of growth
in halibut, but intensively produced juveniles
often display faulty pigmentation and incom-
plete eye migration, conditions that are obvi-
ously related to nutrition. One important goal of
research on larval nutrition should be to gener-
ate the knowledge that will enable us to raise
growth and survival rates and lower deformity
rates in intensive aquaculture to levels equal to
those found in semi-enclosed bay conditions.
Results and status
Nutritional content of live feed
In the course of this and the previous pro-
gramme period we have gained a fairly com-
plete understanding of the basic nutritional
content of several live feed organisms. We still
do not know the nutritional requirements of ma-
rine fish larvae, but the nutritional content of
copepods and the requirements of larger fish
can be used as indicators of whether rotifers and
Artemia contain adequate levels of particular
nutrients.
Data from the literature concerning the protein
content of live feeds are highly variable (e.g.
2461 per cent of dry weight in rotifers), and
seem to primarily depend on the analytical
methods employed in individual studies. Differ-
ent colorimetric methods (e.g. Lowry) have a
tendency to underestimate protein because they
are intended only for analyses of water-soluble
proteins, while live feed organisms also contain
proteins that are insoluble in water. Protein can
also be measured by first measuring the nitro-
gen content of the sample and then multiplying
it by a particular factor to indicate nitrogen con-
tent. A factor of 6.25 is usually employed, but in
fact, each individual organism has its own fac-
tor, which needs to be determined by measuring
total amino acid content. This has recently been
done for rotifers, which proved to have a factor
of 4.46 and a protein content of about 40 per
cent of dry weight, irrespective of the protein
content of their diet. The proportion of water-
soluble protein was about 50 per cent, and the
amount of free amino acids was between 5 and
14 per cent of total protein. Changes in diet pro-
duced only minor changes in the amino acid
composition of the rotifers. As was to be ex-
pected, there were stable levels of protein and
amino acids in rotifers because such factors are
largely genetically determined. Such work has
not been done on Artemia or copepods, so the
precise levels of protein in these feed organisms
are still not known. By and large, the amino acid
composition of total protein in Artemia and roti-
fers is satisfactory when we compare it with that
in copepods, the amino acid requirements of
larger fish and the composition of the halibut
larva body.
The lipid content of the stages of copepods that
are eaten by marine fish larvae is 616 per cent
of dry weight. In enriched Artemia it can vary
from 22 to more than 30 per cent. Rotifers that
have not been fat-enriched have a lipid content
similar to that of copepods, but after enrichment
it often lies between 20 and 30 per cent. The fat-
ty acid composition of rotifers reflects that of
their feed, whether it is ingested during cultiva-
tion or in short-term enrichment, and is there-
fore easy to manipulate. Unenriched Artemia do
not contain DHA (docosahexaenoic acid,
22:6n-3), have a low content of EPA (eicosap-
entaenoic acid, 20:5n-3) and a high content of
ARA (arachidonic acid, 20:4n-6), while cope-
pods contain 2040 per cent DHA, 1520 per
cent EPA and <1 per cent ARA. Even if we en-
rich Artemia with marine fats, it is difficult to
obtain the same fatty acid composition as that of
copepods. This is often explained by reference
to Artemias self-metabolism.
Artemia have low levels of thiamine, vitamin A,
iodine and zinc. However, halibut are capable of
converting canthaxanthin, of which there are
high levels in Artemia, to vitamin A, and we es-
timate that feeds need to contain about 500 mg/
kg astaxanthin or canthaxanthin if they are to
meet the vitamin A requirements of halibut lar-
vae. Cod larvae, which also feed on copepods
that lack vitamin A, probably have a similar
mechanism for converting carotenoids. In roti-
fers, levels of vitamin A, manganese and sele-
nium are lower than in copepods and are also
lower than the requirements of larger fish. Roti-
fers contain insufficient carotenoids to enable
these to meet larval vitamin A requirements. The
following nutrients may be present in lower
amounts than in copepods, but equal to or higher
than the requirements of larger fish: vitamin C,
thiamine, vitamin E, iodine, copper and phos-
phorus.
Nutritional requirements of marine fish
larvae
We know little about the real nutritional require-
ments of cod and halibut larvae. These needs may
differ from those of larger fish, since fish larvae
are undergoing metamorphosis at the same time
as they are growing extremely rapidly. Neverthe-
less, their requirements may not be as high as the
level of nutrients in copepods might suggest. The
task of determining nutritional requirements will
be simplified when we can design classical nutri-
tional studies for marine larvae in order to im-
prove our knowledge in this field.
However, some studies of the fatty acid compo-
sition of larvae have been carried out. It was
originally thought that feeds for fish larvae
Thematic area: Feed, Nutrition, Feeding 219
 should contain a high level of DHA and have a
high DHA/EPA ratio, on the basis of the fatty
acid composition of cod roe. Studies of pigmen-
tation in flatfish have since shown that the ratio
of DHA to EPA is not so important, but that a
high level of ARA and thus a low EPA/ARA
ratio has a dramatic negative effect. However, it
is important to maintain a high level of DHA in
order to ensure that this essential fatty acid is
available for the development of the vision and
the nervous system. Just what the minimum
level of content of n-3 fatty acids might be for
cod and halibut larvae is still unknown, but
>20 per cent DHA, > 10 per cent EPA and
< 2 per cent ARA should provide a good feed as
far as fatty acids are concerned. This can easily
be achieved with rotifers, although enriching
Artemia is rather more difficult.
Protein requirements are relatively unknown,
but it has been shown that only a small propor-
tion of the protein in rotifers is utilised for
growth in rapidly growing turbot larvae. The
larvae presumably compensate for their low
protein utilisation by eating more. This must
also be considered in the context of the need of
fish larvae for an energy substrate, since fish
larvae obtain between 60 and 90 per cent of
their energy from amino acid metabolism. Free
amino acids are easily available to fish larvae
because they do not need to be digested before
they are absorbed. We also assume that water-
soluble protein is more available than insoluble
protein, because it is more available for diges-
tion than insoluble protein.
It has been shown that 400500 mg/kg vitamin
C, which is found in enriched Artemia, and also
often in rotifers, will be sufficient to meet the
needs of marine fish larvae.
220 Aquaculture Research: From Cage to Consumption
Enrichment of rotifers and Artemia with
n-3 PUFA, free amino acids, lipid classes
and micronutrients
There are two traditional methods of enriching
rotifers. In the first method, which is known as
long-term enrichment, we combine growth feed-
ing with an enrichment of nutrients in which the
enrichment process takes place throughout the
production phase. The other method, known as
short-term enrichment, consists of modifying
the nutrient content via a brief final feeding
phase. In this case we usually employ a fat emul-
sion to which other nutrients may be added.
There are several commercial sources of feed and
enrichment additives that yield rotifers with an im-
proved fatty acid profile; e.g. yeast and marine oil
or emulsions, algal concentrates and products that
consist of dehydrated cells of heterotrophic single-
celled organisms (Figure 1, Schizochytrium sp.,
e.g. Algamac, Rotimac, or Crypthecodinium
cohnii, Aquagrow Advantage).
To enrich Artemia with polyunsaturated n-3
fatty acids (PUFA), we usually utilise a 24-hour
enrichment period, employing more or less the
same products as those used for rotifers. SIN-
TEFs experience is that MAROL E is an ef-
ficient means of raising the DHA content of
DHA EPA
50
40
mg/g DW
30
20
10
0
START 0.2 0.4 0.8 1.2
145x100//Kap13-fig01.eps
Figure 1. DHA and EPA in rotifers enriched with different
doses (g/ind.) of Thraustochytride cells after 24 h
enrichment at 20 C.

145x100//Kap13-bilde02.eps
Newly hatched Artemia. Photo: NIFES
Artemia (i.e. 42 mg/g, 25 per cent of total fatty
acids; total lipids 25.5 per cent and DHA/EPA
ratio 2.3). The experience of NIFES and the In-
stitute of Marine Research, however, is that tar-
get-oriented Artemia enrichment is extremely
difficult to reproduce. The Pronova oil TG
5010, which has a fatty acid composition simi-
lar to that of MAROL E, can give Artemia a
DHA content that ranges between 10 and 25 per
cent, even when identical emulsification and
enrichment methods are employed. Commer-
cial fry producers also experience wide varia-
tions in the fatty acid composition of their
Artemia, even when they use the same enrich-
ment media. In the literature, values of DHA in
Artemia enriched using emulsions tend to be
between 6 and 10 per cent of total fatty acids.
However, the adoption of heterotrophic algae in
recent years has improved both the general fatty
acid profile and the reproducibility of Artemia
quality in both commercial aquaculture and re-
search. This has made faulty pigmentation less
of a problem in halibut fry production than it
used to be.
145x100//Kap13-bilde03.eps
Cod larva fed on rotifers. Photo: The Institute of Marine
Research
The composition of lipid classes in live feeds is
not directly related to the composition of lipid
classes in the emulsions used for enrichment.
Rotifers enriched with an emulsion rich in wax
esters increased their proportion of triglycer-
ides. Artemia also has a tendency to retain its
original lipid class composition even after 12
hours of enrichment with emulsions of different
lipid classes. This is probably because phospho-
lipids and wax esters are rapidly broken down in
the organisms gut, and the fatty acids that are
liberated are incorporated into triglycerides.
However, phospholipid enrichment may have
other positive effects on the nutritional content
of feed organisms, for example by increasing
their content of phosphorus and nitrogen bases
such as choline, ethanolamine and inositol.
A recently developed method of enrichment is
based on intensive short-term boosting. The
method enriches rotifers or Artemia with 15 g
enrichment feed l-1 for 30120 minutes. When
the feed organisms are enriched at a high feed
concentration, the gut is rapidly filled, while the
short enrichment time reduces the amount of
nutrient metabolism. The short enrichment time
Thematic area: Feed, Nutrition, Feeding 221
 also prevents the tank environment from be-
coming unduly contaminated and thus allows
for higher live feed density. This method offers
new possibilities for modifying the nutrient
content of live feed organisms.
This new enrichment method was used in a study
aimed at increasing the phospholipids and free
amino acids content of live feed, using liposome
loaded with free amino acids. A new method has
been developed for the mass production of large
liposomes with diameters of 28 m. These lipo-
somes are based on soy lecithin (Epikuron),
which forms a phospholipid membrane around a
solution of free amino acids (FAA). The loss of
FAA from the liposomes in seawater was 9.2 per
cent in the course of two hours.
A large rise in the phospholipid content of roti-
fers and Artemia that had been enriched with
liposomes by the boosting method has been
demonstrated (Figure 2). The rotifers displayed
an increase of 298 per cent in FAA content and
120
100
mg PL/gr dry weight
80
60
40
20 37.2
0
Standard
enriched
Artemia
145x100//Kap13-fig02.eps
Figure 2. Effects of boosting enrichment with liposomes on the content of polar lipids in rotifers previously enriched with
Algamac 2000 , and on Artemia nauplii previously enriched with DHA-Selco
222 Aquaculture Research: From Cage to Consumption
the increase in individual FAA matched the
concentrations of the same AA in the liposomes
very well. The effect in Artemia was less pro-
nounced but still significant, with an increase of
50 per cent (Figure 3).
A combination of boosting and the use of lipo-
somes for enrichment might provide a good tool
for further studies of nutrition and physiology
in fish larvae. In this regard, we can envisage a
further development whereby marine phospho-
lipids replace soya lecithin. Another aspect that
ought to be investigated concerns how long
phospholipid and FAA levels are maintained in
the live feed organism in the fish larvae feeding
situation, since both of these nutrients undergo
rapid metabolism in the feed organisms.
A number of studies have also been carried out
on enrichment of Artemia and rotifers with
micronutrients. Both Artemia and rotifers can
be sufficiently enriched with thiamine by add-
ing 200 mg thiamine HCl per kg emulsion.
74.8 73.2
30.4
Boosting with Standard Boosting with
liposome 2gr/liter enriched liposome 1gr/liter
30 min 60 min
Rotifer
 60
Rotifers enriched with Algamac 2000
Rotifers enriched with Algamac 2000
+ boosting 1 g 60 min epikuren
50
Individual FAA (nmol mg DM)
-1

40

30

20

10

0
pro
val

ile

ala

cys

aaba
gaba
arg *
tyr

lys *
his *
thr

phe *
met *

ser

gly *

tau *
leu

glu

gln

asn

asp
45x100//Kap13-fig03.eps

Figure 3. Effects of Epikuron-based liposome boosting on FAA content of rotifers. The liposomes were filled with a solution
of amino acids.

