Aquaculture research, tập 41, số 3, 2010

Aquaculture Research, 2010, 41, 321

doi:10.1111/j.1365-2109.2010.02479.x

Editorial

The special issue of Aquaculture Research is comprised of some of the papers presented at the XIII
International Symposium on Fish Feeding and
Nutrition, which was held in Floriano¤polis, Brazil,
from 1 to 5 June 2008. Since its inception in 1984,
the International Symposium continue provide excellence in ¢sh nutrition research and the opportunity for communication among researchers. New
knowledge in ¢sh nutrition research plays an important role in the development of global aquaculture as
well as allows for the production of safe and healthy
food for human consumption. The widespread interest in the subject of ¢sh and crustacean nutrition was
marked by the enthusiasm of the 445 participants
from 37 countries.
The scienti¢c programme of the symposium encompassed a workshop on ‘Sustainable aquafeeds for
the third millennium’, followed by 296 oral and poster contributions in the following eight sessions:
Protein; Nutrient Requirement and Availability; Nutrition and Gene Expression; Nutrition and Health;
Environmental Quality and Feeding Strategies; Fish

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd

Quality and Food Safety; Feed Ingredients and Feed
Processing; and Broodstock and Larvae. Seven invited lecturers, and four review papers from these
presentations are published in this special issue. Selected contributions were submitted for peer review
and the resulting manuscripts are published here.
The participants earned our appreciation, especially those who gave oral presentations, posters and
invited papers. The planning and organizing of this

symposium was a considerable undertaking for
which scienti¢c and local organizing committees
met with enthusiasm, energy and strong commitment. Our special thanks are due to all the anonymous reviewers for the assistance in reviewing all
the papers for this proceeding. We hope the articles
presented in this special issue will contribute towards the advancement of our knowledge in the fascinating ¢eld of ¢sh nutrition.
De¤bora M. Fracalossi, Organizing
Committee Chair
Santosh P. Lall, Scienti¢c Committee Chair

321


Aquaculture Research, 2010, 41, 322^332

doi:10.1111/j.1365-2109.2009.02174.x

REVIEW ARTICLE
Protein and amino acid nutrition and metabolism in
fish: current knowledge and future needs
Sadasivam J Kaushik & Iban Seiliez
INRA, UMR 1067, Nutrition, Aquaculture & Genomics Unit, 64310 Saint-Pe¤e-sur-Nivelle, France
Correspondence: S. J. Kaushik, INRA, UMR 1067, Nutrition, Aquaculture & Genomics Unit, Saint-Pe¤e-sur-Nivelle, France.
E-mail:

Abstract
Optimising the amino acid supply in tune with the
requirements and improving protein utilization for
body protein growth with limited impacts on the
environment in terms of nutrient loads is a generic
imperative in all animal production systems. With

the continued high annual growth rate reported for
global aquaculture, our commitments should be to
make sure that this growth is indeed re£ected in provision of protein of high biological value for humans.
The limited availability of ¢sh meal has led to some
concerted e¡orts in ¢sh meal replacement, analysing
all possible physiological or metabolic consequences.
The rising costs of plant feedstu¡s make it necessary
to strengthen our basic knowledge on amino acid
availability and utilization. Regulation of muscle protein accretion has great signi¢cance with strong
practical implications. In ¢sh, despite low muscle
protein synthesis rates, the e⁄ciency of protein
deposition appears to be high. Exploratory studies
on amino acid £ux, inter-organ distribution and particularly of muscle protein synthesis, growth and
degradation and the underlying mechanisms as
a¡ected by dietary factors are warranted. Research
on speci¢c signalling pathways involved in protein
synthesis and degradation have been initiated in order to elucidate the reasons for high dietary protein/
amino acid supply required and their utilization.

Keywords: proteins, amino acids, ¢sh, nutrition,
metabolism
Introduction
Protein supply from seafood contributes signi¢cantly to
human needs in several geographic areas, especially in

322

the developing world as well as in the emerging economies of the world. At a global level, about 45% of all ¢sh
consumed by humans, totalling about 48 millions
tonnes is farm raised (FAO 2007). Aquaculture thus

plays a vital role in supplying products known to have
a high biological value to humans (Bender & Haizelden
1957) besides providing healthy long-chain w3 polyunsaturated fatty acids (Sargent1997).
From a quantitative point of view, e⁄ciency of protein utilization and muscle protein growth are the most
crucial issues. Although ¢sh are generally considered
better converters of dietary protein, compared with terrestrial vertebrates, given the global context of rapid development of aquaculture and the increasing costs and
dearth of protein-rich feedstu¡s, there is an impending
necessity for improvements in dietary protein utilization, achievable only by optimising dietary supply in
tune with the di¡erent physiological needs of organisms. This then necessitates a full understanding of the
physiological basis for the requirements and e⁄cient
exploitation of available sources to meet such needs.

Protein/energy nutrition of fish: general
considerations
Critical assessment of protein requirements have
already been made by a number of authors over the
past two decades (Cowey & Luquet1983; Bowen1987;
Cowey 1994, 1995). The general observation is that
¢sh require a higher level of dietary protein than terrestrial farmed vertebrates. The general contentions
which have very often been put forward with regard
to this high protein requirement of farmed ¢sh are as
follows: (i) ¢sh have high ß apparent ý protein needs,
the basal energy needs of ¢sh are lower than those of

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd


Aquaculture Research, 2010, 41, 322^332 Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez

terrestrial animals, due to the aquatic mode of life,
poikilothermy and ammoniotelism. Based on comparisons of protein e⁄ciency ratios in a number of
farmed animals, it becomes clear that ¢sh and terrestrial animals di¡er only in relative protein concentration in the diet required for achieving maximum
growth rate and that there were no or little absolute
di¡erences in protein requirements (Cowey & Luquet
1983). As already pointed out by Bowen (1987), ¢sh
di¡er from terrestrial animals only in the relative protein concentration in the diet required for maximum
growth rate and such di¡erences are explained by
the lower energy requirement of ¢sh.
The contribution of proteins/amino acids towards
meeting the energy requirements of ¢sh is considered high. Much progress has however been achieved
through optimising the digestible protein (DP) to digestible energy (DE) ratios by reducing the dietary DP
levels with or without concomitant increase in the
dietary non-protein DE supply (Cho & Kaushik 1990;
Cho & Bureau 2001). A decrease in DP/DE ratios has
indeed proven to be extremely e⁄cient in improving
protein utilization and decreasing nitrogenous loses
in most farmed ¢sh (Kaushik & Cowey 1991; Cho &
Bureau 2001). Of the dietary non-protein DE sources,
in most species, fats are well utilized both at the digestive tract level and at a post-absorptive level
(Sargent,Tocher & Bell 2002) whereas dietary carbohydrates require heat treatment to improve its digestibility and supply of DE (Bergot & Breque1983;Wilson
1994). Increasing the dietary fat levels has indeed
been bene¢cial in bringing down the DP/DE ratios
having clear bene¢cial e¡ects in terms of nitrogen
utilization in most ¢n¢sh (Lee & Putnam 1973;
Kaushik & Oliva-Teles 1985; Hillestad & Johnsen
1994; Manuel Vergara, Robaina, Izquierdo & Higuera
1996; Satoh, Alam, Satoh & Kiron 2004). The latitude
of action however appears variable depending on the
species, some species bene¢ting more from higher

dietary non-protein energy than others. In all such
cases, a major issue however is the increased fat
deposition linked with changes in lipogenic enzyme
activities (Dias 1999; Regost 2001). Even if digestible
carbohydrates are made available, the metabolic utilization of absorbed glucose is limited in most ¢sh
(Moon 2001; Panserat & Kaushik 2002) and the net
energy supply is reduced (Bureau 1997; Hemre,
Mommsen & Krogdahl 2002) although there are differences between species (Furuichi & Yone1982; Panserat, Medale, Blin, Breque, Vachot, Plagnes.Juan,
Gomes, Krishnamoorthy & Kaushik 2000; Shiau &
Lin 2001; Enes, Panserat, Kaushik & Oliva-Teles

2008 in press). The lack of control of amino acid catabolism as a¡ected by dietary protein levels is indeed
considered to be one major reason for the high protein requirements of ¢sh (Cowey & Walton1989). This
is somewhat comparable to what is found in the carnivorous cat, where the high protein requirement is
considered to be a consequence of the high obligatory
nitrogen losses incurred in the conversion of nitrogen from indispensable amino acids (IAA) to dispensable amino acids (DAA) in the liver and to a slow rate
of catabolism of IAA (Taylor, Morris, Kass & Rogers
1998).

IAA requirements
Quantitative data on amino acid requirements for all
10 IAA are available only for a limited number of species (National Research Council 1993; Wilson 2002;
Lall & Anderson 2005; Tibaldi & Kaushik 2005).
Given the large number of species of farmed ¢n¢sh
and shrimp, we should admit that it is indeed di⁄cult
to establish the quantitative requirements for all the
10 IAA for each of the species concerned. Measured
amino acid requirements of di¡erent species,
expressed as a proportion of the diet, show also an
apparently high degree of variation (Cowey 1994;

Mambrini & Kaushik 1995b;Wilson 2002). One major
explanation for this apparent variability in IAA requirement data are linked to methodology issues
(Cowey 1995): (i) the mode of expression of data (relative to dietary dry matter or dietary DP or DE level, or
in absolute terms per unit metabolic mass per day,
etc.), (ii) the composition and type of diet used and
whether the ¢sh were able to reach their near maximum growth potential, (iii) the criterion used for the
estimation of requirement and (iv) the statistical
method used for analysing numerical data on
dose^response. Besides conventional dose^response
curves using di¡erent response criteria such as
growth, nitrogen utilization, direct or indirect methods of measurement of amino acid oxidation, metabolic responses, new approaches such as single
amino acid deletion or reduction (Fournier, Gouillou
Coustans, Metailler,Vachot, Guedes,Tulli, Oliva-Teles,
Tibaldi & Kaushik 2002; Green & Hardy 2002; Rollin,
Mambrini, Abboudi, Larondelle & Kaushik 2003) or
diet-dilution techniques (Liebert & Benkendor¡
2007) have also been attempted in ¢sh with results
con¢rming data obtained by conventional methods.
A close analysis of reliable data available however
points towards some degree of homogeneity between

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332

323


Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez Aquaculture Research, 2010, 41, 322^332

100

Wt Gain, %Max

Wt Gain, %Max

100
75
50
25
0
1

2
3
Lys, %diet

4

25

5

0

Wt Gain, % Max

80
60

4

6 8 10 12 14
Lys, % CP

40
20
0
0.5

1.0
1.5
Met, %diet

2.0

80
60
40

Figure 1 Analysis of literature data on lysine
and methionine requirements of di¡erent species
of ¢sh and shrimp.