Iodine has been used both in its available form
I- (as sodium iodide) and in an oil containing
bound iodine, Lipiodol. Both methods offer
good iodine enrichment of Artemia. We found a
linear increase in iodine concentration in roti-
fers with increasing amounts of Lipiodol added
to the feed of the rotifers. Using Lipiodol for
enrichment is simpler than using water-soluble
compounds, but there is some uncertainty as to
whether Lipiodol is converted to I- in the feed
organisms and/or larvae, thus becoming avail-
able for the synthesis of thyroid hormone.
Enrichment of Artemia and rotifers with vita-
min A is difficult to reproduce, probably be-
cause vitamin A is unstable when it is exposed
to light, oxygen and high temperature in the
enrichment tank, while good results have been
obtained with vitamin C and E enrichment. It is
possible to produce rotifers with graded levels
of certain of these nutrients, opening up the pos-
sibility of studies of nutrition in cod larvae.
Formulated feeds for larvae
One important goal of research on nutrition in
marine larvae is to generate knowledge on
which to base the development of formulated
feeds that can be used as early as possible in the
larval phase. This will reduce the need for live
feed organisms, which are both labour-intensive
and complicated to culture.
In cod farming, current practice is usually to
move to formulated feed at about 25 to 30 days
after hatching, i.e. at the end of the phase of

Thematic area: Feed, Nutrition, Feeding 223

 feeding with rotifers. Artemia are thus little
used at present in cod farming. Halibut are
transferred to formulated feed somewhat later.
Most currently available feeds are microbound,
which is to say that the components of the feed
are bound together by a polymer that forms a
network within the particles. Various types of
capsules based on alginate, chitosan or cross-
linked protein are also available. Experiments
have also been carried out on fat encapsulation.
The most serious bottleneck holding back the de-
velopment of formulated feed for larvae is the
high rate of leaching during administration,
which is due to the tiny particle size, which gives
the particles an extremely high surface/volume
ratio. A test of four commercial feeds and test
feeds showed that 1840 per cent of the protein
leached out in the course of two minutes in sea-
water. The leaching rate of small water-soluble
molecules such as FAA, water-soluble vitamins
and minerals may be as high as 90 per cent in the
course of two minutes. Leaching occurs most
rapidly in microbound feeds, but encapsulated
feeds, whose particles are often treated with wa-
ter, may suffer high rates of leaching during feed
processing, and micronutrients in the end prod-
uct may be completely leached out. Although
commercial feeds also leach, the production of
cod fry with an early transition to formulated
feeds has been successful. Nevertheless, feeds
with lower leaching rates would offer a better
guarantee that the nutritional requirements of the
larvae are met, and probably also result in im-
proved growth and survival rates.
The latest development in formulated feeds is
the production of complex particles, in which
water-soluble nutrients are encapsulated in or-
der to prevent leaching, after which the capsules
are incorporated into larger feed particles. Both
224 Aquaculture Research: From Cage to Consumption
liposomes and lipid spray beads can be used for
this purpose.
With financial support from the Research Coun-
cil of Norway, Ewos Innovation and Maripro
have developed two different formulated feeds
for fish larvae; both production processes have
been patented. Promal, from Ewos, is based on
marine phospholipids that encapsulate water-
soluble nutrients in order to prevent leaching
and supply the larvae with what is believed to be
an optimal combination of fats. This feed is al-
ready available on the market. Minipro, from
Maripro, is a feed whose nutrients are encapsu-
lated with the aid of a polymerising agent.
Digestibility of protein sources used in
formulated feeds for larvae
The digestibility of the source of protein is a de-
cisive factor in determining how efficiently the
dietary protein can be utilised by the fish larvae.
Very little is known about protein digestibility
in fish larvae, and data based on studies of adult
fish are not directly relevant because of differ-
ences in the digestive systems of larvae and
adult fish. Among other factors, the lack of a
stomach at the larval stage means that ingested
protein is neither exposed to the denaturing
conditions imposed by the gastric acids nor pre-
digested by pepsin before entering the mid-gut.
Live feed, which usually contains large
amounts of soluble nitrogen, produces the best
growth and survival rates under farming condi-
tions. Earlier experiments on carp larvae
showed that higher levels of soluble protein in
the feed improved growth and survival rates. It
is possible that the soluble protein is more
readily available to proteases and also that it
may be more easily absorbed through pinocyto-
sis. The soluble protein content of the source of
protein may therefore have an important influ-
ence on the digestibility of the protein.
The results of an in vitro study of digestion
showed that live feed and hydrolysed cod fillet
contained most soluble protein. The study was
based on fractionating soluble and insoluble
components, which were subsequently exposed
to a mixture of alkaline proteases. The protein
under digestion was later sampled for analysis
at various points in time. There was no anterior
gastric component (pepsin under acidic condi-
tions) in the experimental design. These exper-
iments showed that the soluble fraction was
consistently more readily digested than the in-
soluble fraction. Overall digestibility was best
for casein, while live feeds were also high (84
86 per cent) and roe meal was lowest at 48 per
cent. Meal was generally relatively indigestible,
even though its soluble fraction was itself high-
ly digestible.
A preliminary stage of pepsin hydrolysis of cod
fillet produced an increase in the soluble frac-
tion, but did not result in higher overall digest-
ibility in vitro. If increased solubility also
increases leaching from particles of feed, this
could reduce the availability of protein in fish
larvae feeds.
The production process may affect protein di-
gestibility. One study showed that the protein
digestibility of the raw materials was higher
than in the final feed, which had been produced
using a protein encapsulation technique.
Effects of fat quality of feeds on growth
and survival rates of larvae and quality of
juveniles
Lipid quality is characterised by its fatty acid
and lipid class composition. It appears that ma-
rine fish larvae need to ingest part of their fat in
the form of phospholipids if they are to utilise it
optimally, although the optimal levels of
phospholipids for cod and halibut larvae are not
known. Phospholipids must therefore be added
to formulated feeds, and it is an open question
whether live feeds, which contain phospho-
lipids in the form of cell membranes, possess
them in sufficient amount. As mentioned above,
it is difficult to enrich live feed with phospho-
lipids.
Phospholipids in microdiets have been shown to
improve growth and survival rates in cod larvae
when the feed is used in a dietary regime to-
gether with rotifers until around 26 days after
hatching. Larvae fed microdiets containing 40
per cent phospholipids had better survival rates
than those on microdiets that contained either
30 or 50 per cent phospholipids. Phospholipids
from marine sources resulted in better growth
and survival rates than soya lecithin.
Marine fish larvae have an incompletely devel-
oped gut. As in the case of protein, it may be
supposed that the enzymes that digest fat have a
limited capacity, and that partially digested fat
in the form of diacylglycerol (DAG) and
monoacylglycerol (MAG) will be more avail-
able to fish larvae than triacylglycerol (TAG),
which is the form of fat usually added to feed.
A tube feeding experiment, in which individual
halibut larvae were fed radioisotope- labelled
TAG, DAG, MAG or phospholipids, showed a
rate of uptake of around 90 per cent for MAG
and 20 per cent for TAG. DAG and phospho-
lipid uptake rates were around 4060 per cent.
These results show that the utilisation of fat
added to formulated feeds is capable of being
considerably improved. We also attempted to
enrich Artemia with MAG, DAG and phospho-
lipids, but this produced only insignificant
changes in lipid class composition in Artemia, a
Thematic area: Feed, Nutrition, Feeding 225
 finding that has been confirmed by the results of
other studies.
Digestive tract development in marine
fish larvae
In marine fish, the digestive tract gradually de-
velops from a short, straight, simple and histo-
logically undifferentiated tube, often closed at
both the mouth and anal ends in yolk-sac larvae,
to a segmented tract in juvenile fish. The first
rapid change takes place just ahead of onset of
exogenous feeding, when the digestive tract dif-
ferentiates into three sections: the foregut, mid-
gut and hindgut, which are histologically and
functionally different from each other. At this
stage, gut length is often less than 50 per cent of
body length. The individual segments of the gut
are separated by muscular sphincters or valves.
With few exceptions, the gut is coiled into a
loop before start-feeding. In the subsequent
phase of development the surface of the gut in-
creases in area (thicker gut epithelium, larger
microvilli). During metamorphosis the foregut
differentiates into a functional stomach cavity
with acid and pepsin secretion capability. Si-
multaneously, the pyloric appendices also de-
velop; these increase the absorptive area of the
digestive tract and are important for protein and
fat absorption. Other changes in the gut take
place after metamorphosis; these changes in-
clude an increase in its relative length. In cod,
metamorphosis is less well-defined than in oth-
er species and the development of the stomach
is very slow.
The liver, pancreas and gall bladder with their
outlets form as early as the egg and yolk-sac
stage, and are assumed to be functional by the
time that the yolk-sac has been absorbed. As
mentioned earlier, fish larvae lack a morpho-
logically differentiated and functional stomach
at the time of start-feeding. This means that the
226 Aquaculture Research: From Cage to Consumption
larvae lack a food reservoir, and that the amount
of food entering into the gut is regulated only by
ingestion rates. Nor do fish larvae have the abil-
ity to release pepsinogen and HCl.
Digestive enzymes
Most of the proteolytic enzymes are produced
in the pancreas and are released into the diges-
tive tract. Trypsin levels rise throughout the
yolk-sac stage, but after the yolk has been ab-
sorbed, they fall rapidly before rising again in
older larvae. The fall in trypsin after the yolk-
sac has been used up can be explained by inad-
equate rates of new synthesis or possibly hun-
ger-induced intracellular breakdown of the
enzyme in the pancreas. Absolute levels of
trypsin vary widely between different groups of
larvae. It is still uncertain whether extracellular
digestion based on pancreatic enzymes is suffi-
cient to hydrolyse proteins rapidly enough.
Brush-border membrane (BBM) enzymes are
located in the gut cell membrane that faces the
gut lumen. These enzymes hydrolyse peptides,
which are the result of cleaving by pancreatic
proteases, into free amino acids and peptides
that are small enough to be absorbed. In halibut
the activity of the BBM enzyme leucine amino-
peptidase (LAP) is low and stable until about
day 30 after start-feeding, whereupon it rises
rapidly to its maximum level at 60 days after
start-feeding, only to fall somewhat again. Cod
larvae have a stable level of LAP activity
throughout the start-feeding period, apart from
a peak in activity around day 35 after start-
feeding.
Mechanisms of absorption
Several quantitative studies of the ability of the
gut to digest and absorb nutrients have been
performed in recent years. These studies have
focused on the absorption kinetics of isotope-
labelled whole protein and amino acids (see be-
145x100//Kap13-fig04.eps
Figure 4. Distribution of CCK-producing cells in the gut of fish larvae (based on Kamisaka, 2005).
low). As yet, few details of the absorption pro-
cess are known, and efforts to characterise the
molecular basis of amino acid and peptide
transport in the digestive tract have just begun.
The gene for the peptide transporter in the cod
gut has been cloned, and preliminary studies
suggest that this gene is expressed from start-
feeding onwards. To date, no sequences for
amino acid transporters have been published,
but two of the transporters for basic amino acids
have been partially characterised.
Hormones
Digestion is a complex process, which we as-
sume is closely controlled in larvae in order to
optimise the uptake of nutrients in the natural
environment. In adult fish, peptide hormones
have been shown to play a leading role in regu-
lating gut movement and enzyme secretion.
Cholecystokinin (CCK) plays a central role in
regulating the liberation of bile and enzymes
from the pancreas, as well as in influencing gut
peristalsis and appetite control. The amino acid
sequence of CCK is extremely conservative
throughout the vertebrates. Two types have
been demonstrated in teleosts: fish CCK1 and
fish CCK2, and we find variations in the sixth
position from the C-terminal end.
The teleosts are a complex group, and we have
found distinct differences between patterns of
distribution and the development of CCK-pro-
ducing cells at the larval stage (Figure 4). In lar-
vae that develop from pelagic eggs and which
Thematic area: Feed, Nutrition, Feeding 227
 have a coiled gut when they begin to eat (e.g.
halibut and tuna), it is difficult to demonstrate
the presence of CCK-producing cells in the gut
until some time after the larvae have started
feeding. In such species, the CCK-producing
cells initially develop in the area of the appendi-
ces and the forepart of the midgut, and are never
found in the rear two-thirds of the gut. In view
of the leading role played by CCK in regulating
gut functions, we may therefore ask how well
the digestive process is controlled during the
start-feeding phase. In larvae with a straight gut,
such as herring and ayu, CCK-producing cells
are present ahead of start-feeding and through-
out the whole length of the gut. A point of inter-
est in this connection is that such larvae are
easier to start-feed. We also find CCK-produc-
ing cells in the brain, and during the early stag-
es, the amount of CCK in the nervous system is
predominant.
Preliminary studies of herring larvae indicate
that CCK is involved in the secretion of trypsin.
One study showed that the presence of proteins
in the digestive system led to higher levels of
CCK in larvae, while the presence of FAA did
not have the same effect. Higher levels of CCK
correlated with increased trypsin activity in the
larvae. In this study, the gut was also supplied
with saline in such amounts as to overfill it. This
did not lead to changes in the level of CCK, al-
though trypsin activity increased after a while.
This indicates that distension of the gut wall via
stretch receptors is not a triggering factor in the
CCK-based regulation of trypsin release.
A higher percentage of added protein hydroly-
sate in the diet of sea bass larvae lowered
trypsin secretion and CCK levels. Taken togeth-
er, these data indicate that dietary protein con-
tent, in combination with protein and peptide
228 Aquaculture Research: From Cage to Consumption
chain lengths and intraluminal proteolytic ac-
tivity, regulates CCK levels.
Protein digestion, absorption and
metabolism
The results of in vivo experiments on fish larvae
based on tube feeding with radio-labelled amino
acids show that FAA are absorbed two to five
times as fast as whole protein (Figure 5), depend-
ing on the type of protein concerned, species and
developmental stage. Peptides appear to be ab-
sorbed somewhat more slowly than FAA. So far,
our experiments on halibut, herring and Senegal
sole suggest that both essential and non-essential
amino acids are absorbed rapidly and efficiently
if they are given in free form. Even if FAA are in-
troduced into the gut in extremely high concen-
trations, losses of intact amino acids to the
surrounding water seems to be low.
Recent data support earlier assumptions that the
ability of larvae to digest protein improves after
metamorphosis (Figure 6). In halibut, about 30
per cent of a protein administered via a tube was
absorbed at the larval stage, while after meta-
morphosis, absorption efficiency had risen to 60
per cent.
About 60 per cent of the protein was also ab-
sorbed at the larval stage if it had been prehy-
drolysed with pepsin. It was also demonstrated
that the capacity to digest protein decreases as
the amount of protein in the gut increases. This
may be related both to digestive capacity and to
the evacuation rate of the gut.
Essential amino acids are incorporated into tis-
sue proteins to a greater extent than non-essen-
tial amino acids (8090 per cent as against 20
30 per cent). Non-essential amino acids tend to
be used more as a substrate in energy metabo-
lism. This indicates that fish larvae can deal