20
0
–20

di¡erent species. Taking lysine and sulphur amino
acids as an example, an attempt was made to do a
meta- analysis of requirement data. As the initial
sizes and growth rates and experimental conditions

vary between studies, a standardized response as
the maximum gain in mass in a given study was
used. For analysing the dose^response, the four-parameter nutrition kinetics analysis (Mercer 1982) was
used. Based on data from several studies on requirements of di¡erent species for lysine and sulphur
amino acids (methionie1cystine) the di¡erent parameters were computed. Calculations were made
using data on dietary amino acid levels expressed
either as percent of the diet or as percentage of crude
protein (% CP). The corresponding dose^response
curves are presented in Fig. 1.
Because the main purpose of dietary protein/amino acid supply is for increasing whole body protein
accretion, calculation of daily amino acid increment
was used for estimating the IAA requirements of
carp and trout (Ogino 1980). Since then, this method
has been used by a number of authors for getting at
least a rough estimate of IAA requirement pro¢le of
several species of ¢sh (Kaushik, Breque & Blanc 1991;
Mohanty & Kaushik 1991; Ng & Hung 1995; Kaushik
1998; Kim & Lall 2000; Gurure, Atkinson & Moccia
2007) or shrimp (Teshima, Alam, Koshio, Ishikawa
& Kanazawa 2002). From these and several other studies, it appears that the ideal protein would be the one
that re£ects the whole body IAA pro¢le of the corresponding species. The whole body protein bound
amino acid pro¢les are however very much similar
between di¡erent species and the amino acids depos-

324

2

100

100
Wt Gain, % Max

50

0
0

– 20

75

2

4
Met, %CP

6

8

Table 1 Whole body amino acid composition of di¡erent
¢n¢sh and crustaceans (expressed as g16gN À 1)Ã
Amino acid

Finfish

Ala
Arg
Asp

Cys
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val

6.17
6.16
9.19
0.96
14.29
6.81
2.47
4.29
7.20
7.38
2.75
4.10
4.37
4.15

4.39
1.01
3.02
4.73

Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ

Shrimp
0.82
0.98
0.85
0.26
2.49

1.69
0.63
0.92
0.70
0.89
0.45
0.47
1.13
0.47
0.54
0.29
0.62
0.53

4.86
6.59
8.37
0.78
12.55
5.03
1.85
3.56
6.13
6.42
2.18
3.44
3.13
3.27
3.18
0.90

3.30
3.95

Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ
Æ

0.56
1.20
0.34
0.11
1.20
1.32
0.17
0.32

0.47
0.51
0.17
0.27
0.39
0.29
0.14
0.19
0.40
0.61

ÃData from a large number of sources including Deshimaru &
Shigeno 1972; Wilson & Cowey (1985); Penìa£orida (1989);
[65]Mambrini & Kaushik (1995b); [1]Akiyama et al. (1997);
[54]Kim & Lall (2000); Alam, Teshima, Yaniharto, Ishikawa &
Koshio (2002).

ited during growth are also similar between di¡erent
teleosts as well as crustaceans (Table 1). It has clearly
been shown that there is possibly more apparent
variability in AA requirement pro¢les than in the
whole bodyAA pro¢les of di¡erent teleosts (Akiyama,
Oohara & Yamamoto 1997). It is however reassuring
that a study by Green and Hardy (2002) con¢rmed
that the requirement pro¢le as proposed by National

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332

Aquaculture Research, 2010, 41, 322^332 Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez

From needs to feeds: developing low or
non-fish meal diets
One of the major issues a¡ecting the aquaculture industry is the availability of protein-rich feedstu¡s.
Under intensive ¢sh farming conditions, ¢sh meal

200
AA loss (mg /kgBW/d)

Research Council (1993) was found to result in the
best nitrogen utilization, compared with that of other
pro¢les based on whole body protein or from regression analyses. The question remains as to whether
the ideal protein really re£ects that of the whole body
AA pro¢le.
Few studies have also looked into the potential of
some of the DAA and to the ratios between dietary
indispensable to dispensable amino acids (IAA/DAA
ratio) (Hughes 1985; Mambrini & Kaushik 1994). By
feeding rainbow trout with diets containing varying
IAA/DAA ratios and using a number of criteria on
protein utilization, a ratio of 57:43 was found to be
the most suitable (Green, Hardy & Brannon 2002).
Similarly, in gilthead seabream, a dietary IAA/DAA
ratio of 1.1 was found to be better than a ratio of 0.8
(Gomez-Requeni, Mingarro, Kirchner, CalduchGiner, Medale, Corraze, Panserat, Martin, Houlihan,
Kaushik & Perez-Sanchez 2003).
Data available today on amino acid requirements
do not make a clear distinction between needs for
the maintenance and growth components. The only

complete set of data on maintenance requirements
for IAA was made available for rainbow trout (Rodehutscord, Becker, Pack & Pfe¡er 1997). Some other
studies have estimated the maintenance needs for individual amino acids in ¢sh (Mambrini & Kaushik
1995a). In a comparative study (Fournier et al. 2002),
the maintenance requirements for arginine was
determined in trout, seabass, seabream and turbot.
Obligatory nitrogen or amino acid losses under protein-free feeding conditions can be assumed to re£ect
the minimum physiological needs for IAA (Young &
El-Khoury 1995). Measurement of amino acid losses
in ¢sh or shrimp under protein-free feeding or fasted
conditions can provide valuable information on the
obligatory amino acid losses. Drawing from whole
body amino acid losses when fed a protein-free diet
over 28 days (Fournier et al. 2002), we could calculate
that there are both quantitative and qualitative di¡erences in endogenous losses in amino acids between
rainbow trout and turbot (Fig. 2), strongly suggesting
di¡erences in protein degradation rates and tissues
involved.

Turbot Trout
150
100
50
0

Figure 2 Pro¢le of whole body amino acid losses
(mg kg À 1 BWday À 1) in turbot and in rainbow trout fed a
protein-free diet over 4 weeks (unpublished data from the
study by Fournier et al. 2002).

Figure 3 Lysine and sulphur amino acid contents of selected protein sources compared with the requirements
for these amino acids by ¢sh.

and ¢sh oil are the most common feedstu¡s supplying the essential nutrients (amino acids, fatty acids,
minerals and trace elements) vital for growth, health,
reproduction and physiological well-being of farmed
¢sh.While the marine capture ¢sheries remains constant, the demand for such feedstu¡s derived from
capture ¢sheries is on the increase and the costs are
rocketing. In this context, replacement of ¢sh meal by
alternative protein sources remains a major thrust
area of research and much has been accomplished
in reducing the level of ¢sh meal in all species (Gatlin,
Barrows, Brown, Dabrowski, Gaylord, Hardy, Herman, Hu, Krogdahl, Nelson, Overturf, Rust, Sealey,
Skonberg, Souza, Stone, Wilson & Wurtele 2007;
Lim,Webster & Lee 2008). Fishmeal is unique in that
it is not only an excellent source of high quality protein having an ideal IAA pro¢le for ¢sh and shrimp.
An example of data on lysine and sulphur amino acid
contents of di¡erent plant protein sources in comparison with that of the requirements of ¢sh is illustrated
in Fig. 3. Fish meal is also a good source of essential
fatty acids, minerals and trace elements. In choosing
alternatives to ¢sh meal, it is then necessary to look

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332

325


Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez Aquaculture Research, 2010, 41, 322^332

at the amino acid pro¢le, but also at other macro and
micronutrients.
In terms of DP supply, compared with ¢shmeal,
there are few ingredients which have similar high
protein levels, but however with di¡erent amino acid
pro¢les. It is now clear that not a single ingredient
can totally replace ¢sh meal but that one needs to resort to a mixture of ingredients mimicking the amino
acid pro¢le of ¢sh meal. It is essential when dealing
with alternate protein sources to have precise quantitative data on amino acid availability and the biological value. It is also imperative that we choose
ingredients whose potential antinutritional factors
(Tacon 1997; Francis, Makkar & Becker 2001; Kaushik
& Hemre 2008) are limited or reduced with respect to
the species concerned. We have shown in rainbow
trout that soyprotein concentrate can totally replace
¢shmeal resulting in equivalent growth and nutrient
utilization, provided some additional methionine is
supplied (Kaushik, Cravedi, Lalles, Sumpter, Fauconneau & Laroche 1995). Non-¢shmeal diets incorporating a mixture of di¡erent protein sources are well
utilized by rainbow trout (Watanabe, Verakunpiriya,
Watanabe,Viswanath & Satoh 1998) but much less so
by yellowtail (Watanabe, Aoki, Watanabe, Maita,
Yamagata & Satoh 2001). In European seabass
(Kaushik, Coves, Dutto & Blanc 2004) as well as gilthead seabream (Benedito-Palos, Saera-Vila, CalduchGiner, Kaushik & Perez-Sanchez 2007; De Francesco,
Parisi, Perez-Sanchez, Gomez-Requeni, Medale,
Kaushik, Mecatti & Poli 2007), there is much potential
to reduce the level of ¢sh meal to a signi¢cant level in
¢n¢sh diets. Similarly, much progress has also been
made to develop non-¢shmeal diets for shrimp
(Amaya, Davis & Rouse 2007).
There are misgivings on the potential bene¢ts of
supplementing diets with free amino acids (Dabrowski

& Guderley 2002). Even very early (Nose, Arai, Lee &
Hashimoto 1974; Plakas, Tanaka & Deshimaru 1980),
di¡erences between postprandial free amino acid levels between ¢sh fed a protein diet or amino acid based
diet. In rainbow trout, di¡erences in uptake between
protein-bound and free amino acids have also been demonstrated with postprandial blood free amino acid
peaks appearing later for protein-bound amino acids
(Cowey & Walton 1988). They also showed that incorporation of labelled carbon residues into glycogen and
lipid from an amino acid diet was greater than from a
whole protein diet, whereas incorporation of radioactivity into tissue protein was higher with the latter. In
both cyprinids (Nose et al. 1974; Murai, Akiyama &
Nose 1983) and in shrimp (Lim 1993), adjustment of

326

dietary pH improves the utilization of diets with
high levels of synthetic amino acids. Increasing
the number of meals was proposed to improve
amino acid utilization (Yamada, Tanaka & Katayama
1981) in carp. In order to reduce absorption of free
amino acids while digestion of intact protein
occurs, coating amino acids with agar for instance
has been found to be e⁄cient resulting in improved
nitrogen utilization (Cho, Kaushik & Woodward
1992; Fournier et al. 2002). These necessary precautions de¢nitely improves utilization of amino acids
supplied in the free form even at very high levels.
There are indeed several reports showing that
crystalline amino acids are well utilized both with
semi-puri¢ed (Cho et al.1992; Rodehutscord, Mandel,
Pack, Jacobs & Pfe¡er 1995; Fournier et al. 2002) and
practical diets in several species of ¢sh (Kaushik et al.

1995, 2004; Williams, Barlow & Rodgers 2001;
Yamamoto, Shima & Furuita 2004; De Francesco
et al. 2007).
In plant-protein-based diets, (Espe, Lemme, Petri &
El-Mowa¢ 2006) showed that amino acids are utilized as well as protein bound amino acids even at a
10% incorporation level.