145x100//Kap13-fig05.eps
Figure 5. Absorption of protein at different degrees of
hydrolysis by halibut larvae (based on Tonheim et al,
2005).
with and regulate amino acid metabolism to a
greater extent than was previously believed.
Amino acid retention can be improved if the
amino acid profile of the feed is improved by
the addition of peptides. Studies have shown
that lipid absorption has little influence on ami-
no acid absorption. Methods for tagging amino
acids in Artemia (largely protein) with isotopes
have also been developed, allowing the rate of
amino acid uptake, transfer and metabolism in
145x100//Kap13-fig06.eps
Figure 6. Comparison of the absorption of intact and hydrolysed protein in halibut larvae and juveniles (Tonheim et al, 2005).
tissue to be followed. In larvae of Senegal sole,
about 30 per cent of the amino acids in Artemia
were taken up and transferred to tissue within
an hour of being consumed. In juveniles that
had developed a stomach, around 55 per cent of
the amino acids were transferred within the
same period of time. This shows that the diges-
tion of live feed is more efficient when it is pre-
digested in a functional stomach, even though
the difference is less than might have been ex-
pected.
Effects of hydrolysed protein in the feed
on cod and halibut larvae
Cod larvae fed on formulated feed have higher
survival rates when some of the protein in the
feed is given in the form of hydrolysate. Exper-
iments have been done in which as much as 40
per cent of the protein was given as pepsin hy-
drolysate, and survival rates rose in line with the
proportion of hydrolysate. In halibut, on the
other hand, a gradual decline in survival rates
was noted as the proportion of pepsin hydroly-
sate in the feed protein was raised to 45 per cent.
These results are not in agreement with the ef-
fect of hydrolysis on the uptake of tube-admin-
istered protein by halibut larvae (Figures 5 and
6), but they can be explained by the leaching of
Thematic area: Feed, Nutrition, Feeding 229

145x100//Kap13-bilde04.eps
Atlantic halibut juvenile. Photo: ystein Sle, University of
Bergen
water-soluble protein from feeds that contain a
high proportion of hydrolysate. Halibut are
slow to tackle feed in the water, and the feed
particles remain suspended for a long time be-
fore they are consumed. Cod, on the other hand,
are very rapid feeders, with the result that there
is less leaching to the water. Furthermore, the
protein requirements of halibut are probably
higher than those of cod (see below), and pro-
tein leaching is thus more critical for halibut.
Hydrolysed protein had no effect on growth rate
in either species.
Effects of nutrition on metamorphosis in
halibut
A survey of the literature shows that vitamin A,
fatty acid composition and thyroid hormone all
influence pigmentation in flatfish. Iodine is es-
sential for the synthesis of thyroid hormone.
Even though Artemia lack vitamin A, halibut
larvae appear to obtain a sufficiency of vitamin
A through the conversion of canthaxanthin from
Artemia. It is generally agreed that fatty acid
composition affects pigmentation in halibut, a
supposition that is supported by the fact that the
better enrichment methods that have reached
230 Aquaculture Research: From Cage to Consumption
the market recently have improved pigmenta-
tion in commercially farmed halibut juveniles.
We do not know to what extent thyroid hormone
affects pigmentation, but we believe that the
process of pigment cell development involves
interactions among the three above-mentioned
factors, and possibly others as well. We are thus
now in a position to avoid faulty pigmentation,
but we do not know the mechanisms involved in
pigmentation. An understanding of this process
might prevent the reappearance of faulty pig-
mentation in the event of changes in fish farm-
ing procedures.
Incomplete eye migration is still a recurring
problem in halibut farming. The literature on
this problem is sparse, even though the problem
is also found in other flatfish, but some hypo-
theses have been put forward. Thyroid hormone
controls metamorphosis in flatfish, as it does in
other vertebrates. Iodine deficiency may result
in reduced thyroid hormone synthesis. Iodine
levels are 50700 times as high in copepods as
they are in Artemia. Marine organisms usually
obtain the iodine they need from seawater, but
iodine is found as I-, its available form, only in
the photic zone. Fish farms with deep water in-
lets may therefore find themselves pumping in
seawater that is low in available iodine. In con-
junction with a low iodine content in the feed,
this may result in iodine deficiency, reduced
thyroid hormone synthesis and incomplete
metamorphosis. There are also certain indica-
tions that a more intense supply of energy to the
larvae, for example through feeding with on-
grown Artemia (i.e. Artemia that have been fed
on fish meal for four days) may improve eye mi-
gration. Better eye migration has also been
found in a group of larvae whose level of fat was
four times as high as that of the control group.
Juveniles with incomplete eye migration are
often doubly pigmented.
Optimal composition of macronutrients
in weaning feeds for cod and halibut
We carried out a series of experiments on cod
and halibut with the aim of identifying the opti-
mal composition of macronutrients in weaning
feeds and feeds for small juveniles. The protein
requirement of halibut was less than 60 per cent.
A search of the literature and unpublished data
indicated that the requirement was around 58
per cent. Raising fat content from 5 to 25 per-
cent did not increase growth rate. Raising the
proportion of carbohydrates from 0 to 15 per
cent resulted in a strong increase in the liver in-
dex and a rise in liver glycogen concentration.
At the highest carbohydrate levels we also
found glycogen and glucose in other tissues
than the liver. We concluded that feed for hali-
but fry ought to contain less than five per cent
carbohydrate.
The protein requirement for maximal growth of
cod is 41 per cent, and the fat content of the feed
can be varied between 15 and 25 per cent. When
the fat content of the feed in one study was re-
duced to below 15 per cent, cannibalism in-
creased. With more than 25 per cent fat in the
feed, the livers fat storage capacity was a limiting
factor. Cod fry appear to tolerate and even to ben-
efit by carbohydrate levels of up to 15 per cent. A
high level of protein in the feed (>63 per cent) re-
duced growth in 0.21.0-g fry. Our hypothesis is
that this effect is due to late development of the
stomach, and thus limited protein digestion, a hy-
pothesis that is supported by the fact that pepsin
activity in stomachs of cod larvae and juveniles
continues to increase until the fish weigh 5 g.
Conclusions and perspectives
for the future
We now know the basic nutrient content of
Artemia, rotifers and copepods and we know
enough to be able to control the fat composition
of rotifers. We need to continue to study the
question of a reproducible and satisfactory fat
composition in Artemia and to control the con-
tent of potentially rate-limiting micronutrients
in both of these feed organisms. When this has
been done, we will be in a position to design
classical nutrition studies aimed at identifying
the needs of larvae for particular nutrients.
More knowledge in this area will enable us to
improve survival and growth rates and reduce
the incidence of deformities in both cod and
halibut. In both species, it is essential to identify
the causes of faulty development, since a better
understanding of this area will prevent such
anomalies from recurring when farming condi-
tions and possibly also feeds are altered. We
know that malpigmentation in halibut is a func-
tion of nutrition, but just what causes develop-
mental anomalies in cod and impaired eye-
migration in halibut is not known. Where for-
mulated feeds are concerned, it will be impor-
tant to identify means of preventing water-
soluble micronutrients and amino acids, pep-
tides and protein from leaching into the water
column. We also need to examine the potential
for improving the digestibility of protein and fat
sources used in formulated feeds for larvae.
The development of enzymes involved in protein
digestion has been partly characterised. A Cana-
dian group has made important progress on cod in
this area. In halibut, we still lack data on both pan-
creatic enzymes and pepsin. Generally speaking,
extremely little is known about mechanisms of up-
take of whole protein, peptides and amino acids,
how these are modified in the course of develop-
ment, and how they are influenced by the feed
eaten by larvae at different stages of development.
We know little about fat digestion and absorption
in cod and halibut, and it would be useful to start
Thematic area: Feed, Nutrition, Feeding 231
 characterising digestive enzymes, uptake mecha-
nisms and the uptake process itself. We have just
begun to study how digestion in fish larvae is
regulated, and we also still lack sufficient know-
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Mette Srensen1,2), Rune Waagb3) and Rolf Erik Olsen4)

1) Akvaforsk The Institute of Aquaculture Research, 2) Aquaculture Protein Centre,

3) NIFES (National Institute of Nutrition and Seafood Research), 4) Institute of Marine Research

Feed Resources Feed Technology

Aquaculture is the fastest growing food-producing sector in the world. The na-
tional and international aquaculture industry considers the availability of suit-
able feed resources to be one of the greatest challenges to future growth. The
world's marine fish resources are limited, and although a large proportion of
these are used for aquaculture, sustainable alternatives must be sought lower in
the food chain, both in the sea and on land. We have already seen a global short-
age in the supply of marine oils, and there has therefore been a particular focus
on the supply and quality of fat sources in feed for salmonids. Although the fat
sources do not affect the growth of the fish to a great extent, aspects such as well-
being and health, quality of the end product, and not least safety are highly rele-
vant. Research related to feed resources and technology has largely concerned
studies of biological suitability and optimisation of existing production processes.

Thematic area: Feed, Nutrition, Feeding 235

 The marine fish resources that have been used
in the production of fishmeal and oil are at
present very limited and to some extent overex-
ploited. Evaluating the use of other available in-
gredients for feed from the sea and from land
has thus been recognised as essential for further
growth in the aquaculture industry. An investi-
gation into feed resources concluded that there
were many potential future resources applicable
in fish farming under certain conditions. In this
chapter, we will try to summarise the research
in feed resources and feed technology carried
out under the Aquaculture programme. On the
basis of great differences in the challenges and
approaches relating to different feed resources,
we divided this chapter into research on marine
resources, on plant resources, and on micro-
organisms produced in culture. In evaluating
the suitability of raw materials, the industry
must take into account issues other than price
and availability. Documentation and research
must be undertaken to investigate the nutritional
quality of new raw materials, their technical
quality, their safety for fish, the environment
and the consumer, and the logistics involved. It
is also vital to confirm that the feed resources
provide seafood in line with ethical standards
and the consumers needs and demands. The
section on research into feed technology prima-
rily covers alternative production methods for
feed and processing of feed ingredients.
Marine feed resources
By-products and by-catch
One way to increase the availability of marine
ingredients for fish feed is to improve the util-
isation of marine by-products and by-catch. In
the fishing industry, up to 50 per cent may end
up as by-products in the form of trimmings,
heads, viscera, skin, backbones, etc., and na-
tionally this comprises about 600,000 tonnes
236 Aquaculture Research: From Cage to Consumption
per year. Although Norway at present utilises
up to 75 per cent of its by-products, from a glo-
bal perspective there is great room for improve-
ment. Every year large quantities of fish also
disappear as by-catch, especially from the
trawler fleet. This resource has great potential
for utilisation. Further, we know that the annual
production of biomass at lower trophic levels is
large, while only a fraction of this biomass is
used by fish and marine mammals.
One of the greatest challenges involved in the
use of trimmings and by-catch is logistics and
intermediate storage of the products before pro-
duction of feedstuffs or feed. Fermentation and
ensiling are the methods assumed to have the
greatest potential, even though these processes
may change the quality of the feedstuffs. Fish
silage has also been used as raw material for
further processing into fishmeal and fish oil.
The results show that when the quality of the
raw materials is good, it is possible to produce
fish silage products of excellent quality. Poor
quality raw materials, however, result in signif-
icant deterioration in the silage quality, and in
meal and oil produced from the silage. In partic-
ular, rancidification of the fat may cause prob-
lems. Changes in technical properties have also
been observed in extruded feed based on fish-
meal mixed with fish silage. The feed becomes
less expanded and more compact. Although this
will reduce the fat-holding capacity of high-
energy feed, there are advantages in using silage
in fishmeal production. Growth trials on salmon
have shown that feed with up to 20 per cent sil-
age in the production process yields the same
growth, colour and feed conversion as feed
based on LT (low temperature) fishmeal. New
methods for production of feed ingredients
from silage, by-products or by-catch make an
important contribution to improving the
exploitation of marine raw materials. Various
145x100//Kap14-fig01.eps
Meganyctiphanes norvegica (Photo: Line Anfinsen, Institute of Marine Research)
technologies are described in the section on
feed technology.
Krill and plankton
At a lower trophic level, krill and plankton pro-
vide another significant source of marine feed
ingredients. Although we still do not have a
complete overview of biomass and production,
it is assumed that in Northern waters, primarily
the Norwegian Sea and Barents Sea, the volume
amounts to several hundred million tonnes.
Euphasiid and Calanus spp probably have a
production capacity high enough to allow prof-
itable harvesting. There are also strong indica-
tions that only a fraction of this production is
used by fish and mammals in the ocean. Utilisa-
tion of these products could provide raw mate-
rials for the aquaculture industry far into the
future.
Feeding trials with salmon, cod and halibut
given feed based on three krill species and an
amphipod from Northern waters as well as
Antarctic krill have confirmed that krill can re-
place fishmeal as the protein source of at least
4060 per cent of the protein in the feed, with-
out challenging growth, feed conversion or
quality. For higher inclusion levels growth may
be comparable with fishmeal, but the feed con-
version ratio has a tendency to increase, which
means that the fish must eat more feed to
achieve the same growth. The reason for this is
still not clear, but there are strong indications
that the fish get a mild form of diarrhoea, due to
the high levels of chitin in krill meal.
Although krill and amphipods contain the
essential fatty acids EPA (20:5n-3) and DHA
(22:6n-3), they are, with few exceptions, prima-
rily protein sources. A shortage of marine fat
for fish in aquaculture is a major concern in the
coming years. Here, Calanus finmarchicus is a
relevant resource. The biomass of Calanus in
the ocean outside Norway is conservatively es-
Thematic area: Feed, Nutrition, Feeding 237
 a) b)

145x100//Kap14-fig01.eps

Figure 1a. Levels of dioxins (PCDD/F) and dioxin-like PCBs (DLPCB) in farmed salmon fed with feed based on fish oil (FO) or
vegetable oil (VO) during a complete production cycle (Berntssen et al., 2005).
Figure 1b.Correlation between observed levels of dioxins and dioxin-like PCB congeners in commercially farmed salmon,
and predicted levels based on known levels in feed and a simple first-order kinetics model (Berntssen, 2007. Pers. comm.).

timated to constitute several hundred million
tonnes. Depending on the time of year, this
plankton may contain large quantities of fat.
This fat is, however, in the form of wax esters,
and not triglycerides as in fish oils. Since these
may be toxic in large quantities, they are not
suitable for human consumption. If fish can
convert wax esters to healthy fat, use of
Calanus in fish feed could make a significant
contribution of marine fatty acids for human
consumption. These issues are the subject of a
project in which fish oil is replaced with wax
from Calanus finmarchicus in feed for salmon,
cod and halibut. Although the project is in the
start-up phase, we already know that salmon
utilise wax esters about as well as fat from fish
oil.
A challenge with krill as a feedstuff is its high
fluoride content, which in certain species may
be as high as 6 g/kg dry weight. The EUs upper
limit of 150 mg fluoride/kg feedstuff currently
prevents the aquaculture sector from using
these meals as a protein source in fish feed. It is,
however, known that most of the fluoride from
the krill meal in the feed is not absorbed by fish
in seawater, and very low concentrations are de-
posited in the fillet. It therefore remains to as-
sess the implications of elevated dietary
fluoride for the health of the fish. Results from
such trials will improve the scientific basis for
establishing more appropriate limits, making it
possible to use krill meal safely as a future feed
resource. Preliminary indications show that cer-
tain krill meals also contain concentrations of
selected undesirable metals above the upper
limits stipulated in feed legislation. Despite
higher concentrations, feeding trials indicate
that replacing fishmeal with krill meal in fish
feed indeed reduces the levels of these metals in
cod and salmon fillets.
By-products from mussels
The mussel industry in Norway has consider-
able production potential, and by-products from
mussels are a potential feed resource. There is
significant wastage in todays production due to
rejection of small sizes, breakage during pro-
duction, attached barnacles, and the content of
algae toxins, which in certain cases are discov-
ered after harvesting. Controlled disposal of by-
products involves substantial expenses for the

238 Aquaculture Research: From Cage to Consumption

producers. Utilising these products as ingredi-
ents in fish feed would both reduce the costs of
disposal and provide a market for the whole
mollusc. In a research project, the possibility of
using mussels in feed for salmon and cod was
investigated, using mussels both with and with-
out detected diarrhoea toxins. Mussels were
processed through boiling, cleaning and drying,
and then ground to a fine powder. The protein
and fat content, as well as the composition of
amino acids in the mussel meal, were compar-
able with fishmeal. Feeding experiments in-
cluding up to 8 per cent mussel meal in feed for
salmon showed no change in palatability, de-
spite the fact that mussel meal contains some
flavour-promoting free amino acids in higher
concentrations than fishmeal. Short-term trials
with feed supplemented with pure toxins
showed that salmon tolerated toxin levels which
are regarded as threshold values for human con-
sumption, although this affected certain clinical
blood parameters. At these toxin levels, the
growth of the fish was also reduced. This indi-
cates that mussel meal can be used in fish feed,
but meal with toxins should be avoided.
Undesirable compounds in traditional
and new marine raw materials
Feed ingredients are unfortunately also sources
of a number of undesirable substances, which
have drawn attention relative to the production
of safe seafood. Certain marine oils may in-
clude substantial quantities of persistent org-
anic pollutants (POPs), such as dioxins, PCBs
(polychlorinated biphenyls) and brominated
flame retardants. The problem with these com-
pounds is that they are stored in the fat, and bio-
accumulate in the food chain. Thus, oily farmed
fish may retain the dietary undesirables in the
muscle. With the use of plant oils, it has been
shown that the level of dioxins and dioxin-like
PCBs can be reduced by eight to 12 times com-
pared with fish fed diets based on marine oils.
Studies have also been conducted in which as-
similation and excretion rates for 29 different
chemical forms of dioxins, furans, and dioxin-
like PCBs have been estimated. The results of
such trials can be used to predict the end-point
level of POPs in commercially farmed salmon
when the levels in feed are known (Figure 1).
While fish oil is the principal source of many
organic pollutants, fishmeal is the principal
source of undesirable elements, such as arsenic
and mercury. The toxicity of these metals and
the transfer from feed to fish is determined by
the chemical form. In marine feed ingredients,
both arsenic and mercury occur in organic
forms. The organic form of mercury (methyl
mercury) is toxic, while the organic forms of ar-
senic (like arsenobetaine) are not toxic. Experi-
ments have been conducted to evaluate the
dietary upper limit for methyl mercury toxicity,
using early biomarkers specifically selected for
dietary exposure. Increased intestinal cell pro-
liferation, indicated by PCNA immunohis-
tochemistry, was observed in salmon fed with 1
mg methyl mercury per kg of feed for two
months. After four months, reduced cell prolif-
eration and histopathological changes (shorter
and thicker villi) were observed in the intestine
(Figure 2).
Arsenobetaine is known to be non-toxic to ani-
mals and humans. While a great deal is known
about the assimilation and excretion of arseno-
betaine in humans, the mechanism in fish is still
unclear. To study deposition of the compound in
fish, salmon were fed with isotope-marked arse-
nobetaine. The results showed that the com-
pound is distributed throughout the fish, with
the greatest accumulation in the muscle. This is
in contrast to mammals and humans, where ar-
senobetaine is not stored in any organs, but is
Thematic area: Feed, Nutrition, Feeding 239
 a)
145x100//Kap14-fig01.eps
Figure 2. Pronounced increase in cell proliferation, shown immunohistochemically coloured with PCNA, in intestine of
salmon after feeding for two and four months with feed supplemented with methylmercury (Berntssen et al., 2004).
rapidly excreted in the urine. By analysis of to-
tal quantity of arsenic in feed ingredients, feed
and fish fillets, potentially high levels of arseno-
betaine found in marine raw materials could be
misinterpreted as hazardous arsenic. It is there-
fore important to analyse and report the exact
chemical species of the undesirable substances.
Research results within undesirable substances
are very important to the operating conditions
for the aquaculture sector, since the authorities
in the EU and in Norway take such results into
account when the legislation on feedstuffs is to
be revised or new safe upper limits are to be de-
termined.
Feed ingredients from plant
sources
There is currently a general national ban on the
use of slaughter trimmings from land animals in
fish feed due to the fear of spreading bovine
spongiform encephalopathy. The search for al-
ternative protein and fat ingredients for fish
feed has therefore resulted in a strong focus on
the use of plant ingredients, as well as of ingre-
dients based on micro-organisms.
Research on the use of plant and microbial
sources in feed for fish is concentrated on how
240 Aquaculture Research: From Cage to Consumption
b) c)
these affect the commercial value, the nutri-
tional and technical quality of the feed, and the
actual processing of the feed. In addition,
processes which could reduce the content of
anti-nutrients in plant protein sources are being
explored, especially with regard to any negative
effect on the intestine of the fish and potential
implications on feed conversion efficiency,
physiology and well-being.
Plant fat sources in salmon feed
Various plant oils have been used as a substitute
for fish oil in feeding trials with salmon, either
added as pure oils (for example soybean oil,
rapeseed oil or linseed oil) or as oil mixtures.
The main conclusion is that salmon tolerates a
relatively high content of plant oils in the feed,
without any impact on growth or feed utilisation
in salmon of different sizes. The concerns on
the use of plant oils in feed for fish has primar-
ily focused on changing the composition of fat-
ty acids in the fish fillet and how this affects
other quality characteristics of the product. Fish
is traditionally associated with a high content of
healthy long-chain n-3 fatty acids (the fishy
fatty acids EPA and DHA). The level of these
fatty acids is reduced in the fish fillet when the
fish is fed with plant oils, and the level of C18
fatty acids increases. It has therefore been con-
sidered important to focus the research on
health aspects of both the farmed fish and the
consumer in relation to the replacement of ma-
rine oils with plant oils in fish feed, with a spe-
cial focus on effects related to immunology,
lipid oxidation, undesirable substances (diox-
ins) and cardiovascular diseases (the from
fjord to table concept). Research has shown
that changes take place in the cardiovascular
system of salmon corresponding to the develop-
ment of atherosclerosis in humans, and the de-
gree of changes is related to the size or age of
the fish. No clear associations have, however,
been found between the fatty acid composition
in the feed and the degree of cardiovascular
changes in the fish. Although results from on-
going research projects are still being pro-
cessed, the results so far confirm that plant oil
can make up a considerable proportion of the
energy in the feed without affecting the health
of the salmon.
A clinical trial on nutrition in humans showed a
therapeutic effect associated with increasing the
content of marine n-3 fatty acids in the salmon
fillet, so that the daily intake averaged 3 g. De-
spite a lower content of marine n-3 fatty acids in
salmon fed with plant oil, patients eating this
salmon showed a neutral or positive response in
key markers related to the development of car-
diovascular diseases. This means that there are
also factors other than n-3 fatty acids in salmon
fillets that are beneficial in prevention of these
diseases. In the same clinical study, the intake
of environmental pollutants was also studied,
and the results from this study may provide a
contribution to the debate about product man-
agement and risk related to the intake of oily
fish.
Use of plant oils as feedstuffs may introduce
new undesirable substances that usually occur
only in low concentrations in the marine envir-
onment, such as the pesticide endosulfan. The
existing upper limit for endosulfan in fish feed
is considerably lower than for other livestock. It
is based on the naturally low concentrations of
the pesticide in marine feed resources and the
high sensitivity of the fish to water exposure.
Trials are being conducted with the objective of
determining toxic concentrations for endosul-
fan in fish feed.
Plant protein ingredients in feed for
salmon and cod
Plant protein sources comprise a large group of
potential ingredients. Soybeans dominate the
world market because of supply, price and nu-
tritional quality. There are two key challenges
regarding the use of soybean for fish. First, soya
contains indigestible components and anti-
nutrients that are harmful to the fish. It has been
shown that soybean meal in feed for Atlantic
salmon causes inflammation-like reactions in
the posterior intestine, which may be unfavour-
able because of reduced immunological func-
tion of the intestine and reduced feed conver-
sion efficiency. The second major challenge is
related to the use of genetically modified (GM)
soya. Globally, the production of GM plants is
increasing primarily glyphospate tolerant
soya, maize and cotton. The use of GM ingredi-
ents is regulated by Norwegian and EU feed
legislation, with regard to both approved prod-
ucts and mandatory labelling of products that
include GM ingredients. Because of the gener-
ally negative attitude to GM products in public
opinion, the aquaculture sector wishes to use
GM-free ingredients (nGM) in fish feed, and
these currently are more expensive than GM
products. Results from a project which aimed to
study the health risks associated with GM
plants in salmon feed showed that DNA from
GM soya passes unchanged through the intesti-
nal tract. The DNA sequences were not found in
Thematic area: Feed, Nutrition, Feeding 241
 tissue from the intestine, liver, brain or muscle
of the salmon. Another study investigated
whether incorporation of 12 per cent of GM
soya and maize into salmon feed resulted in
measurable changes in the intestinal mucosa in
relation to fish given feed based on GM-free
ingredients and fishmeal. There were no differ-
ences between GM and nGM ingredients, but
feed based on soya induced changes in intest-
inal cells compared with maize and fishmeal-
based feed. The main conclusion of a PhD the-
sis on GM ingredients in salmon feed is that in-
corporation of low levels of GM plant materials
(soya and maize) can be regarded as safe, both
for fish and for consumers. Changes in spleen
size which may be attributable to the use of GM
feed have, however, caused concern, prompting
a cautious attitude to GM feedstuffs for fish un-
til more research has been conducted.
Cod is a relatively new species in the aquacul-
ture context, and several projects have been un-
dertaken to study feed efficiency and product
quality in relation to the choice of ingredients
and chemical composition of cost-effective
feed. Methodology used to determine the di-
gestibility of main nutrients may influence the
result, and attempts have therefore been made
to standardise this for cod. The main conclusion
from this research is that cod have a greater
ability than salmon to adapt to plant feed ingre-
dients. Even the use of relatively high levels of
soybean meal in the diets for cod resulted in
good growth, health, and product quality. In sin-
gle trials, however, minor histological changes
in the intestine were observed. The digestibility
of the fat was reduced somewhat with the use of
25 per cent soybean meal. The cod seem to
compensate for this with a higher intake of feed.
The finding that cod utilises soya-based feed
better than salmon is probably explained by the
longer gastrointestinal tract in cod, less sensit-
242 Aquaculture Research: From Cage to Consumption
ive digestive enzymes, as well as the presence
of a fermentation chamber with high micro-
bial activity in the distal part of the intestine,
which may participate in digestion. There have
been some concerns that maize in diets for cod
could discolour its white flesh. Research so far
has shown that maize gluten caused a yellow
hue in the skin, while the muscle was not dis-
coloured.
Micro-organisms as a feedstuff
for fish
Bacteria
Today, substantial volumes of natural gas are
produced on the Norwegian continental shelf.
In addition to serving as fuel, gas can be used as
an energy and carbon source for heterotrophic
biomass production. Bioprotein (BPM) is a
promising new feed ingredient produced in
Norway by Norferm AS; however, production
was, at least temporarily, discontinued in 2006.
The single cell protein was produced using aer-
obic fermentation of bacteria, primarily
Methylococcus capsulatus. Natural gas was the
energy source, while ammonia, water, phos-
phate, and minerals were added to the process.
The product was spray-dried and had a chemi-
cal composition of 70 per cent protein, 10 per
cent fat and about 7 per cent ash, and an amino
acid profile fairly similar to a high-quality fish-
meal. The fat consists primarily of phospho-
lipids in which the fatty acid composition is
dominated by the fatty acids C16:0 and C16:1.
BPM is legally approved for inclusion at 33 per
cent in feed for salmon in seawater and 19 per
cent in feed for salmon in freshwater. The main
conclusion of a PhD thesis studying use of BPM
in diets for Atlantic salmon, rainbow trout and
Atlantic halibut was a slight reduction in appar-
ent digestibility coefficients with increasing in-
clusion level of BPM replacing LT fishmeal in
the diet. The growth rates and feed efficiency diets in which BPA and BPM replaced up to
ratio, however, were highest in Atlantic salmon 250 g kg-1 of crude protein from high-quality
and rainbow trout fed the highest levels of BPM fish meal.
in the diet, 36 per cent and 27 per cent respec-
tively (Figure 3). BPM in diets for Atlantic hal- Microalgae
ibut can be included up to 9 per cent without Microalgae form the basis for most of the
negative effects. Atlantic halibut fed up to 18 aquatic food chains and therefore play a key
per cent BPM in the diet showed reduced feed role in the nutrition of fish. For example, micro-
intake, growth rates and feed efficiency ratio. algae are currently used as feed for zooplank-
ton, which are in turn used for start-feeding of
BPM bacterial protein meal autolysate (BPA) is marine fish fry such as halibut, turbot and cod
produced from BPM under high pressure by hy- (see separate chapters). Microalgae may also
drolysis catalysed by endogenous enzymes and have a potential for direct use as a raw material
is characterised by a high content of free amino in fish feed. The chemical composition of
acids and low molecular weight proteins. A microalgae varies depending on the species, but
higher digestibility for most indispensable ami- it is foremost as a net producer of long-chain
no acids in the BPA compared to BPM has been polyunsaturated n-3 fatty acids EPA (20:5 n-3)
reported in feeding experiments with mink. No and DHA (22:6 n-3) that microalgae are inter-
such differences in digestibility of nitrogen and esting. The concentration of unsaturated fatty
amino acids were observed in rainbow trout fed