Amino acid utilization
Regulation of feed intake by dietary amino acid balance has been little studied.Whether a dietary amino
acid de¢ciency or excess leads to increases in voluntary feed intake over a long term has not been analysed in depth. A preliminary analysis shows that
single amino acid de¢ciencies (arginine, leucine, luysine, methionine) lead to decreased feed intake (de la
Higuera 2001). In European seabass, responses
appear to di¡er depending on the amino acid, tryptophan de¢ciency exerting the most signi¢cant depression in voluntary feed intake (Tibaldi & Kaushik 2005).
Further insight on the consequences of marginal amino acid de¢ciencies linked with dietary DP levels on
short or long-term feed intake is needed to optimize
dietary amino acid supply and utilization.
Nutritional regulation of amino acid metabolism
has already been dealt with in detail in a number of
in-depth reviews (Walton 1985; Cowey & Walton
1989; Dabrowski & Guderley 2002). At the hepatic
level, dietary protein levels appear to exert little e¡ect
on amino acid catabolism whereas there is a relatively good response of enzymes of amino acid metabolism to the corresponding amino acid intakes. The
lack of control by dietary protein levels on amino acid

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332


Aquaculture Research, 2010, 41, 322^332 Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez

oxidation in ¢sh contrasts with what is generally
seen in mammals and this is considered to be the major reason explaining teleosts’adaptation to high dietary protein levels.
Somatic growth involves irreversible transformation of dietary substrates and tissue energy stores
into tissue and organs. There is de¢nitely a good correlation between somatic growth rate and instantaneous protein synthesis rates (Haschemeyer & Smith
1979; Smith 1981; Fauconneau 1985; Carter & Houlihan 2001). Protein synthesis rates di¡er between tissues and the lowest instantaneous fractional protein
synthesis rates are seen in the white muscle and the
highest values in active tissues such as the liver or the
digestive tract (Fauconneau 1985; Fauconneau &
Arnal 1985; McMillan & Houlihan 1989b) like in
terrestrial vertebrates (Fig. 4). The e⁄ciency of deposition of synthesized protein is however high in the
muscle of ¢sh (Fauconneau 1985; Peragon, Ortegagarcia, Barroso, de la Higuera & Lupianez 1992;
Carter & Houlihan 2001). Nevertheless, while muscle
is the largest component of the lean body mass in ¢sh
as in most other vertebrates and muscle protein accounts for close to 50% of the body protein, muscle
protein synthesis rates represent only about 20% of
the whole body protein synthesis (Fig. 5).
A good correlation between metabolic rate and
protein turnover rates appears to exist across di¡erent species (Young 1991). Inclusion of data from rainbow trout ¢ts well to this general scheme (Fig. 6),
despite the low metabolic rates reported in ¢sh. The
energetic cost of protein synthesis in ¢sh is considered to be several fold higher than in mammals (Dabrowski & Guderley 2002) and protein oxidation
accounts for the most important source of energy in
¢sh (Weber & Haman 1996). Exerting control over
this oxidation and knowledge on the quantitative
contribution of individual amino acids towards

35
30
25
20
15

10
5
0

Ks, %/day
Fed

Fasted

Re-fed

Figure 4 Fractional rates of protein synthesis (%/day) in
various tissues of rainbow trout (redrawn from McMillan
& Houlihan 1989b).

(a)
Blood
Viscera 2%

Liver
1%

Adipose
3%

11%
Gills
3%
Head
13%

Muscle
55%

Fins
1%
Skin
11%

(b)

(c)
Other
25%

Other
42%

Muscle
22%

Muscle
50%
Liver
14%

Liver
1% Dig.Tract

Dig.Tract

39%

7%

Figure 5 Relative importance of tissue size (a) compared
with protein content (b) and protein synthesis rates in different tissues (c) in rainbow trout.

800

Mouse

Metabolic rate,
kJ/kg/d

700
600
500

Chickens

400

Rat

300
200
100
0
0

10

20
30
40
Protein turnover, g/kg/d

50

Figure 6 Relation between protein turnover and metabolic rates across species (data for terrestrial animals from
Young 1991 and for ¢sh from Fauconneau 1985).

this metabolic expenditure are areas worth further
investigation.
Fasting followed by re-feeding induces an increase
in protein turnover (Smith 1981; McMillan & Houlihan 1989a). The role of insulin, and insulin-like
growth factor 1 (IGF-1) as mediators of the anabolic
drive is well described in mammals (Millward 1989,
1995). Current knowledge in higher animals shows
that this is accomplished by stimulation of the mammalian target of rapamycin (mTOR), a cell signalling
pathway involved in the regulation of initiation of
mRNA translation (Garami, Zwartkruis, Nobukuni,
Joaquin, Roccio, Stocker, Kozma, Hafen, Bos &

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332

327

Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez Aquaculture Research, 2010, 41, 322^332

Thomas 2003; Tee, Manning, Roux, Cantley & Blenis
2003; Zhang, Cicchetti, Onda, Koon, Asrican, Bajraszewski, Vazquez, Carpenter & Kwiatkowski 2003). In
mammals, amino acids as well as insulin are known
to act as regulators of this TOR signalling pathway
(By¢eld, Murray & Backer 2005; Nobukuni, Joaquin,
Roccio, Dann, Kim, Gulati, By¢eld, Backer, Natt, Bos,
Zwartkruis & Thomas 2005; Kim, Goraksha-Hicks,
Li, Neufeld & Guan 2008; Sancak, Peterson, Shaul,
Lindquist, Thoreen, Bar-Peled & Sabatini 2008).
Although the mechanisms of regulation are complex
and little understood, we have recently been able to
show, in rainbow trout, that insulin and amino acids
regulate TOR signalling as in mammals (Seiliez, Panserat, Skiba-Cassy, Fricot, Vachot, Kaushik & Tesseraud 2008b), opening new research perspectives
on the molecular bases of amino acid utilization in
teleosts.
Given the dynamic status of protein turnover implying continuous protein synthesis and degradation
and because muscle protein synthesis rates are low
in ¢sh despite the high e⁄ciency of deposited protein,
it is essential to get full insight on the protein degradation pathways. In ¢sh, we do not yet have a clear
idea of the relative importance of the three major proteolytic systems operating in vivo (lysosome, Ca21 dependent and ubiquitin^proteasome dependent). The
ubiquitin^proteasome route of protein degradation
involves two discrete steps: ¢rst, multiple ubiquitin
molecules covalently attach to the protein substrate
(Ciechanover 1994; Goldberg 1995) and then these
tagged proteins are degraded by the proteasome
(Kornitzer & Ciechanover 2000), resulting in peptides of 7^9 amino acid residues (Voges, Zwickl &
Baumeister 1999). In mammals, the ATP-dependent
ubiquitin^proteasome proteolytic pathway is considered to be the major route of protein degradation involved in skeletal muscle loss and is regulated by the

nutritional status (Attaix & Taillandier 1998; Lecker,
Solomon, Mitch & Goldberg 1999; Jagoe & Goldberg
2001; Lecker, Jagoe, Gilbert, Gomes, Baracos, Bailey,
Price, Mitch & Goldberg 2004). In contrast, in rainbow trout (Oncorhynchus mykiss), the activity of the
proteasome in muscle does not change during starvation-induced muscle degradation (Martin, Blaney,
Bowman & Houlihan 2002). Furthermore, microarray gene expression analysis in atrophying rainbow
trout showed that mRNA levels for the subunits of
the proteasome were either not a¡ected or downregulated (Salem, Kenney, Rexroad III & Yao 2006),
leading to the suggestion that degradation of muscle
proteins in trout occurs by a route distinct from the

328

one observed in mammals (Mommsen 2004). But,
our own recent data show that, in the muscle of rainbow trout, the polyubiquitination step of the ubiquitin^proteasome route is regulated by feeding similar
to what is observed in mammals (Seiliez, Gabillard,
Skiba-Cassy, Garcia-Serrana, Gutierrez, Kaushik,
Panserat & Tesseraud 2008a) and supports the idea
that we have to reconsider the role of this proteolytic
route in muscle protein degradation and its nutritional regulation.

Future research
Given the general context of aquaculture and the importance of dietary protein/amino acids in aquafeeds, a number of areas need our attention.We need
to strengthen our understanding of the consequences of marginal amino acid imbalances under
minimal dietary DP levels capable of maximum
growth and physiological well being. Given the relatively high contribution of excess amino acids for energy, we need to gain knowledge on the relative
contributions or preferential utilization of individual
amino acid oxidation to overall metabolic demands.
Similarly, understanding the role of individual amino
acids directly or indirectly through hormonal factors

in eliciting the anabolic drive in the regulation of
muscle growth is necessary.While we know that signi¢cant ‘protein-sparing’ and reduction in nitrogenous losses are achieved by decreasing the DP/DE
ratios, we need to get more insight on the underlying
mechanisms especially as a¡ected by dietary factors.
Comprehensive data should bring forth more similarities than di¡erences between terrestrial animals
and aquatic organisms in their nitrogen metabolism
and utilization.

References
Akiyama T., Oohara I. & Yamamoto T. (1997) Comparison of
essential amino acid requirements with A/E ratio among
¢sh species (Review paper). Fisheries Science 63, 963^970.
Alam M.,Teshima S.I.,Yaniharto D., Ishikawa M. & Koshio S.
(2002) Dietary amino acid pro¢les and growth performance in juvenile kuruma prawn Marsupenaeus japonicus. Comparative Biochemistry and Physiology (Part B
Biochemistry and Molecular Biology) 133B, 289^297.
Amaya E.A., Davis D.A. & Rouse D.B. (2007) Replacement of
¢sh meal in practical diets for the Paci¢c white shrimp
(Litopenaeus vannamei) reared under pond conditions.
Aquaculture 262, 393^401.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332


Aquaculture Research, 2010, 41, 322^332 Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez

Attaix D. & Taillandier D. (1998) The critical role of the ubiquitin^proteasome pathway in muscle wasting in comparison to lysosomal and Ca21-dependent systems. In:
Advances in Molecular and Cell Biology (ed. by E.E. Bittar &
A.J. Rivett), pp. 235^266. JAI Press, Stamford, CT, USA.
Bender A.E. & Haizelden S. (1957) Biological value of the proteins of a variety of ¢sh meals. British Journal of Nutrition

11, 42^43.
Benedito-Palos L., Saera-Vila A., Calduch-Giner J.A., Kaushik
S. & Perez-Sanchez J. (2007) Combined replacement of ¢sh
meal and oil in practical diets for fast growing juveniles of
gilthead sea bream (Sparus aurata L.): networking of systemic and local components of GH/IGF axis. Aquaculture
267, 199^212.
Bergot F. & Breque J. (1983) Digestibility of starch by rainbow
trout: e¡ects of the physical state of starch and of the intake level. Aquaculture 34, 203^212.
Bowen H. (1987) Dietary protein requirements of ¢shes-A
reassessment. Canadian Journal of Fisheries & Aquatic
Sciences 44, 1995^2001.
Bureau D.P. (1997) The partitioning of energy from digestible
carbohydrates by rainbow trout (Oncorhynchus mykiss).
University of Guelph, Guelph, ON, Canada, 170pp.
By¢eld M.P., Murray J.T. & Backer J.M. (2005) hVps34 is a nutrient-regulated lipid kinase required for activation of p70
S6 kinase. 280, 33076^33082.
Carter C.G. & Houlihan D.F. (2001) Protein synthesis. In: Fish
Physiology.Vol. 20. Nitrogen Excretion (ed. by P.A.Wright &
P.M. Anderson), pp. 31^75. Academic Press, New York,
NY, USA.
Cho C.Y. & Bureau D.P. (2001) A review of diet formulation
strategies and feeding systems to reduce excretory and
feed wastes in aquaculture. Aquactic Research 32,
349^360.
Cho C.Y. & Kaushik S.J. (1990) Nutritional energetics in ¢sh: energy and protein utilization in rainbow trout (Salmo gairdneri).World Review of Nutrition and Dietetics 61,132^172.
Cho C.Y., Kaushik S. & Woodward B. (1992) Dietary arginine
requirement of young rainbow trout (Oncorhynchus mykiss). 102, 211^216.
Ciechanover A. (1994) The ubiquitin^proteasome proteolytic pathway. Cell 79,13^21.
Cowey C.B. (1994) Amino acid requirements of ¢sh ^ a critical appraisal of present values. Aquaculture 124, 1^11.
Cowey C.B. (1995) Protein and amino acid requirements: a

critique of methods. 11,199^204.
Cowey C.B. & Luquet P. (1983) Physiological basis of protein
requirements of ¢shes. Critical analysis of allowances. In:
IV International Symposium on Protein Metabolism and
Nutrition. Les Colloques INRA, No. 16 (ed. by M. Arnal, R.
Pion & D. Bonin), pp. 365^384.
Cowey C.B. & Walton M.J. (1988) Studies on the uptake of
(14C) amino acids derived from both dietary (14C) protein
and dietary (14C) amino acids by rainbow trout, Salmo
gairdneri Richardson. Journal of Fish Biology 33,
295^305.

Cowey C.B. & Walton M.J. (1989) Intermediary metabolism.
In: Fish Nutrition, 2nd edn, (ed. by IJEH.) pp. 259^329. Academic Press, NewYork, NY, USA.
Dabrowski K. & Guderley H. (2002) Intermediary metabolism.
In: Fish Nutrition (ed. by J.E. Halver & R.W. Hardy),3rd edn,
pp. 309^365. Elsevier, Amsterdam, the Netherlands.
De Francesco M., Parisi G., Perez-Sanchez J., Gomez-Requeni
P., Medale F., Kaushik S.J., Mecatti M. & Poli B.M. (2007)
E¡ect of high-level ¢sh meal replacement by plant
proteins in gilthead sea bream (Sparus aurata) on growth
and body/¢llet quality traits. Aquaculture Nutrition 13,
361^372.
de la Higuera M. (2001) E¡ects of nutritional factors and feed
characteristics on feed intake. In: Food Intake in Fish (ed.
by D. Houlihan, T. Boujard & M. Jobling), pp. 250^268.
Iowa State University Press, State Avenue Ames, IA, USA.
Deshimaru O. & Shigeno K. (1972) Introduction to the arti¢cial
diet for prawn Penaeus japonicus. Aquaculture 1, 115^133.
Dias J. (1999) Lipid Deposition in Rainbow Trout (Oncorhynchus mykiss) and European Sea Bass (Dicentrarchus labrax): Nutritional Control of Hepatic Lipogenesis. University

of Porto, Portugal & University of Bordeaux I, France,
190pp., annexes.
Enes P., Panserat S., Kaushik S. & Oliva-Teles A. (2008) Nutritional regulation of hepatic glucose metabolism in ¢sh
Fish Physiol. Biochemistry, in press.
Espe M., Lemme A., Petri A. & El-Mowa¢ A. (2006) Can
Atlantic salmon (Salmo salar) grow on diets devoid of ¢sh
meal? Aquaculture 255, 255^262.
FAO. (2007) The State of World Fisheries and Aquaculture
2006. FAO, Rome, p.162.
Fauconneau B. (1985) Protein synthesis and protein deposition in ¢sh. In: Nutrition and Feeding in Fish (ed. by C.B.
Cowey, A.M. Mackie & J.G. Bell), pp. 17^45. Academic
Press, London, UK.
Fauconneau B. & Arnal M. (1985) In vivo protein synthesis
in di¡erent tissues and the whole body of rainbow trout
(Salmo gairdnerii R.). In£uence of environmental temperature. A 82,179^187.
Fournier V., Gouillou Coustans M.F., Metailler R., Vachot C.,
Guedes M.J., Tulli F., Oliva-Teles A., Tibaldi E. & Kaushik
S.J. (2002) Protein and arginine requirements for maintenance and nitrogen gain in four teleosts. BritishJournal of
Nutrition 87, 459^469.
Francis G., Makkar H. & Becker K. (2001) Antinutritional
factors present in plant-derived alternate ¢sh feed ingredients and their e¡ects in ¢sh. Aquaculture 199, 197^227.
Furuichi M. & YoneY. (1982) Changes in activities of hepatic
enzymes related to carbohydrate metabolism of ¢shes
in glucose and insulin-glucose tolerance tests. 48,
463^466.
Garami A., Zwartkruis F.J., Nobukuni T., Joaquin M., Roccio
M., Stocker H., Kozma S.C., Hafen E., Bos J.L. & Thomas G.
(2003) Insulin activation of Rheb, a mediator of mTOR/
S6K/4E-BP signaling, is inhibited by TSC1 and 2. 11,
1457^1466.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332

329


Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez Aquaculture Research, 2010, 41, 322^332

Gatlin D.M., Barrows F.T., Brown P., Dabrowski K., Gaylord
T.G., Hardy R.W., Herman E., Hu G., Krogdahl A., Nelson
R., Overturf K., Rust M., Sealey W., Skonberg D., Souza
E.J., Stone D., Wilson R. & Wurtele E. (2007) Expanding
the utilization of sustainable plant products in aquafeeds:
a review. Aquatic Research 38, 551^579.
Goldberg A.L. (1995) Functions of the proteasome: the lysis
at the end of the tunnel. Science 268, 522^523.
Gomez-Requeni P., Mingarro M., Kirchner S., CalduchGiner J.A., Medale F., Corraze G., Panserat S., Martin
S.A.M., Houlihan D.F., Kaushik S.J. & Perez-Sanchez J.
(2003) E¡ects of dietary amino acid pro¢le on growth performance, key metabolic enzymes and somatotropic axis
responsiveness of gilthead sea bream (Sparus aurata).
Aquaculture 220,749^767.
Green J.A. & Hardy R.W. (2002) The optimum dietary essential
amino acid pattern for rainbow trout (Oncorhynchus mykiss),
to maximize nitrogen retention and minimize nitrogen excretion. Fish Physiology and Biochemistry 27, 97^108.
Green J.A., Hardy R.W. & Brannon E.L. (2002) The optimum
dietary essential: nonessential amino acid ratio for rainbow trout (Oncorhynchus mykiss), which maximizes nitrogen retention and minimizes nitrogen excretion. Fish
Physiology and Biochemistry 27, 109^115.
Gurure R., Atkinson J. & Moccia R.D. (2007) Amino acid
composition of Arctic charr, Salvelinus alpinus (L.) and

the prediction of dietary requirements for essential amino
acids. Aquaculture Nutrition 13, 266^272.
Haschemeyer A.E.V. & Smith M.A.K. (1979) Protein synthesis
in liver, muscle and gill of mullet (Mugil cephalus L.)
in vivo. 156, 93^102.
Hemre G.I., MommsenT.P. & Krogdahl A. (2002) Carbohydrates
in ¢sh nutrition: e¡ects on growth, glucose metabolism
and hepatic enzymes. Aquaculture Nutrition 8,175^194.
Hillestad M. & Johnsen F. (1994) High energy/low protein
diets for Atlantic salmon: e¡ects on growth, nutrient retention and slaughter quality. Aquaculture 124, 109^116.
Hughes S.G. (1985) Evaluation of glutamic acid and glycine
as sources of nonessential amino acids for lake trout (Salvelinus namaycush) and rainbow trout (Salmo gairdneri).
81A, 669^671.
Jagoe R.T. & Goldberg A.L. (2001) What do we really know
about the ubiquitin^proteasome pathway in muscle atrophy? Current Opinion in Clinical Nutrition and Metabolic
Care 4, 183^190.
Kaushik S.J. (1998) Whole body amino acid composition of
European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata) and turbot (Psetta maxima) with an
estimation of their IAA requirement pro¢les. Aquatic Living Resources 11, 355^358.
Kaushik S.J. & Cowey C.B. (1991) Ammoniogenesis and dietary factors a¡ecting nitrogen excretion. In: Nutritional
Strategies & Aquaculture Waste (ed. by C.B. Cowey & C.Y.
Cho), pp. 3^19. Univerity of Guelph, Guelph, Canada.
Kaushik S.J. & Hemre G.-I. (2008) Plant proteins as alternative sources for ¢sh feed and farmed ¢sh quality. In: Im-

330

proving Farmed Fish Quality and Safety (ed. by O. Lie), pp.
300^327.Woodhead Publishing Limited, Cambridge, UK.
Kaushik S.J. & Oliva-Teles A. (1985) E¡ect of digestible energy
on nitrogen and energy balance in rainbow trout. Aquaculture 50, 89^101.

Kaushik S.J., Breque J. & Blanc D. (1991) Requirements for
protein and essential amino acids and their utilization by
Siberian sturgeon (Acipenser baeri). In: Acipenser. Actes du
colloque (ed. by P.Williot), pp. 25^39. Cemagref, Bordeaux,
France.
Kaushik S.J., Cravedi J.P., Lalles J.P., Sumpter J., Fauconneau
B. & Laroche M. (1995) Partial or total replacement of ¢sh
meal by soybean protein on growth, protein utilization,
potential estrogenic or antigenic e¡ects, cholesterolemia
and £esh quality in rainbow trout, Oncorhynchus mykiss.
Aquaculture 133, 257^274.
Kaushik S.J., Coves D., Dutto G. & Blanc D. (2004) Almost total replacement of ¢sh meal by plant protein sources in
the diet of a marine teleost, the European seabass, Dicentrarchus labrax. Aquaculture 230, 391^404.
Kim E., Goraksha-Hicks P., Li L., Neufeld T.P. & Guan K.L.
(2008) Regulation of TORC1 by Rag GTPases in nutrient
response. Nature Cell Biology 10, 935^945.
Kim J. & Lall S.P. (2000) Amino acid composition of whole
body tissue of Atlantic halibut (Hippoglossus hippoglossus),
yellowtail £ounder (Pleuronectes ferruginea) and Japanese
£ounder (Paralichthys olivaceus). Aquaculture 187, 367^
373, 2000.
Kornitzer D. & Ciechanover A. (2000) Modes of regulation of
ubiquitin-mediated protein degradation. Journal of Cellular Physiology 182, 1^11.
Lall S.P. & Anderson S. (2005) Amino acid nutrition of salmonids: dietary requirements and bioavailability. In: Cahiers Options Me¤ diterrane¤ ens (ed. by D. Montero, B. Basurco,
I. Nengas, M. Alexis & M. Izquierdo), pp. 73^90. CIHEAM,
Zaragoza, Spain.
Lecker S.H., Solomon V., Mitch W.E. & Goldberg A.L. (1999)
Muscle protein breakdown and the critical role of the ubiquitin^proteasome pathway in normal and disease states.
Journal of Nutrition 129, 227S^237S.
Lecker S.H., Jagoe R.T., Gilbert A., Gomes M., Baracos V., Bailey J., Price S.R., MitchW.E. & Goldberg A.L. (2004) Multiple

types of skeletal muscle atrophy involve a common program of changes in gene expression. Federation of American Societies for Experimental Biology Journal 18, 39^51.
Lee D.J. & Putnam G.B. (1973) The response of rainbow trout
to varying protein/energy ratios in a test diet. Journal of
Nutrition 103, 916^922.
Liebert F. & Benkendor¡ K. (2007) Modeling lysine requirements of Oreochromis niloticus due to principles of the diet
dilution technique. Aquaculture 267, 100^110.
Lim C. (1993) E¡ect of dietary pH on amino acid utilization
by shrimp (Penaeus vannamei). Aquaculture 114, 293^303.
Lim C., Webster C.D. & Lee C.-S. (2008) Alternative Protein
Sources in Aquaculture Diets. Haworth Press, New York,
NY, USA.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332