Specific growth rate (SGR)

120 a a
ab
b b
100
SGR (% of control group)

a
a
80

60 b

40
Salmon

20 Trout
Halibut
0
0 9 18 27 36
Dietary BPM inclusion level (%)

145x100//Kap14-fig01.eps

Figure 3. Specific growth rate (% of body weight per day) of salmon, trout and halibut fed diets containing increasing levels
of bacterial protein meal (BPM). The values are standardised to per cent of the specific growth rate of the control group of
each species. Significant differences within each species are denoted with different letters. No significant differences were
found in trout. (Aas, 2006..)

Thematic area: Feed, Nutrition, Feeding 243

 acids is affected by incubation factors including
food supply and temperature.
Nannochloropsis oceanica is an alga species
that both satisfies the requirement as a high
PUFA source, and can be produced efficiently
in significant quantities. A number of trials have
been conducted, aimed at optimising produc-
tion conditions such as light, temperature, CO2
and food supply. The results have shown that N.
oceanica can be produced at 100 g/m2/day,
which results in a potential of approximately 30
kg dry algae mass/m2/year using additional
light. The content of polyunsaturated fatty acids
can be doubled or tripled by reducing the water
temperature from 25 to 10 oC a few days before
harvesting. N. oceanica has also proved to grow
very well in 200-litre pilot reactors of the
Biofence type. The project indicates that it is
possible to develop the production of micro-
algae into a new industry. This is, however, a
new research field and a major effort is required
before such production can be initiated.
Similarly, biotechnological production of the
lipid-rich marine micro-organisms
Traustochytrids through large-scale fermenta-
tion may represent valuable contributions as
feed resources and a source of essential fatty
acids for the aquaculture sector. They may be
used both in marine start-feeding and as an in-
gredient in growth feed to complement the long
chained n-3 fatty acids EPA and DHA which
plant oils lack. The microbes contain 47 per
cent fat on a dry matter basis and can be used di-
rectly or fractionated into oil and a meal frac-
tion rich in phospholipids. In the continuation
of the ongoing chemical characterisation, the
products will be evaluated as feedstuffs for fish
and as an enrichment media for rotifers for use
in the production of marine larvae.
244 Aquaculture Research: From Cage to Consumption
Feed technology
Most fish feed production in Norway today is
based on extrusion technology. The process in-
volves mixing, shearing and heating under ele-
vated pressure, before the extrudate is forced
through a die to produce an expanded pelleted
feed. Great effort has been made to optimise the
extrusion process in relation to the type of in-
gredients to produce a high-quality pellet that
readily absorbs oil in a vacuum coater. This is
and will continue to be an ongoing initiative
from the feed producers, especially when new
raw materials with different technical character-
istics are introduced. Optimisation of feed tech-
nology is aimed at improving technical quality,
increasing the availability of nutrients and re-
ducing the content of anti-nutrients in the feed.
Research has shown promising results in reduc-
ing the heat stable anti-nutritive factor phytate
under conditions that can be directly applied in
an extrusion line. More research is, however,
needed to develop a fully integrated online tech-
nology for this purpose.
During the past decade, alternative production
methods for fish feed have been launched in-
volving the direct use of by-products and indus-
trial fish, without separating the fat and protein
phases into fish oil and meal. The techniques
were developed to improve the exploitation of
by-products, and also represent energy-saving
production methods. One of the methods is
based on the use of microwave technology to
heat (and thus disinfect) and bind pellets
through protein coagulation. Another technique
uses the gelling properties of certain binding
agents, which are mixed with minced fish and
activated in an acid bath, known as the Gelly-
Feed technique. The advantage of both technol-
ogies is that feed can be processed locally, with
local resources and players. It is also possible to
vary the feed ingredients to a greater extent, and
145x100//Kap14-fig01.eps
GellyFeed production plant from production of preserved paste to finished pellets (Photo: GellyMar)
to choose between producing fresh moist feed
and drying the produced feed. On the other
hand, the supply of raw materials for alternative
production of fish feed is unpredictable, an ef-
fect which is intensified in a large-scale system.
The concept of feed produced using microwave
technology has been refined into the Sea Grain
process, in which an alternative type of fat-rich
fishmeal is manufactured from marine by-prod-
ucts and from pelagic species using microwave
heating.
GellyFeed technology
GellyFeed is a patented process for the produc-
tion of fish feed, developed by Norsk Hydro.
The process comprises two stages. The first
stage is preservation of by-products from the
fish industry by adding an alkaline preservative.
This results in a fish paste with a firm consist-
ency and storage stability, which satisfies the
EUs new hygiene requirements. Bacteria and
viruses are inactivated. The unique aspect of
alkaline preservation is that the enzyme activity
normally present in by-products from fish and
in traditional silage is also inactivated. This
results in a low level of protein autolysis, con-
tributing to good water-binding properties. In
addition, an osmosis effect is achieved, caus-
ing the water to be absorbed into the pellet so
that the consistency of the paste becomes firm.
The second stage of the GellyFeed process en-
tails making pellets from the preserved paste.
Before pelleting, the paste is processed in a
mixer into which alginate and other additives
such as vitamins and antioxidants are incorpo-
rated. Finally, the mixture is pelleted and the
final structure is set by immersing the pellet in
an acidic bath. The acid bath neutralises the
alkaline pellet and immediately starts a gelling
Thematic area: Feed, Nutrition, Feeding 245
 process that makes the pellet water-stable, non-
sticky, and durable. This results in a pellet
which may consist of 97 per cent marine ingre-
dients. The finished pellet has a pH of 45 and
a dry matter content of between 3040 per cent.
The chemical composition can be adjusted to
satisfy the nutrient requirements by adjusting
the ingredient composition. For a leaner feed, a
mixture of white-fish paste and oily-fish paste is
used. If only herring trimmings are used, the re-
sult is a salmon feed with a high content of fat
and protein. The digestibility of the main nutri-
ents for salmon is good, and often better than
commercial dry feed, with a digestibility of 96
per cent for fat, 81 per cent for protein and 87
per cent for energy. The salmon also showed
improved feed conversion. Arrangements are
being made for local production under licence
with feed factories that produce less than
10,000 tonnes per year, that is, considerably
smaller units than in the dry-feed industry.
Sea Grain technology
The process of manufacturing Sea Grain prod-
ucts involves mincing, emulsifying and drying
the marine by-products or raw materials on the
basis of a patented process. By-products from
herring, capelin, sand eel and blue whiting are
used in the production of Sea Grain, which is
then used in extruded feed for salmon smolt.
These Sea Grain products have low water con-
tent and are therefore easy to store. Depending
on the ingredients, the products varied between
3741 per cent fat and 5056 per cent protein on
a dry matter basis, but it is possible to adjust the
composition further using other combinations
of ingredients. The results of the trials have
shown that the Sea Grain products are well
suited to salmon feed, without negative effects
on growth and health of the fish. The composi-
tion of the products has, however, varied some-
what, primarily in the quantity of soluble
246 Aquaculture Research: From Cage to Consumption
protein, which has been shown to result in dif-
ferences in appetite, growth, and feed conver-
sion.
The idea behind the Sea Grain concept is
future on-board production of high-quality fat-
rich meal with better utilisation of by-products
and by-catch, lower transport costs for dry
products, and sustained quality of these re-
sources. A challenge related to the fat-rich
products is the lower potential for removal of
contaminants with the use of, for example, by-
products with a high content of organic pollut-
ants and other contaminants in production (see
below).
Technology for modification and refining
of marine resources
New national and international legislation im-
poses restrictions on the use of by-products.
Many available raw materials with a low level
of utilisation or high content of undesirable sub-
stances must be modified or refined if they are
to be used as feedstuffs or supplements in fish
feed.
Marine by-products are valuable raw materials,
and efforts are being made to achieve increased
utilisation of these in the manufacture of new
consumer products, biochemicals and feed-
stuffs. In volume terms, however, it is the pro-
duction of feedstuffs, in the form of silage
concentrates, fish oils and meal products con-
taining protein, for which there is processing
capacity and a market large enough to absorb
the largest quantities of by-products. As men-
tioned before, a knowledge base has been devel-
oped for commercial application of fish silage
in the fishmeal industry, and it is estimated that
some 20 per cent of silage could be used in tra-
ditional fishmeal and fish oil production with no
deterioration in quality.
Hydrolysis of by-products using industrial en-
zymes is another way to improve the efficiency
of utilisation of by-products and other industrial
fish. This process can be compared with silage.
The protein is hydrolysed into smaller peptides
and amino acids, and can be used as a readily
available feed supplement for farmed fish.
Feeding trials with salmon have shown that 18
24 per cent of the fishmeal protein can be re-
placed with fish protein hydrolysate. The ad-
vantage of hydrolysis with industrial enzymes
is the increased predictability and reproducibil-
ity of the products, and it is a significantly mild-
er and less time-consuming treatment than the
ensiling process.
For certain marine raw materials, the content of
undesirable pollutants will exceed the upper
limits specified in the legislation. The focus on
organic pollutants such as dioxins and PCBs has
been strong, especially for industrial fish caught
in overtaxed areas such as the North Sea, the
Baltic Sea and Skagerak, and for by-products
from the white fish industry. Feeding trials with
salmon have shown deposition of PCB of 8090
per cent and deposition of dioxins of 50 per cent
in salmon (Figure 1). The international legisla-
tion will be revised to include PCBs, and one
should be prepared for the prospect of require-
ments for removal of contaminants from raw
materials. Research on several cost-effective
cleaning techniques for fishmeal and fish oil has
been evaluated and tested. The organic pollut-
ants are fat-soluble, and simply reducing the fat
component of the fishmeal will be an adequate
decontamination technique for certain raw
materials (3040 per cent efficiency). Other raw
materials will require more drastic cleaning.
One of the initiatives tested entailed a new prin-
ciple for removal of pollutants based on the ex-
traction of press cake from fishmeal production
with soybean oil. The process has been shown
to be able to remove up to 90 per cent of pollut-
ants in the laboratory, and this is better than or
equivalent to what can be achieved with solvent
extraction of fishmeal. Solvent extraction is not
desirable since it may change the nutrient com-
position of the finished meal. Removing con-
taminants with soybean oil, however, results in
an undesirable change in the fatty acid compo-
sition of the fishmeal. This may be solved in the
extraction process by replacing soybean oil
with marine oil, which is itself purified after-
wards. These are regarded as processes that can
easily be integrated into the existing production
of fishmeal and fish oil.
Activated carbon is an effective technique for
removing dioxins from fish oil, either alone or
in combination with a deodorisation technique
(vacuum pump distillation). Dioxin-like PCB
(DLPCB) compounds are less efficiently re-
moved, while brominated flame retardants (PB-
DE) cannot be removed using this method.
Alternatively, molecular distillation appears to
enable highly efficient removal of dioxins,
dioxin-like PCBs and brominated flame retar-
dants in fish oil (more than 90 per cent). The
process also reduces the content of fat-soluble
vitamins, while the fatty acid composition and
oxidation status are not changed. The research
concludes that technology for removing pollut-
ants exists, but that it is a challenge to find cost-
effective solutions that take into account the
other qualities of the raw materials and, in turn,
the quality of the seafood products.
Perspectives
Fishmeal and fish oil have traditionally been the
dominant ingredients in salmon feed. The
aquaculture industry has already experienced
limitations in the marine resource base, and fur-
ther pressure on these resources is expected
Thematic area: Feed, Nutrition, Feeding 247
 with reallocation of a higher proportion directly
to human consumption. This has necessitated
an intensified research effort to find sustainable
alternatives that can provide a sound basis for
further growth and profitability in the aquacul-
ture sector. It is especially urgent to find alter-
native fat sources of acceptable quality which
take into account economic factors, production
capacity, the aquaculture environment, the
health of the fish, and not least the production of
a safe and healthy product. Research has gone a
long way towards confirming that there are
many alternative fat and protein raw materials
among marine crustaceans, plant products and
single cell organisms. Short-term challenges are
related to the influence of the fat sources on the
seafood fatty acid composition and content of
harmful and undesirable substances. For protein
ingredients, the main challenges are related to
anti-nutrients and undesirable substances,
which often depend on the degree of refine-
ment. Research related to modelling and the de-
velopment of feeding strategies shows that with
the alternative raw materials of today there are
opportunities for full or partial replacement of
fishmeal and fish oil in aquaculture. The use of
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Thematic area: Feed, Nutrition, Feeding 251
 Bjrn Bjerkeng1), Trine Ytrestyl1) and Rolf Erik Olsen2)

1) Akvaforsk The Institute of Aquaculture Research 2) Institute of Marine Research

Metabolism and Uptake of Carotenoids in

Farmed Fish

Considerable progress in the determination of factors that influence carotenoid

pigmentation of salmonid fishes has been made in recent years. Comparative tri-
als with red-fleshed Atlantic salmon and white-fleshed Atlantic halibut have re-
vealed that gastrointestinal absorption of dietary carotenoids does not influence
the amount of carotenoids deposited in the muscle tissue of the latter species.
After intraperitoneal injection of astaxanthin in Atlantic salmon, the concentra-
tion was 20 times higher in the blood and 15 times higher in the muscle than was
the case after adding astaxanthin to the diet, indicating that the potential for
muscle uptake in Atlantic salmon is considerably higher than previously antici-
pated. Similar indications have come from studies on binding astaxanthin to
muscle protein fractions. Comparative studies have shown that fish species with
white muscle may absorb substantial amounts in the blood, and that their mus-
cle proteins may associate with astaxanthin. This is evidence for regulation of
astaxanthin uptake at the muscle cell level.

252 Aquaculture Research: From Cage to Consumption

Appearance is among the most important prop-
erties of food items including fish, and colour is
perhaps the most conspicuous feature of food
appearance. Preferences for food items with a
certain colouration are learned and elicit psy-
chological expectations based on experience,
tradition and customs, and also trigger an emo-
tional response in the consumer. Muscle colou-
ration is among the most important quality
criteria for salmonid fishes [1]. In a recent real-
choice experiment it was found that consumers
were willing to pay significantly more for fillets
of Atlantic salmon (Salmo salar) that had nor-
mal or above normal redness than for paler fil-
lets [2]. However, there are large differences in
muscle carotenoid concentrations among
salmonid fish species. Muscle concentrations of
astaxanthin may range as high as 59 mg kg-1 in
wild sockeye salmon (Oncorhynchus nerka)
[3], whereas the muscle carotenoid concentra-
tion in large rainbow trout (O. mykiss) may
reach 25 mg kg-1 [45].
Consumers show preference for pink-coloured
products of salmonid fishes. The red colour
contributes strongly to the pleasure of eating
salmon and may have a signalling effect with
respect to quality [67], and carotenoids act as
an antioxidant in such products [8]. Colour may
therefore be used as a direct estimate of quality
since it is required to be within a certain range
for a given product. Correct colour determines
consumer preference for and prices of salmon
products. It is therefore important to have
knowledge of variation in colouration among
individual fish and between various salmonid
fish species, and of the factors that govern this
variation. Colour may also directly indicate
quality by signalling the degree of maturity of
vegetables and fruits. Astaxanthin has a minor
influence on taste, and may be added to prod-
ucts of salmonid fishes without negative effects
during processing [9].
Animal colours, including those in seafood,
have different characteristics and may be divid-
ed into two main categories. The first category
includes physical or structural colours, and is
not due to true pigments. Such colours are due
to light reflections in structural elements of the
material. Tyndall blues and diffraction colours
in animals are caused by light scattering in col-
loid systems of solid material in gel. Iridescent
or metallic blue is caused by light interference
with thin laminates. Such colours can be found
in the external ornaments, also those of com-
mercially important salmonid fishes. The sec-
ond main category consists of true pigments,
and includes carotenoids, lipofuscins, quinones,
flavonoids and anthocyanins, indoles, tetrapyr-
rols, flavins, purins and pterins, and inorganic
pigments. Combinations of the colour catego-
ries, for instance Tyndall blue and yellow pig-
ments, may give rise to green colours. In
addition, the translucency of the material is of
importance for the appearance.
All aspects of colouration may be altered in fish
products post mortem, and certain aspects may
be manipulated during farming or processing.
The main focus in aquaculture has been on mus-
cle tissue pigmentation of salmonid fishes. The
colour of the muscle tissue of the different
salmonid fishes is caused by uptake, accumula-
tion and metabolic transformation of ingested
carotenoids, which is the topic of this article.
Pigmentation of salmonid fishes and fish in
general is a small field internationally. How-
ever, in Norway, where the farming of Atlantic
salmon in particular is of great economic im-
portance, it is an important topic of research.
Much of the research in the field of salmonid
fish pigmentation has therefore been conducted
Thematic area: Feed, Nutrition, Feeding 253