Aquaculture Research, 2010, 41, 322^332 Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez

Mambrini M. & Kaushik S. (1995a) E¡ect of temperature on
sulfur amino acid requirements for maintenance and
growth of juvenile rainbow trout. In: Proceedings of the
7th International Symposium on Protein Metabolism and
Nutrition (ed. by A.F. Nunes, J.P. Portugal, J.P. Costa & J.R.
Ribeiro), pp. 117 122. EAAP Publication no. 81, Santarem,
Portugal.
Mambrini M. & Kaushik S.J. (1994) Partial replacement of
dietary protein nitrogen with dispensable amino acids in
diets of Nile Tilapia, Oreochromis Niloticus. Comparative
Biochemistry and Physiology A ^ Comparative Physiology
109, 469^477.

Mambrini M. & Kaushik S.J. (1995b) Indispensable amino
acid requirements of ¢sh: correspondence between quantitative data and amino acid pro¢les of tissue proteins.
Journal of Applied Ichthyology 11, 240^247.
Manuel Vergara A., Robaina L., Izquierdo M. & Higuera
M.D.L. (1996) Protein sparing e¡ect of lipids in diets for
¢ngerlings of gilthead sea bream. 62, 624^628.
Martin S.A., Blaney S., Bowman A.S. & Houlihan D.F. (2002)
Ubiquitin^proteasome-dependent proteolysis in rainbow
trout (Oncorhynchus mykiss): e¡ect of food deprivation.
P£ugers Archives 445, 257^266.
McMillan D.N. & Houlihan D.F. (1989a) The e¡ect of refeeding on tissue protein synthesis in rainbow trout. Physiological Zoology 61, 429^441,1988.
McMillan D.N. & Houlihan D.F. (1989b) Short-term responses of protein synthesis to re-feeding in rainbow
trout. Aquaculture 79, 37^46, 1989.
Mercer L.P. (1982) The quantitative nutrient^response relationship. Journal of Nutrition 112, 560^566.
Millward D.J. (1989) The nutritional regulation of muscle
growth and protein turnover. Aquaculture 79, 1^28,
1989.
Millward D.J. (1995) A protein-stat mechanism for regulation of growth and maintenance of the lean body mass.
Nutrition Research Reviews 8, 93^120.
Mohanty S. & Kaushik S.J. (1991) Whole body amino acid
composition of Indian major carps and its signi¢cance.
Aquatic Living Resources 4, 61^64.
Mommsen T.P. (2004) Salmon spawning migration and
muscle protein metabolism: the August Krogh principle
at work. Comparative Biochemistry and Physiology Part B:
Biochemistry and Molecular Biology 139, 383^400.
Moon T.W. (2001) Glucose intolerance in teleost ¢sh: fact or
¢ction? Comparative Biochemistry and Physiology Part B:
Biochemistry and Molecular Biology 129, 243^249.
Murai T.H., AkiyamaY. & Nose T. (1983) E¡ects of dietary pH

and electrolyte concentration on utilization of crystalline
amino acids by ¢ngerling carp. 49,1377^1380.
Ng W.K. & Hung S.S.O. (1995) Estimating the ideal dietary
indispensable amino acid pattern for growth of white
sturgeon, Acipenser transmontanus (Richardson). Aquaculture Nutrition 1, 85^94.
Nobukuni T., Joaquin M., Roccio M., Dann S.G., Kim S.Y.,
Gulati P., By¢eld M.P., Backer J.M., Natt F., Bos J.L.,

Zwartkruis F.J. & Thomas G. (2005) Amino acids mediate
mTOR/raptor signaling through activation of class 3
phosphatidylinositol 3OH-kinase. Proceedings of the
National Academy of Sciences of the United States of America
102,14238^14243.
Nose T., Arai S., Lee D.L. & HashimotoY. (1974) Note on amino acids essential for growth of young carp. Bulletin of the
Japanese Society of Scienti¢c Fisheries 40, 903^908.
National Research Council. (1993) Nutrient Requirements of
Fish. National Academy of Sciences, Washington, DC,
USA.
Ogino C. (1980) Requirements of carp and rainbow trout for
essential amino acids. 46, 171^174.
Panserat S. & Kaushik S. (2002) Re¤gulation nutritionelle du
me¤tabolisme glucidique chez les poissons: exemple de la
truite arc-en-ciel (Oncorhynchus mykiss), faible utilisatrice
des glucides alimentaires. INRA Productions Animales 15,
109^117.
Panserat S., Medale F., Blin C., Breque J.,Vachot C., Plagnes.Juan E., Gomes E., Krishnamoorthy R. & Kaushik S.
(2000) Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp. American Journal of Physiology Regulatory
Integrative and Comparative Physiology 278, R1164^
R1170.
Penìa£orida V.D. (1989) An evaluation of indigenous protein

sources as potential component in the diet formulation
for tiger prawn, Penaeus monodon, using essential amino
acid index (EAAI). Aquaculture 83, 319^330.
Peragon J., Ortegagarcia F., Barroso J.B., de la Higuera M. &
Lupianez J.A. (1992) Alterations in the fractional proteinturnover rates in rainbow-trout liver and white muscle
caused by an amino-acid-based diet and changes in the
feeding frequency. Toxicological and Environmental Chemistry 36, 217^224.
Plakas S.M.K.,Tanaka Y. & Deshimaru O. (1980) Changes in
the levels of circulating plasma free amino acids of carp
(Cyprinus carpio) after feeding a protein and an amino
acid diet of similar composition. Aquaculture 21, 307^332.
Regost C. (2001) E¡ects of Lipids on the Nutritional, Physical
and Organoleptic Quality of the Flesh in BrownTrout (Salmo
trutta) and Turbot (Psetta maxima). University Rennes I,
Rennes, France 193pp.
Rodehutscord M., Mandel S., Pack M., Jacobs S. & Pfe¡er E.
(1995) Free amino acids can replace protein-bound amino
acids in test diets for studies in rainbow trout
(Oncorhynchus mykiss). Journal of Nutrition 125,
956^963.
Rodehutscord M., Becker A., Pack M. & Pfe¡er E. (1997) Response of rainbow trout (Oncorhynchus mykiss) to supplements of individual essential amino acids in a
semipuri¢ed diet, including an estimate of the maintenance requirement for essential amino acids. Journal of
Nutrition 127,1166^1175.
Rollin X., Mambrini M., Abboudi T., LarondelleY. & Kaushik
S.J. (2003) The optimum dietary indispensable amino acid

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332

331

Protein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez Aquaculture Research, 2010, 41, 322^332

pattern for growing Atlantic salmon (Salmo salar L.) fry.
BritishJournal of Nutrition 90, 865^876.
Salem M., Kenney P.B., Rexroad C.E. III & Yao J. (2006) Microarray gene expression analysis in atrophying rainbow
trout muscle: a unique non-mammalian muscle degradation model. Physiological Genomics 28, 33^45.
SancakY., Peterson T.R., Shaul Y.D., Lindquist R.A.,Thoreen
C.C., Bar-Peled L. & Sabatini D.M. (2008) The Rag GTPases
bind raptor and mediate amino acid signaling to mTORC1.
Science 320, 1496^1501.
Sargent J.R. (1997) Fish oils and human diet. British Journal
of Nutrition 78, S5^S13.
Sargent J.R.,Tocher D.R. & Bell G.J. (2002) The lipids. In: Fish
Nutrition, 3rd edn, ed. by J.E. Halver & R.W. Hardy), pp.
181^257. Academic Press and Elsevier Science, New York
, NY, USA and Amsterdam, the Netherlands.
Satoh S., Alam M.A., Satoh K.-I. & Kiron V. (2004) E¡ects
of dietary lipid and phosphorus levels on nitrogen
and phosphorus excretion in young yellowtail
Seriola quinqueradiata: a preliminary observation.
pp. 1082^1088.
Seiliez I., Gabillard J.-C., Skiba-Cassy S., Garcia-Serrana D.,
Gutierrez J., Kaushik S., Panserat S. & Tesseraud S.
(2008a) An in vivo and in vitro assessment of TOR signaling cascade in rainbow trout (Oncorhynchus mykiss).
American Journal of Physiology Regulatory Integrative and
Comparative Physiology 295, R329^R335.
Seiliez I., Panserat S., Skiba-Cassy S., Fricot A., Vachot C.,
Kaushik S. & Tesseraud S. (2008b) Feeding status

regulates the polyubiquitination step of the ubiquitin^
proteasome-dependent proteolysis in rainbow trout
(Oncorhynchus mykiss) muscle. Journal of Nutrition 138,
487^491.
Shiau S.Y. & Lin Y.H. (2001) Carbohydrate utilization and its
protein-sparing e¡ect in diets for grouper (Epinephelus malabaricus). Animal Science 73, 299^304.
Smith M.A.K. (1981) Estimation of growth potential by measurement of tissue protein synthetic rates in feeding and
fasting rainbow trout, Salmo gairdnerii Richardson. Journal of Fish Biology 19, 213^220.
Tacon A.G.J. (1997) Fishmeal replacers: review of antinutrients within oilseeds and pulses. A limiting factor for the
aquafeed green revolution? In: Feeding Tomorrow’s Fish
(ed. byA.G.J. Tacon & B. Basurco), pp.153^182. Cahiers Options Me¤ diterrane¤ ennes Zaragoza, Spain.
Taylor T.P., Morris J.G., Kass P.H. & Rogers Q.R. (1998) Maximal growth occurs at a broad range of essential amino
acids to total nitrogen ratios in kittens. Amino Acids 15,
221^234.
Tee A.R., Manning B.D., Roux P.P., Cantley L.C. & Blenis J.
(2003) Tuberous sclerosis complex gene products,Tuberin and Hamartin, control mTOR signaling by acting as a
GTPase-activating protein complex toward Rheb. Current
Biology 13,1259^1268.
Teshima S., Alam M.S., Koshio S., Ishikawa M. & Kanazawa
A. (2002) Assessment of requirement values for essential