145x100//Kap15-fig01.eps
Figure 1. Chemical structures of optical RS isomers of
astaxanthin, (3R,3R)- 1a, (3R,3S)- 2a and (3S,3S)-
astaxanthin 3a.
in Norway, where continuity and expertise have
been accumulated over a period of more than 30
years. This article focuses on publicly funded
research during the past decade, as well as pro-
vides references to important literature.
Carotenoids in seafood
The conspicuous colour of seafood is often
caused by the presence of carotenoids. All ani-
mals depend on a dietary supply of carotenoids.
Carotenoids are usually tetraterpenes that are
biosynthesised by photosynthesising organisms
and certain bacteria and fungi. Carotenoids add
colour to important aquaculture species, for ex-
ample, the muscle tissue of salmonid fish spe-
cies, the exoskeleton and muscle epithelium of
crustaceans, the external colour of the integu-
ment of fishes and the gonads of molluscs. It is
highly desirable that fillets of Atlantic salmon
have a rich pink colour, and for this reason it is
necessary to supplement the feed with caro-
254 Aquaculture Research: From Cage to Consumption
145x100//Kap15-fig01.eps
Figure 2. Chemical structures of the quantitatively most
important geometrical isomers of astaxanthin, all-E- (a),
9Z- (b), 13Z- (c), and 15Z- (d) (3R,3R)-astaxanthin. Chiral
centra are indicated with an asterisk for structure a.
tenoids. Astaxanthin (3,3-dihydroxy-,-caro-
tene-4,4-dione) and canthaxanthin (,-
carotene-4,4-dione) produced by chemical
synthesis are, either alone or in combination,
the most commonly used carotenoids for pig-
mentation of salmonid fishes. Astaxanthin pos-
sess three optical R/S isomers (Figure 1) and
272 possible geometrical E/Z isomers, whereof
the quantitatively most important are the all-E-,
9Z-, 13Z- and 15Z-isomers (Figure 2). The most
important alternative sources of astaxanthin are
the yeast Phaffia rhodozyma, the microalgae
Haematococcus pluvialis or products based on
crustaceans.
Supplementation of fish feed with carotenoids
is expensive, and previously represented up to
1520 per cent of total feed costs. Recently, it
has become practice to use considerably less
carotenoids in the feed. Only about 515 per
cent of the dietary carotenoids are utilised for
muscle pigmentation. The low degree of utilisa-
tion is partly due to a low absorption rate in the
gastrointestinal tract, deposition in other organs
and metabolic transformation into colourless
compounds that may eventually be excreted.
Certain fish species, such as salmonid fishes,
are capable of transforming the 4(4)-ketocaro-
tenoids astaxanthin and canthaxanthin into vita-
min A1 and A2 (retinol and 3,4-didehydro-
retinol) [1012]. The bioavailability of caro-
tenoids is altered with age and physiological
status in salmonid fishes. Small fish preferen-
tially deposit carotenoids in the skin, whereas
deposition in the muscle increases as the fish
grow [13]. At the onset of sexual maturation,
sex hormones induce a relocalisation of caro-
tenoids from muscle to skin, which contributes
to the characteristic nuptial colouration [14].
The colour is of great importance since male
sockeye salmon (Oncorhynchus nerka) selec-
tively prefer spawning with female models of a
deeper red colour [15]. Carotenoids are bioac-
tive compounds. Thus the astaxanthin content
of rainbow trout eggs is correlated to fertilisa-
tion and hatching rates, which indicates that as-
taxanthin is required for optimum reproduction
[16]. High dietary astaxanthin concentrations
may also reduce cataract in Atlantic salmon
[17]. Astaxanthin and canthaxanthin have been
regarded as vitamins, and a positive effect on
growth of salmon larvae has served as a basis
for the recommendation that all fish diets
should contain at least 10 mg of these caro-
tenoids per kg feed [18].
Uptake of carotenoids
Uptake of carotenoids from the gastrointestinal
tract is critical for their utilisation. A character-
istic feature of the uptake process is that maxi-
mum concentrations in the blood are found up
to 30 hours after force feeding [19]. A common
measure of gastrointestinal absorption is the ap-
parent digestibility coefficient (ADC), which
estimates the difference in the amount of a nu-
trient present in the diet and the amount excret-
ed in the faeces. For carotenoids to be absorbed
and enter the systemic circulation they must be
released from the matrix that surrounds them,
be incorporated into mixed micelles, enter
through the brush border membrane and be
transported through the enterocytes, as well as
incorporated into the chylomicrons and lipopro-
teins. Typical values for the ADC of astaxanthin
and canthaxanthin are between 30 and 60 per
cent, but higher or lower values may be found
depending on carotenoid source, dietary dose
and feed composition. Astaxanthin dipalmitate
and esterified carotenoid are more poorly uti-
lised than unesterified astaxanthin [20]. This
also appears to be true for natural astaxanthin
esters of the green algae Haematococcus pluvi-
alis [21]. Astaxanthin is about 1.4 times more
efficient as a carotenoid source than canthaxan-
thin for muscle pigmentation of rainbow trout
[22,23]. However, in Atlantic salmon canthax-
anthin is a better source for muscle pigmenta-
tion than astaxanthin [2426]. The blood
concentration of carotenoids has been regarded
as a good indicator of their bioavailability in
salmonid fishes [2729]. In Atlantic salmon it
was found that more canthaxanthin than astax-
anthin was taken up when the dietary inclusion
level was similar to but higher than 30 mg/kg.
When the diet was supplemented with both
carotenoids the interaction between them
caused a lower utilisation of both, but the reduc-
tion was highest for astaxanthin [26]. A study of
the ADC, muscle accumulation and relative
concentrations of astaxanthin isomers in rain-
bow trout showed that the E/Z isomers were ab-
sorbed from the gastrointestinal tract to a differ-
ent degree, while this was not true for the
optical R/S isomers [30,31]. The absorption of
the geometrical isomers descended in the fol-
lowing order: All-E-astaxanthin > 13Z-astaxan-
thin > 9Z-astaxanthin.
Thematic area: Feed, Nutrition, Feeding 255
 Increasing the dietary lipid content causes an
increased deposition of astaxanthin in the mus-
cle of Atlantic salmon [22,23,32]. The compo-
sition of dietary fatty acid may also influence
uptake and deposition. This is indicated by
higher astaxanthin concentrations in Atlantic
salmon fed diets supplemented with capelin or
Peruvian oil high in polyunsaturated fatty acids
compared to Atlantic salmon fed diets supple-
mented with herring or sandeel oils [33,34].
The effect of lipid sources and bile acids have
been studied in dorsal aorta cannulated Atlantic
salmon [35]. The salmon were fed diets supple-
mented with herring oil, soy lecithin, lard, or
herring oil supplemented with taurocholin. Soy
lecithin caused a poorer uptake of astaxanthin
compared to herring oil, whereas lard had a pos-
itive effect on the uptake. Dietary supplementa-
tion with taurocholin did not significantly affect
the blood concentration of astaxanthin. Thus, it
appears that alteration of the mixed micelle
structure in the intestine does not improve the
astaxanthin uptake. The positive effect of lard
on the blood concentration of astaxanthin may
have been affected by the increased concentra-
tions of 16:0, 18:1n-9 or 18:2n-6 fatty acids or
the lower content of the 20:1n-9 and 22:1n-9
fatty acids of lard compared to herring oil, caus-
ing an increased solubility of astaxanthin in the
intestinal micelles.
Recently, the authors have shown that feed in-
take [36] and temperature [37] influence the
ADC of carotenoids. Groups of Atlantic salmon
weighing about 2 kg were fed either a full or a
restricted ration that corresponded to about 100
per cent and 40 per cent of apparent satiation,
respectively, followed by a switch in rations af-
ter 14 days. The control group was fed a full ra-
tion during the entire experiment. The feed
intake corresponded to 0.45 per cent and 0.16
per cent of the biomass for the full and restrict-
256 Aquaculture Research: From Cage to Consumption
ed ration, respectively. A negative correlation
was found between feed intake and ADC. The
ADC was 1.5 times higher in the fish fed to 40
per cent of apparent satiation compared to the
fish fed the full ration. However, due to the rel-
atively low feed intake in the fish fed the re-
stricted diet, the total amount of digested
astaxanthin only represented about 50 per cent
of that digested by the fish fed the full ration.
Estimates based on the results from this trial in-
dicate that a low retention of astaxanthin at a
high feed intake level may cause a reduction in
the muscle concentration of astaxanthin in
rapidly growing salmon [36]. This may further
explain observations of poor pigmentation in
periods of extreme thermal growth. In a trial
with small Atlantic salmon (approximately
500 g), a 10 per cent lower ADC of astaxanthin
was found at 8 C compared to 12 C [37].
Species differences in muscle
pigmentation
Several different causes may explain the differ-
ences in carotenoid pigmentation found in dif-
ferent wild fish species. For instance, different
fish species may have different carotenoid di-
gestibility and gastrointestinal absorption rate
of ingested carotenoids. The catabolic transfor-
mation (metabolism) of carotenoids may be dif-
ferent, and there may be differences in ingested
doses and foraging habits as well. Most fish
species are capable of absorbing a certain
amount of carotenoids, but most of them depos-
it these carotenoids or their metabolites in the
integument. At present it is virtually unknown
to what extent the gastrointestinal absorption
rate, catabolic transformation rate, ingestion of
different doses, or other factors are responsible
for the differences in muscle carotenoid pig-
mentation between salmonid and other fish spe-
cies that normally have white flesh. Thus, the
ADCs of individual astaxanthin E/Z isomers
and total astaxanthin were determined in a com-
parative experiment with Atlantic salmon and
Atlantic halibut (Hippoglossus hippoglossus)
fed the same diet supplemented with 66 mg as-
taxanthin/kg consisting of 75.3 per cent all-E-,
2.6 per cent 9Z-, and 21.5 per cent 13Z-astaxan-
thin, respectively [38]. The results showed that
the ADC of total astaxanthin was in fact signi-
ficantly higher in the Atlantic halibut than in the
Atlantic salmon after 56 days and 112 days of
feeding (77.8 and 45.9 per cent, and 71.7 and
59.2 per cent, respectively). A possible explana-
tion for this difference in ADC could be that
specific growth rate of the latter species is 3.5
times higher. Furthermore, the ADC of all-E-as-
taxanthin was significantly higher than the
ADC of 9Z-astaxanthin in both species. The ex-
periment showed that astaxanthin digestibility
and gastrointestinal absorption is not a limiting
factor for utilisation of astaxanthin in Atlantic
halibut compared to Atlantic salmon, and that
other factors must be identified to explain the
absence of astaxanthin deposition in the muscle
of Atlantic halibut. Despite the higher ADC in
Atlantic halibut, the plasma concentration of as-
taxanthin was considerably lower than in Atlan-
tic salmon (the total carotenoid concentration
was 0.7 and 4.0 mg l-1, respectively). This may
be due to different catabolic transformation
rates, astaxanthin transport in the blood, and up-
take at the muscle cell level. Blood uptake of
geometrical E/Z isomers in humans following
ingestion of a meal containing 100 mg astaxan-
thin indicates that the absorption process in hu-
mans is different than that in fish, since a
selective accumulation of astaxanthin Z-iso-
mers takes place [39]. As in fish, the uptake of
astaxanthin was lower, only 25 per cent based
on the AUC (area under the concentration time
curve), following administration of astaxanthin
fatty acyl esters compared to unesterified astax-
anthin [40].
To investigate whether the carotenoid transport
capacity of the blood could represent an impor-
tant limitation to muscle supply of carotenoids,
experiments involving intraperitoneal injection
of an astaxanthin preparation were performed to
study astaxanthin uptake in the blood and depo-
sition in various tissues and organs [41,42]. Dif-
ferent doses of commercial water-dispersible
astaxanthin were suspended in a phosphate
buffer and injected into the intraperitoneum of
Atlantic salmon, rainbow trout and Atlantic cod
(Gadus morhua) to determine uptake and depo-
sition of the individual geometrical E/Z isomers
of astaxanthin, as well as the idoxanthin con-
centration of plasma, muscle, liver, kidney and
skin as an indicator of metabolic transforma-
tion. In the first trial [41], duplicated groups of
rainbow trout (initial weight 550 g) were inject-
ed with astaxanthin, and were either subse-
quently fed a diet supplemented with a low level
of astaxanthin (10 mg kg-1), a diet supplement-
ed with 60 mg astaxanthin kg-1 (fed control), or
a diet supplemented with 10 mg astaxanthin/kg-
1
(0-group) for eight weeks. In the second trial
[42], duplicated groups of Atlantic salmon (ini-
tial weight 370 g) and Atlantic cod (initial
weight 210 g) were injected with 0, 12.5, 25 or
50 mg astaxanthin, respectively. Subsequently,
all fish were fed a diet supplemented with a low
level of astaxanthin (10 mg kg-1) for four
weeks. In plasma the astaxanthin concentration
curve increased in a dose dependent fashion,
and was positively linearly correlated with the
injected dose in both Atlantic salmon and At-
lantic cod. Individual concentrations up to 90,
50 and 20 mg astaxanthin l-1 were detected in
the plasma of Atlantic salmon, Atlantic cod and
rainbow trout, respectively. In comparison, an
in vitro investigation of the saturation level of
canthaxanthin in the lipoproteins of rainbow
trout revealed that the binding capacity of the
lipoproteins was about 100 times as high as the
Thematic area: Feed, Nutrition, Feeding 257
 concentration obtained by feeding the fish a diet
supplemented with 100 mg canthaxanthin per
kg-1 [43]. Thus, the transport capacity of the
blood of salmonids and other fish species does
not limit the utilisation of carotenoids.
Metabolism of carotenoids in salmonid
fishes
Salmonid fishes possess enzyme systems that
can transform ingested astaxanthin and can-
thaxanthin or certain other carotenoids into
vitamin A1 and A2 as mentioned previously.
Recent findings show that Atlantic halibut pos-
sess this capability as well [44], and suggest
that this metabolic conversion may be more
widespread among fish than previously recog-
nised. The gastrointestinal epithelium and liver
are presumably the quantitatively most impor-
tant organs for catabolic transformation of caro-
tenoids, even though reductive metabolites
have been detected in the kidney, spleen, go-
nads, skin and retina. The quantitative impor-
tance of the various organs for metabolic
transformation has not yet been precisely deter-
mined, and requires the identification of the
genes and enzymes of different metabolic path-
ways and the metabolites that are produced.
However, the authors have found that metabolic
products are formed rather rapidly because the
reductive astaxanthin metabolite idoxanthin
(3,3,4-trihydroxy-,-carotene-4-one) was that govern carotenoid metabolism in salmonid
detected in the plasma 6 hours after force-feed-
ing Atlantic salmon with radioactively labelled
astaxanthin [19]. Idoxanthin is formed by re-
duction of one of the keto-groups of astaxan-
thin, whereas 4-hydroxyechinenone (4-
hydroxy-,-carotene-4-one) is a correspond-
ing reductive metabolite of canthaxanthin
(Figure 3). Considerable differences between
species are found with regard to idoxanthin ac-
cumulation in the blood and various tissues.
Whereas idoxanthin comprises 40 to 60 per cent
258 Aquaculture Research: From Cage to Consumption
of the total carotenoids of the muscle in Arctic
charr (Salvelinus alpinus; 1.1 kg) [45,46], the
corresponding figure for Atlantic salmon is
about 10 per cent [38], while idoxanthin is bare-
ly detectable in rainbow trout [30]. Recently,
the authors isolated and quantified 4-hydroxy-
echinenone in the muscle of Atlantic salmon
0+ and 1+ smolts fed a diet supplemented with
canthaxanthin [47]. We found that the (4S)-
isomer (Figure 3b) accumulated selectively (81
per cent 4S- and 19 per cent 4R-isomer, re-
spectively, of total amount of 4-hydrox-
yechinenone), which strongly indicates that
canthaxanthin is reduced selectively by the
enzyme involved. A selective reduction of can-
thaxanthin in favour of the (4S)-hydroxy-
echinenone has previously been reported for
laying hens [48] and skin of rainbow trout [13].
The concentration of 4-hydroxyechineone in
the muscle was relatively low (<3.1 per cent of
total carotenoids) and diminished with increas-
ing fish size and age. This corresponds to find-
ings of idoxanthin in Arctic charr and Atlantic
salmon, in which the concentration of idoxan-
thin was higher in small fish than large fish, and
indicates that the metabolic capacity to trans-
form absorbed astaxanthin decreases with age
and size in salmonid fishes [45,49]. However,
the metabolic capacity may increase at the onset
of sexual maturation [50]. Although the factors
fishes have not been identified, it has been
shown that hormones may be involved, since
sex hormones (11-ketotestosterone and 17-
estradiol) stimulate metabolic transformation
and redistribution, which take place during sex-
ual maturation [51]. The idoxanthin content of
the muscle of Atlantic salmon is partly geneti-
cally determined and has a heritability coeffi-
cient of 0.4 [52]. No effects of temperature and
individual feed intake were detected on astax-
anthin metabolism measured as idoxanthin con-

145x100//Kap15-fig01.eps
Figure 3. Structural formulas of the metabolites a)
idoxanthin (3,3,4-trihydroxy-,-carotene-4-one) and b)
4-hydroxyechinenone (4-hydroxy-,-carotene-4-one).
centration in plasma in an experiment with
small Atlantic salmon (500 g) [37]. Large indi-
vidual variation in the plasma concentration of
idoxanthin was, however, found (<0.570 per
cent of the total carotenoid content). In groups
of Atlantic salmon that were fed restrictively,
fish with visible damage to the pectoral fins had
a higher idoxanthin concentration in the plasma
than individuals without fin damage, and indi-
cates that stress may influence carotenoid
metabolism. Stress has previously been sug-
gested to cause increased metabolism of caro-
tenoids in salmonid fishes [49].
Changes in pigmentation pattern during
sexual maturation
It is well recognised that sexual maturation
causes dramatic changes in the pigmentation
pattern of salmonid fishes [14]. In addition to a
redistribution of the carotenoids from the mus-
cle to the skin and gonads, high metabolic activ-
ity causes a 25 per cent drop in the total body
burden of carotenoids in female rainbow trout
compared to sexually immature fish of the same
age and size, while the drop in male rainbow
trout is about 80 per cent of the total body bur-
den [5]. In a trial with Arctic charr, it was found
that intraperitoneal implants containing hor-
mones influenced pigmentation. Thus, Arctic
charr supported with implants containing 11-
ketotestosterone or 17-estradiol had higher
carotenoid concentrations in the skin than Arc-
tic charr supported with implants containing
3,5,3-triiodo-L-thyronine or with implants that
did not contain hormones; in addition, idoxan-
thin comprised a higher proportion of the total
carotenoids in the blood [51]. In Arctic charr a
considerably higher proportion of idoxanthin
(15 per cent of total carotenoids) was found in
the muscle of sexually maturing fish compared
to immature fish of the same age and size [50].
This trial also showed that astaxanthin com-
prised only a small part of the total carotenoids
(<20 per cent of total carotenoids), whereas the
major carotenoid was idoxanthin (57 per cent of
total carotenoids). Furthermore, it was shown
that crustaxanthin (3,4,34-tetrahydroxy-,-
carotene) isomers comprised about 20 per cent
of the total carotenoids in the gonads, and that
the reduction in astaxanthin was selective in
favour of the sterically most hindered
3,4(3,4)-cis glycolic isomers, whereof the
3,4,34-di-cis; 3,4-trans 34-cis; 3,4,34-di-
trans glycolic isomers comprised 40, 45, and 16
per cent of the total crustaxanthin, respectively.
In the skin, astaxanthin esters represented about
Thematic area: Feed, Nutrition, Feeding 259
 85 per cent of the total carotenoids. The bio-
chemical basis for selective accumulation of
various carotenoids in different tissues and or-
gans still remains to be elucidated. However, se-
lective accumulation should be related to tissue
specific metabolism, uptake or excretion.
Metabolism of E/Z and R/S
isomers
Some features regarding the distribution of geo-
metrical E/Z isomers and optical R/S isomers
have implications for a selective metabolic
transformation. Whether rainbow trout were fed
diets high or low in all-E-astaxanthin (97 per
cent vs. 64 per cent of total astaxanthin) a selec-
tive accumulation of 13Z-astaxanthin was
found in the liver (4859 per cent of total astax-
anthin), whereas 9Z-astaxanthin only com-
prised about 2.5 per cent of the total astaxanthin
[31]. 9Z-Astaxanthin comprised only about 2.5
per cent of the total astaxanthin of the blood.
This indicates that a selective metabolic trans-
formation of the various geometrical E/Z iso-
mers takes place, presumably in the liver. A
selective accumulation of 13Z-astaxanthin in
the liver has also been found in Atlantic salmon
and Atlantic halibut [38]. A slight selective ac-
cumulation of (3S,3S)-astaxanthin took place
in skin and kidney tissue [31]. This may indi-
cate that a selective stereochemical conjugation
takes place during detoxification reactions
(Phase II) in the kidney, by potential formation
of glucuronides or sulphates. In the skin, the se-
lective accumulation is probably related to the
esterification process, similar to the one that
takes place during gastrointestinal hydrolysis.
A selective accumulation of (3S,3S)-zeaxan-
thin, a metabolite of astaxanthin, in the skin of
rainbow trout supports this view [5].
260 Aquaculture Research: From Cage to Consumption
Accumulation of carotenoids in
muscle and other tissue
Considerable progress has recently been made
with respect to the binding of astaxanthin in the
muscle tissue of salmonid fishes. Even though
astaxanthin is liposoluble, it has long been re-
cognised that it is primarily associated with the
protein, not the lipid, fraction of muscle tissue
[53]. A newly developed method has enabled
the determination of astaxanthin binding pro-
teins in fish muscle tissue [54]. An ultracentri-
fugation method and the use of sodium cholate
for dispersion of astaxanthin complexes were
developed to separate unbound astaxanthin by
filtration over a 30 kDa filter. After fractional
extraction of the muscle proteins, the various
fractions that bound astaxanthin could be iden-
tified. Fractional extraction of protein-bound
astaxanthin using different brine concentrations
and pH have also indicated that a water soluble
extract had the highest affinity to astaxanthin,
i.e. the highest ratio between astaxanthin and
protein, and indicated that a sarcoplasmaprotein
was involved in the binding of astaxanthin [55].
By using fractional extraction of muscle pro-
teins from Atlantic salmon, Atlantic halibut and
haddock, it was shown that certain fractions had
a greater ability to bind to astaxanthin than oth-
er fractions, but the same protein fractions from
all three species were able to associate with
astaxanthin [56]. Recent work has provided
evidence that the primary binding protein of
astaxanthin in the muscle of Atlantic salmon is
a-actinin; a-actinin isolated from Atlantic hali-
but also combined with astaxanthin in about the
same stoichiometric ratio [57]. These experi-
ments indicate that the astaxanthin-binding ca-
pacity of the muscle proteins does not limit the
deposition of astaxanthin in the muscle; rather,
a high metabolic transformation rate may limit
the retention of ingested astaxanthin.
Smolt production regimes may influence caro-
tenoid uptake and deposition of carotenoids.
This was investigated in an experiment with At-
lantic salmon transferred to seawater either as
0+ or 1+ smolts, and fed a diet supplemented
with 30 mg astaxanthin and 30 mg canthaxan-
thin per kg for 35 weeks [47]. The 0+ smolts
had a significantly higher carotenoid concentra-
tion in the muscle than the 1+ smolts (approxi-
mately 10 per cent) after correcting for weight
differences. This indicates that the growth pat-
tern may influence carotenoid utilisation, possi-
bly due to the reduction in ADC caused by
increased feed intake during more rapid growth
[36]. Similarly, continuous illumination during
the seawater phase may cause better pigmenta-
tion than the natural photoperiod in autumn
smolts [58].
Extremely high plasma concentrations of astax-
anthin have been achieved in Atlantic salmon,
rainbow trout and Atlantic cod by intraperito-
neal injection of astaxanthin (50100 mg)
[41,42]. The concentrations were up to 20 times
145x100//Kap15-fig01.eps
Figure 4. Atlantic cod intraperitoneally injected with 0 or 100 mg astaxanthin, respectively. (Photo: Arild Linseth,
Akvaforsk, Sunndalsra)
higher than levels that can be achieved by feed-
ing Atlantic salmon diets supplemented with
astaxanthin. In both Atlantic salmon and Atlan-
tic cod the astaxanthin accumulated in the mus-
cle in a dose-dependent manner. Individual
concentrations up to 30 mg kg-1 were detected
in Atlantic salmon, which represent about 15
times higher concentrations than can be
achieved by feeding fish of a similar size diets
supplemented with astaxanthin. A good linear
correlation was found between plasma and
muscle concentrations of astaxanthin, which in-
dicates that increased uptake causes increased
muscle deposition and that muscle is not satu-
rated at this level. Only about 1 mg astaxanthin
kg-1 was present in Atlantic cod injected with
astaxanthin despite the high plasma concentra-
tions (about 20 mg l-1). A patent has been ap-
plied for for a method of injecting astaxanthin
into salmonid fishes. In conclusion, neither the
binding capacity of the muscle for astaxanthin,
nor plasma transport appears to be factors that
limit the efficient utilisation of astaxanthin in
Atlantic salmon. Furthermore, it appears obvi-
Thematic area: Feed, Nutrition, Feeding 261
 ous that receptors related to the uptake of astax-
anthin in the muscle have a species-specific
distribution in different tissues, which may ex-
plain the differences observed in uptake be-
tween salmonid fish species and white-fleshed
fish species, although the latter may have a
good capability of depositing carotenoids in the
skin and gonads (Figure 4).
Concluding remarks
Considerable progress has been made in re-
search on carotenoids and salmonid fish spe-
cies. The authors are convinced that a
breakthrough in determination of factors that
govern gastrointestinal uptake and uptake in
liver and muscle cells is imminent. Work with
muscle cells in culture and proteom studies has
been initiated and, provided projects receive
financial support, it will contribute to such a
breakthrough. This in turn may create tools to
investigate variation in various populations and
may contribute to improving selective breeding
programmes for commercially important spe-
cies.
Small fish exhibit a relatively poor utilisation of
carotenoids 0+ smolts utilise carotenoids bet-
ter than 1+ smolts. Carotenoid utilisation de-
pends on seasonal variations, and carotenoid
digestibility depends on feed intake and temper-
ature. Nevertheless, it may be of practical im-
portance to provide the correct dosage for the
different stages of production. There may be
more optimal dosage regimes than simply feed-
ing fish a constant dose throughout the produc-
tion cycle. Research on pigment strategies has
provided models that estimate economically
optimal pigmentation regimes, or the lowest to-
tal amount of carotenoid required to achieve a
given carotenoid level in the muscle at slaugh-
ter. By using data on feed intake, growth, mus-
262 Aquaculture Research: From Cage to Consumption
cle carotenoids, carotenoid digestibility and
retention, and so on, mathematical models can
be produced to suggest optimal regimes. The re-
sults indicate that the expenditure on astaxan-
thin can be reduced considerably compared to
few years ago, while still achieving accepted
final concentrations in the muscle (7 mg kg-1)
of the slaughtered fish (+4 kg). Pigmentation
models for farmed Atlantic salmon have recent-
ly emerged. Nonlinear regression metaanalysis
of published experimental data has served as a
basis for the development of a pigmentation
model [59]. An excellent relationship was
found between modelled astaxanthin concen-
tration in the muscle and the explanatory vari-
ables dietary astaxanthin concentration and fish
size. Based on this model the authors developed
a mathematical programming model designed
to optimise dietary astaxanthin concentrations
throughout the grow-out period that results in
well-pigmented salmon at minimal cost [60].
These models are rather simple, and other para-
meters that influence carotenoid utilisation as
mentioned above have not been taken into ac-
count, although they may improve the models.
A dynamic ordinary differential equation model
was recently developed to further the under-
standing of the fundamental processes that reg-
ulate the absorption and metabolism of
carotenoids in Atlantic salmon [61]. The model
mimics feeding experiments well and indicates
that uptake over the muscle membrane is pos-
sibly an important source of variation and that a
dynamic model can be instrumental in deter-
mining underlying determinants of complex
traits.
Quality issues comprise an important aspect of
pigmentation of products of salmonid fishes.
Documentation of the effects of process param-
eters such as salting technique, temperature and
duration of processing on the stability of astax-

145x100//Kap15-fig01.eps
Figure 5. Extraordinary decolouration of an Atlantic
salmon fillet (right-hand side) after injection-salting.
(Photo: Sveinung Birkeland, Norconserv, Stavanger)
anthin, colouration, texture and more during
processing of cold-smoked salmon has recently
been provided [6266]. Although important in-
formation on factors that influence the quality
of smoked salmon during processing has al-
ready been published, work is still needed,
among other things, to avoid problems with pig-
mentation as shown in Figures 5 and 6 [67].
A considerable amount of work has been done
to develop alternative astaxanthin sources. We
have recently studied the stability of astaxan-
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