332

amino acids in the prawn, Marsupenaeus japonicus (Bate).
Aquatic Research 33, 395^402.
Tibaldi E. & Kaushik S.J. (2005) Amino acid requirements of
Mediterranean ¢sh species. Cahiers Options Me¤ diterrane¤ ennes 63, 59^65.
Voges D., Zwickl P. & Baumeister W. (1999) The 26S
proteasome: a molecular machine designed for
controlled proteolysis. Annual Review of Biochemistry 68,

1015^1068.
Walton M.J. (1985) Aspects of amino acid metabolism in teleost ¢sh. In: Nutrition and Feeding in Fish (ed. by C.B. Cowey,
A.M. Mackie & J.G. Bell), pp. 47^67. Academic Press, London, UK.
Watanabe T.,Verakunpiriya V.,Watanabe K.,Viswanath K. &
Satoh S. (1998) Feeding of rainbow trout with non-¢sh
meal diets. Fisheries Science 63, 258^266, 1997.
Watanabe T., Aoki H.,Watanabe K., Maita M.,YamagataY. &
Satoh S. (2001) Quality evaluation of di¡erent types of
non-¢sh meal diets for yellowtail. Fisheries Science 67,
461^469.
Weber J.M. & Haman F. (1996) Pathways for metabolic fuels
and oxygen in high performance ¢sh. Comparative Biochemistry & Physiology 113, 33^38.
Williams K., Barlow C. & Rodgers L. (2001) E⁄cacy of crystalline and protein-bound amino acids for amino acid enrichment of diets for barramundi/Asian seabass (Lates
calcarifer Bloch). Aquatic Research 32, 415^429.
Wilson R. (1994) Review: utilization of dietary carbohydrate
by ¢sh. Aquaculture 124, 67^80.
Wilson R.P. (2002) Amino acids and proteins. In: Fish Nutrition, 3rd edn, ed. by J.E. Halver & R.W. Hardy), pp.143^179.
Academic Press, NewYork, NY, USA.
Wilson R.P. & Cowey C.B. (1985) Amino acid composition of
whole body tissue of rainbow trout and Atlantic salmon.
Aquaculture 48, 373^376.
Yamada S., Tanaka Y. & Katayama T. (1981) Feeding experiments with carp fry fed an amino acid diet by increasing
the number of feedings per day. 47,1247.
Yamamoto T., Shima T. & Furuita H. (2004) Antagonistic effects of branched-chain amino acids induced by excess
protein-bound leucine in diets for rainbow trout (Oncorhynchus mykiss). Aquaculture 232, 539^550.
Young V. & El-Khoury A. (1995) Can amino acid requirements for nutritional maintenance in adult humans be
approximated from the amino acid composition of body
mixed proteins? 92, 300^304.
Young V.R. (1991) Some basic aspects of protein^energy interrelationships. In: Protein^Energy Interactions. Proceedings of an IDECG Workshop (ed. by N.S. Scrimshaw & B.
Schˇrch), pp. 16^29. UNU Lausanne, Lausanne, Switzerland.

Zhang H., Cicchetti G., Onda H., Koon H.B., Asrican K., Bajraszewski N., Vazquez F., Carpenter C.L. & Kwiatkowski
D.J. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts
PI3K-Akt signaling through downregulation of PDGFR.
112,1223^1233.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 322^332


Aquaculture Research, 2010, 41, 333^344

doi:10.1111/j.1365-2109.2009.02426.x

Important antinutrients in plant feedstuffs for
aquaculture: an update on recent findings regarding
responses in salmonids
—shild Krogdahl1,2, Michael Penn1,2, Jim Thorsen1,2, StÔle Refstie1,3 & Anne Marie Bakke1,2
1

Aquaculture Protein Centre, Centre of Excellence, —s, Norway,

2

Norwegian School of Veterinary Science, Oslo, Norway,

3

No¢ma Marin, SunndalsÖra, Norway

Correspondence: — Krogdahl, Norwegian School of Veterinary Science, PO Box 8146 Dep., NO-0033 Oslo, Norway.

E-mail:

Abstract
This review presents an overview of antinutritive factors (ANFs) relevant for ¢sh nutrition. The sources of
ANFs and the possibilities of reducing the impact of
ANFs are brie£y mentioned. Proteinase inhibitors,
lectins, saponins and oligosaccharides are given a
more thorough presentation regarding mechanisms
of action and the state of knowledge regarding e¡ects
on gut function in ¢sh and upper safe dietary levels.
Thereafter, selected results from recent works addressing the involvement of T cells and proteinaseactivated receptors in soybean-induced enteritis are
summarized. Our conclusions are as follows: we are
only beginning to understand e¡ects of ANFs in ¢sh;
strengthening of the knowledge base is urgently
needed to understand the e¡ects and to ¢nd the
means to overcome or modify these e¡ects; interactions between the e¡ects of ANFs appear to be very
important; the microbiota may modify the e¡ects of
ANFs; not only salmonids are a¡ected; not only soybeans contain ANFs of biological importance in ¢sh;
and with increased knowledge, we can develop better
diets for optimal nutrition, health and economy in
aquaculture.

Keywords: ¢sh, antinutrients, digestive physiology,
immunology, gut health

Introduction
Nature has equipped many plants with the ability to
synthesize a variety of chemical substances with the

r 2010 The Authors

Journal Compilation r 2010 Blackwell Publishing Ltd

apparent function of protecting them from becoming
food for microbes, insects and higher animals. Consequently, many of these compounds may exert
harmful e¡ects when ingested by humans and animals. Such substances are often called antinutritive
factors (ANFs), although they may also have bene¢cial e¡ects, such as being antioxidative, immunostimulatory or prebiotic, depending on the amount
ingested. Possible harmful e¡ects include reduced palatability, less e⁄cient utilization of feed nutrients for
growth, altered nutrient balances of the diets, inhibition of growth, intestinal dysfunction, altered gut
micro£ora, immune modulation, goitrogenesis, pancreatic hypertrophy, hypoglycaemia or liver damage.
The species of animal, its age, size, gender, state of
health and plane of nutrition and any stress factors
may modify these responses.
In his textbook on toxic constituents of plant foods,
Liener (1980) wrote the following: ‘What has only recently been realized is that although there might not
be an immediate violent reaction to a certain food
component there might still be a slow cumulative adverse e¡ect resulting in overt disease or less than optimal health. This poses a great challenge, since
knowledge of these e¡ects is gained slowly and with
di⁄culty, particularly if the causative principles remain unidenti¢ed’. Liener’s statement is still valid
and relevant. During the last 10^15 years, inclusion
of plant feedstu¡s in ¢sh feed has increased markedly
and consequently so has exposure to ANFs. Antinutritive factors are novel to most cultivated ¢sh species,
particularly carnivorous species. Fish nutritionists
should keep in mind Liener’s warning that ‘there

333


An update on antinutrients in aquaculture feeds — Krogdahl et al.

may be slow cumulative adverse e¡ects resulting in

overt disease or less than optimal health’. It is not unlikely that ANFs are involved in the aetiology of diseases that are emerging in the aquaculture industry
today related to gut function and the immune apparatus, for example enteritis, low protein and lipid digestibility, diarrhoea and neoplasia.

Important antinutrients
Antinutrients are de¢ned as endogenous compounds
in feedstu¡s that may reduce feed intake, growth, nutrient digestibility and utilization, a¡ect the function
of internal organs or alter disease resistance. In plant
feedstu¡s, these include structural components such
as ¢bres, components storing nutrients and energy
such as phosphorous-rich phytic acid and a-galactoside oligosaccharides, allergens and various inherent
chemical defences against being eaten. This paper
does not intend to provide a comprehensive review
of current knowledge in the area but to present key
information on selected ANFs and to brie£y review
new ¢ndings in the area. Two previous review papers
(Francis, Makkar & Becker 2001; Gatlin, Barrows,
Brown, Dabrowski, Gaylord, Hardy, Herman, Hu,
Krogdahl, Nelson, Overturf, Rust, Sealey, Skonberg,
Souza, Stone, Wilson & Wurtele 2007) provide informative overviews of the general biological e¡ects of
ANFs and summarized the state of knowledge at
their respective times of publication. Among the relevant ANFs are: soluble as well as insoluble ¢bres that
interfere with digestion, absorption and utilisation of
macro- as well as micro-nutrients (van der Kamp,
Asp, Miller Jones & Schrama 2004); phytic acid, which
impairs mineral digestion and contains phosphorus
in a form unavailable to monogastrics (Thompson
1993; Eeckhout & Depaepe 1994); enzyme inhibitors,
which may slow digestion of protein, carbohydrates
and lipids (Krogdahl & Holm 1979; Liener 1980; Krogdahl & Holm1981; Berg-Lea, BrattÔs & Krogdahl1989;
Thompson1993); lectins, which bind to gut cell receptors, possibly stimulating intestinal growth, make the

gut more permeable for increased in£ux of macromolecules and bacteria, stimulate insulin production
and alter metabolism (Grant 1991); saponins, which
interfere with lipid and protein digestion and may increase the permeability of the gut mucosa (Liener
1980; Cuadrado, Ayet, Burbano, Muzquiz, Camacho,
Cavieres, Lovon, Osagie & Price 1995); glucosinolates,
which decrease the uptake of iodine into the thyroid
gland and may lead to goitre unless the iodine level in

334

Aquaculture Research, 2010, 41, 333^344

the diet is increased accordingly (Liener1980); phytoestrogens (iso£avons/coumestan), which may interfere
with the e¡ects of endogenous oestrogen (Mazur &
Adlercreutz 1998); phytosterols, which may interfere
with cholesterol absorption and metabolism (Ostlund, Racette & Stenson 2003); quinolizidine alkaloids, such as lupanin, which may cause nervous
system symptoms and intestinal disorders (Wink,
Schmeller & Latz-Bruning 1998); or oligosaccharides,
which may alter microbiota of the intestinal tract
and increase osmotic pressure in the intestine if not
metabolized by the microbiota (Wiggins 1984; Cummings, Englyst & Wiggins 1986).
Some ANFs are easy to eliminate by processing,
and others are more di⁄cult to eliminate. There are
also examples for which common processing steps,
such as heat treatment, may activate the antinutritional e¡ects. For all ANFs, fermentation or enzyme
treatments directly focusing on inactivation of a speci¢c ANF may reduce content or activity in the feedstu¡. Selective breeding and genetic modi¢cation
may also alter the content. E¡orts are being made in
several laboratories to modify the ANF contents of
important crops such as soybeans and rapeseeds
(Shewry, Tatham & Halford 2001). However, genetic

modi¢cation may also result in unintended alterations in the contents of ANFs (Cellini, Chesson,
Colquhoun, Constable, Davies, Engel, Gatehouse,
Karenlampi, Kok & Leguay 2004). Table1lists the major ANFs present in a variety of feedstu¡s and treatments that may reduce biological activity either by
elimination or by inactivation.

Proteinase inhibitors
Inhibitors of proteinases, i.e. of trypsin, chymotrypsin, elastases and carboxypeptidases, are proteins
that form stoichiometric complexes with the respective enzymes and inhibit their activity in the gastrointestinal (GI) tract. Proteinase inhibitors are found
in most plants (Liener 1980). In addition, enterokinase inhibitors have been described (Bhat, Jacob &
Pattabiraman 1981). The molecular weight of proteinase inhibitors is found in the range between 6000
and 50 000 kDa and their speci¢cities vary considerably. Some inhibit only one type of enzyme, e.g. trypsin or chymotrypsin, and others inhibit two or three.
Some inhibit one enzyme molecule per inhibitor
molecule, and others inhibit two or more. An overview of plants with endogenous proteinase inhibitors
commonly used as food has been provided by Belitz

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344


Aquaculture Research, 2010, 41, 333^344

An update on antinutrients in aquaculture feeds — Krogdahl et al.

Table 1 Antinutrients in common plant ingredients and
treatments that will eliminate or reduce biological activity
Antinutrient

Sources

Type of treatment

Proteinase
inhibitors
Amylase inhibitors
Lipase inhibitor
Lectins

Legumes

Saponins
Sterols

Peas
Beans
All plants
seeds
All plants
All plants
Rape seed,
beans
Legumes
Legumes

Oestrogens
Gossypol

Beans
Cotton seed

Oligosaccharides

Quinolozidine
alkaloids
Goitrogens

Legumes
Lupins

Heat, methionine
supplementation
Heat
Heat
Heat, supplementation
with specific carbohydrates
Mineral supplementation
Dehulling
Dehulling, restriction of heat
treatment
Alcohol extraction
Alcohol/non-polar
extraction, cholesterol
supplementation
Alcohol/non-polar extraction
Non-polar extraction, iron
supplementation
Alcohol/aqueous extraction
Aqueous extraction

Rape seed

Iodine supplementation

Phytic acid
Fibre
Tannins

For all antinutrients, levels may be reduced by fermentation, treatment with enzymes that speci¢cally inactivate the compound,
selective breeding and genetic modi¢cation.

stabilize the molecule and make it relatively stable to
proteolytic breakdown, acid denaturation as well as
heat. This inhibitor may bind one trypsin and one
chymotrypsin simultaneously. Inhibitors with a similar structure can be found in several legumes. The
larger (21000 kDa) Kunitz inhibitor with a speci¢c
site for trypsin may also bind chymotrypsin but in a
less stable complex and only one enzyme molecule is
inhibited at a time.
The e¡ects of proteinase inhibitors have been studied thoroughly in mammals and birds since their
discovery in 1947 (Kunitz 1947). Based on these studies, an understanding of their actions has been developed (Liener 1980). In the intestine, inhibitors (e.g.
a trypsin inhibitor) ¢rst form a rather stable complex
with trypsin, thereby reducing trypsin activity. This
in turn stimulates the secretion of pancreozymin^
cholecystokinin from the gut wall. This hormone
stimulates the secretion of trypsin from pancreatic
tissue and stimulates the gall bladder to empty its
contents into the intestine. Trypsin synthesis in the
pancreas is stimulated, resulting in an increased requirement for protein and for cysteine in particular
as trypsin is very rich in cysteine. In some animals,
proteinase inhibitors cause pancreatic hypertrophy.
Whether this also takes place in ¢sh is not clear.
In studies with salmonids, proteinase inhibitors

have been found to reduce the apparent digestibility
of both protein and lipid (Berg-Lea et al. 1989; Krogdahl, Berg Lea & Olli 1994; Olli & Krogdahl 1994; Olli,
Krogdahl, van den Ingh & BrattÔs 1994). Among the
fatty acids, the longer saturated and monounsaturated were most a¡ected. The e¡ects on the digestibilities correlated with a decrease in trypsin activity and

Figure 1 The amino acid sequence of the Bowman^Birk
inhibitor from soybeans. Amino acids interacting with
trypsin (Ser-Lys) and chymotrypsin (Leu-Ser) are marked
in grey, whereas the seven cystin bridges are shown in
black (adapted from Ikenaka et al.1974).

& Grosch (1999), including their molecular weights
and speci¢cities.
Most plant proteinase inhibitors belong either to
the Kunitz inhibitor family or to the Bowman^Birk
inhibitor, both ¢rst observed in soybeans (Liener
1980). The amino acid structure of the Bowman^Birk
inhibitor from soybeans is shown in Fig. 1. The inhibitor has a molecular weight of about 8000 kDa and
is characterized by the seven disulphide bridges that

Figure 2 E¡ects of level of soybean proteinase inhibitors
in diets for Atlantic salmon on protein, lipid, fatty acid
(C18:0) and dry matter digestibility and on trypsin activity
of chyme in the proximal intestine (Berg-Lea et al.1989).

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344

335

An update on antinutrients in aquaculture feeds — Krogdahl et al.

Aquaculture Research, 2010, 41, 333^344

Figure 3 E¡ects of increasing levels of proteinase inhibitors in rainbow trout diet on trypsin activity and trypsin protein
in intestinal contents. The samples were taken from the mid intestine between the distal most pyloric caecum and the
distal intestine (Olli et al.1994).

presumably also in chymotrypsin, both of which are
inhibited by soybean proteinase inhibitors (Fig. 2).
The results presented in Fig. 3 indicate that the proteinase inhibitors stimulated pancreatic enzyme secretion, causing the enzyme protein level of the
intestinal content (trypsin protein) to increase. However, the activity in the intestinal content was not increased. Enzyme activity appeared to be una¡ected at
the lower inhibitor levels and short-term feeding (12
days), but higher levels decreased the activity. After
longer term feeding, it appeared that the pancreas
no longer managed to compensate for decreased enzyme activity by increased secretion. Enzyme production appeared not to keep up with the increased
demand.
Work by Krogdahl et al. (1994) investigated the effects of increasing levels of dietary proteinase inhibitors on the digestibilities of protein and cysteine in
intestinal segments along the GI tract of rainbow trout.
The results support the ¢ndings of the previous study
(Berg-Lea et al. 1989), indicating that the inhibitors increased the secretion of cysteine-rich pancreatic enzymes into the GI tract. The level of cysteine of the
digesta increased drastically in the pyloric segments
of the intestine in which pancreatic secretions are assumed to enter the GI tract, yielding a negative apparent digestibility in the pyloric region of the intestine.
Many ¢sh studies have been performed with feedstu¡s showing varying proteinase inhibitor activities
(1^30 g kg À 1). From these studies, it is apparent that
there are considerable di¡erences in sensitivity between ¢sh species. Salmonids and Nile tilapia may
be particularly sensitive (Francis et al. 2001). Most ¢sh
species seem to be able to compensate when proteinase inhibitor activities are below 5 g kg À 1 (Olli &
Krogdahl 1994; Olli et al. 1994; Francis et al. 2001).

336

Figure 4 The agglutinating (clumping) e¡ect of a model
lectin on cells. A lectin is a protein with speci¢c a⁄nity for
carbohydrate moieties for example on cell receptors. Each
molecule may bind to more than one carbohydrate moiety
and therefore can bind more than one cell and agglutinate
the cells. The lectin e¡ects depend on the function of the
receptors.

Lectins
Lectins, also called haemagglutinins, are sugar binding proteins apparently present in all organisms with
metabolic and protective functions. Lectins are produced in most plant feedstu¡s, particularly in legumes and cereals (1^20 g kg À 1). They are
monomers, dimers or polymers of the same peptide
chain, or more complex aggregates of di¡erent peptides (Fig. 4). Lectins di¡er in speci¢city to which type
of carbohydrate they bind. Their mode of action is
mediated through binding to glycated cell receptors,
which may cause receptor activation. Lectins with
two or more binding sites will agglutinate cells with
receptors of similar glycation, e.g. blood cells and enterocytes. Because various receptors di¡er in glyca-

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344


Aquaculture Research, 2010, 41, 333^344

An update on antinutrients in aquaculture feeds — Krogdahl et al.

tion, their susceptibility to a particular lectin may differ. Hendriks, van den Ingh, Krogdahl, Olli and Koninkx (1990) studied binding of soybean lectins to
enterocytes from Atlantic salmon. The results
showed that tissues from both the proximal and the
distal intestine bind this lectin. The binding appeared
to be stronger for the cells from the distal than the
proximal intestine. The e¡ects of binding depend on
the receptor’s function. In the gut, some lectins a¡ect
the regulation of transport, intestinal hormone release, proliferation of various mucosal cells and may
also alter nutrient metabolism depending on the
function and signalling of the a¡ected intestinal cell.
Lectins may be transported through the gut mucosa by endo- or transcytosis. Some are very toxic, such
as the castor bean (ricin) and jack bean (concavaline
A) lectins. The kidney bean lectin, another highly
toxic lectin, compromises the mucosal barrier and
exposes it to microbial invasion, which can potentially lead to sepsis and death. Germ-free animals are
therefore not as a¡ected by these lectins as conventionally reared animals (reviewed by Liener 1980).
Lectins may also be health promoters when supplied
at lower dietary levels (Bardocz, Grant, Ewen, Pryme
& Pusztai 1998).
Only two papers have been found in the scienti¢c
literature reporting results of in vivo studies with ¢sh
fed puri¢ed plant lectins (Buttle, Burrells, Good, Williams, Southgate & Burrells 2001; Iwashita,Yamamoto, Furuita, Sugita & Suzuki 2008). Buttle and
colleagues’study was conducted with soybean lectin
included in a diet (35 g kg À 1) fed to rainbow trout for
53 days. The authors concluded that soybean lectin
binds to the distal intestine in particular and contributes to the pathology observed in soybean-fed salmonids ¢rst described by van den Ingh & Krogdahl
(1990) and van den Ingh et al. (1991). However, the effects on the gut histology of soybean lectin alone
lacked most of the typical soybean alterations seen
in soybean-fed salmonids. In the work of Iwashita
et al. (2008), also with rainbow trout, soybean lectins

did not appear to a¡ect gut morphology. The authors
interpreted the results to indicate that the soybean
lectin supplemented in a semi-puri¢ed diet, together
with a mix of soy saponins, soy iso£avones, phytate
and saccharose, caused an increase in the proliferation of ¢brous connective tissue in the lamina propria
of the distal intestine. Supplied alone in the semi-synthetic diet, however, the soy lectin did not cause any
histological alteration in the intestine. Fish fed this
ANF mix showed a reduced gall bladder index
(weight of organ per unit body weight). The e¡ect is

seemingly related to the presence of the soy lectin,
although its signi¢cance, if any, is not known, and
the authors did not discuss it.
The basis for answering the question of whether
lectins are involved in enteritis seen in soybean-fed
salmonids is weak and further investigation regarding interaction with other ANFs is necessary. However, from the existing knowledge, it appears that
lectins play a minor, if any, role.
Present knowledge regarding the e¡ects of di¡erent plant lectins in ¢sh is far from su⁄cient for any
suggestion of upper limits. As various lectins from
di¡erent sources are highly speci¢c in their binding,
upper levels must be determined for each individual
lectin.

Saponins
Saponins are glycosides produced by more than 100
families of plants such as soy, pea, sun£ower and lupin. Soybeans generally contain saponins in the
range of 1^5 g kg À 1, and the level in soybean is generally higher than in other common feedstu¡s (Anderson & Wolf 1995). Saponins are amphipathic
molecules, containing a hydrophobic steroidal or triterpenoid aglycone to which one or more hydrophilic
sugar chains are attached. Glycation varies considerably among saponins and may include glucose, galactose, glucuronic acid, xylose or rhamnose.
The amphipathic nature of saponins is directly related to many of their biological activities. Saponins

form micelles and can intercalate into cholesterolcontaining membranes, forming holes. Saponins also
a¡ect the functions of mammalian intestinal epithelia by increasing the permeability of intestinal mucosal cells, inhibiting active mucosal transport and
facilitating uptake of substances that are normally
not absorbed (Johnson, Gee, Price, Curl & Fenwick
1986), such as allergens (Gee,Wortley, Johnson, Price,
Rutten, Houben & Penninks 1996). Orally administered saponins that are incorporated into cell membranes will eventually be lost in the normal process
of intestinal epithelial replacement (Sjolander & Cox
1998). They can be degraded by acid and alkaline
hydrolysis (Cleland, Kensil, Lim, Jacobsen, Basa,
Spellman, Wheeler, Wu & Powell 1996) and glucosidases of bacterial origin (Gestetner, Birk & Tencer
1968). Saponins are also lost due to binding with cholesterol, forming an insoluble complex that cannot be
absorbed (Malinow, Mclaughlin, Papworth, Sta¡ord,
Kohler, Livingston & Cheeke 1977). It seems that

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344

337


An update on antinutrients in aquaculture feeds — Krogdahl et al.

saponins, at least in some species, may stimulate feed
intake. Several papers, summarized by Francis, Makkar and Becker (2005), report a growth-promoting
e¡ect of saponins from Quillaja saponaria in common
carp and tilapia. However, the reliability of the results
obtained from these experiments has been questioned (Gatlin et al. 2007). At present, it is not clear
whether saponins have a stimulatory e¡ect on
growth, whether di¡erent saponins in ingredients
such as soy or peas would have the same e¡ect or

whether various species of ¢sh would react similarly.
Di¡erent saponins have been shown to vary in their
biological activities (Oda, Matsuda, Murakami, Katayama, Ohgitani & Yoshikawa 2000). Considering
the wide range of dietary habits, it is not unreasonable to expect di¡erences in the biological e¡ects of
dietary content between ¢sh species.
Recent studies have shed light on the involvement
of soybean saponins in the development of the enteritis induced by soybeans in salmonids. There are
strong indications that saponins play a role in the
soya e¡ect on salmonids, but not alone (Knudsen, Jutfelt, Sundh, Sundell, Koppe & Frokiaer 2008). Supplementation of a semi-puri¢ed soy saponin product in a
¢sh meal-based diet (1.7 and 2.6 g kg À 1 diet) did not
alter the intestinal morphology. When the diet contained lupine kernel meal, the saponin supplementation caused histological and pathophysiological
changes similar to those that occur in salmonids fed
soybean meal (SBM). In vitro studies of the gut wall in
the lupine1saponin fed ¢sh showed increased per-

Aquaculture Research, 2010, 41, 333^344

meability. The authors concluded that soybean saponins increased the intestinal epithelial permeability
but did not, per se, induce enteritis. In the work of
Iwashita et al. (2008), saponin supplementation
(3.8 g kg À 1) in a semi-synthetic diet (casein, gelatine,
gelatinized starch and dextrin, pollock oil, vitamins
and minerals) fed to rainbow trout induced histological alterations similar to those observed in soybeanfed salmonids. Figure 5 shows the results of a recent
study in our laboratory comparing the characteristics of the distal intestine of Atlantic salmon fed
either a reference diet (Ref.), a reference diet with puri¢ed soybean saponins added (Saponin) or a diet with
30% extracted SBM. In this experiment, ¢sh fed the
reference diet showed normal characteristics; the
treatment with saponin caused slight histological
changes and also seemed to reduce the activity of
brushborder membrane enzymes. The saponins

alone, however, did not increase chyme trypsin activity as seen in the SBM-fed ¢sh. From these studies, we
conclude that soy saponins play a key role in soybean-induced enteritis, but the pure compound will
not induce enteritis unless some other plant component is also present in the diet.
Our knowledge of the role and e¡ects of saponins
in ¢sh diets requires strengthening, particularly
regarding interactions with other feed components,
possible growth-promoting potentials and especially
regarding e¡ects in species other than salmonids.
Current knowledge does not allow an accurate upper
safe level to be set for the various saponins from dif-

Figure 5 The ¢gure shows response in the distal intestinal regarding tissue fold height, vacuolization and activity of
leucine aminopeptidase (LAP) and regarding trypsin activity of the chyme. The ¢sh were fed either a reference diet based
on ¢sh meal (Ref), reference diet added 2 g soybean saponin (95%) kg À 1 (Saponin), or a diet with 30% soybean meal
(SBM) (Krogdahl et al. unpubl. obs.). The treatment with saponin in general caused moderate histological changes compared to those seen in the soybean fed ¢sh, and also seemed to reduce the activity of brushborder enzymes but did not
increase the chyme trypsin activity. Results within each parameter marked with di¡erent letters were signi¢cantly di¡erent (Po0.05).

338

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344


Aquaculture Research, 2010, 41, 333^344

An update on antinutrients in aquaculture feeds — Krogdahl et al.

Figure 6 The following molecular outline of a-galactosyl homologues of sucrose: ra⁄nose, stachyose and verbascose
which are the main oligosaccharides of legumes and cereals.

ferent sources, but levels up to 1g kg À 1 appear to be
safe (Francis et al. 2001).

Oligosaccharides in legumes and cereals
Oligosaccharides produced by a wide range of
legumes and cereals are a-galactosyl derivatives of
sucrose present at levels in the range of15^80 g kg À 1
(Saini 1989) (see Fig. 6). These oligosaccharides are
not hydrolysed by endogenous enzymes in monogastrics and are therefore of little, if any, energy value for
carnivorous ¢sh. These compounds are osmotically
active and may cause diarrhoea and interfere with
nutrient digestion.
Soluble carbohydrates are present in soybeans in
the range of 12^15%, about half of which is sucrose.
The remainder comprises low-molecular-weight oligosaccharides of the ra⁄nose family: 1^2% ra⁄nose
and 5^6% stachyose. The question has been raised as
to whether the oligosaccharides are involved in the
development of soybean-induced enteritis. A shortterm study with ra⁄nose and soybean molasses in
diets for Atlantic salmon gave no indication of negative e¡ects of ra⁄nose (Krogdahl, Roem & Baeverfjord
1995). However, studies with other oligosaccharides
and other ¢sh species are required to be able to arrive
at conclusions on e¡ects in ¢sh. An upper safe limit
for dietary inclusion cannot be estimated.
Signi¢cant proportions of the dietary oligosaccharides disappear from the intestinal contents in
¢sh (Refstie, Sahlstrom, Brathen, Baeverfjord & Krogedal 2005). Thus, it is likely that an increase in dietary oligosaccharide levels will stimulate the growth
of certain microorganisms and consequently alter
the microbiota. However, no information is available
on the e¡ects of these oligosaccharides on intestinal
microbiota in ¢sh. There are indications that certain

oligosaccharides, such as mannose and fructose oligosaccharides, may alter the micro£ora. If so, this
may explain why results on enteritis di¡er somewhat
between experiments and points to a possible role of
oligosaccharides as prebiotics.

Update on recent findings on immune
responses involved in soybean-induced
enteritis
The in£ammatory response induced by the inclusion
of standard SBM products in the diet of salmonids
(see Fig.7) is characterized by a shortening of the primary and secondary mucosal folds and a widening of
the lamina propria, which is in¢ltrated by a mixed
population of in£ammatory cells identi¢ed as lymphocytes, macrophages, eosinophilic and neutrophilic granular cells, and di¡use immunoglobulin
M (IgM) (Baeverfjord & Krogdahl 1996; Bakke-McKellep, Press, B×verfjord, Krogdahl & Landsverk 2000).
The functional alterations include a reduction in
brushborder enzyme activities (Bakke-McKellep
et al. 2000; Krogdahl, Bakke-McKellep & Baeverfjord
2003), reduced uptake of macromolecules (Uran,
Aydin, Schrama, Verreth & Rombout 2008) and increased permeability (Nordrum, Bakke-McKellep,
Krogdahl & Buddington 2000; Knudsen et al. 2008).
The e¡ects seem to be dose dependent both regarding
histological and functional characteristics (Krogdahl
et al. 2003). At dietary levels below 10% inclusion of
SBM, the e¡ects vary from absent to very distinct. A
recent Norwegian risk evaluation of plant ingredients
in ¢sh feed suggests 5% as an upper limit (Hemre,
Amlund, Aursand, Bakke, Olsen, RingÖ & Svihus
2009).
The work of Uran, Goncalves, Taverne-Thiele,
Schrama,Verreth and Rombout (2008) indicates that

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344

339


An update on antinutrients in aquaculture feeds — Krogdahl et al.

Aquaculture Research, 2010, 41, 333^344

Figure 7 Histological characteristics of distal intestinal mucosa from Atlantic salmon fed a reference diet based on ¢sh
meal (left) and a diet with 410% standard soybean meal (right). The soybean response is characterized by a shortening of
the primary and secondary mucosal folds and a widening of the lamina propria, which is in¢ltrated by a mixed population
of in£ammatory cells identi¢ed as lymphocytes, macrophages, eosinophilic and neutrophilic granular cells, and di¡use
immunoglobulin.

salmonids are not the only species that develop enteritis when fed diets with SBM. The common carp seem
to respond with similar changes. In the carp, however, the symptoms appeared to diminish with time.
The development of soybean-induced enteropathy
clearly involves the immune functions of the GI tract.
A comparison of immunological parameters in ¢sh
with a normal structure and ¢sh showing enteritis
represents a unique tool for the investigation of the
immunological mechanisms in the intestine, as well
as mechanisms that are involved in the protection of
the gut and the organism as a whole. Research
should include means of preventing feed-induced enteropathies. Our recent studies have therefore focused on cells and cell receptors known to be
involved in immune responses in mammals, and that
are active in similar diseases such as celiac disease

(gluten intolerance), Crohn’s disease and ulcerative
colitis in humans.

T-cell receptors (TCRs)
T cells are key leucocytes in cell-mediated immunity.
Various subtypes of T cells, such as helper (TH), cytotoxic (TC), memory (TM), regulatory (Treg) and natural
killer (NKT) T cells characterized in mammals, modulate the immune response depending on the situation. The di¡erent types of T cells di¡er somewhat in

340

their protein expression of the TCR complex, providing a basis for characterization of various T-cell populations. Whether the cells in¢ltrating the lamina
propria of the distal intestine in soybean fed salmonids comprise T cells has remained an unanswered
question due to lack of appropriate markers. However,
recently, we were able to develop a protocol for the
detection of T-cell-like cells in Atlantic salmon using
human antibodies developed against a conserved epitope of the CD3e protein, which is part of the TCR
complex of all known T cells (Bakke-McKellep, Froystad, Lilleeng, Dapra, Refstie, Krogdahl & Landsverk
2007). The results showed that during the SBM-induced enteropathy, many of the cells in¢ltrating the
lamina propria of the distal intestine were lymphocytes with reactivity to the antibody against the
CD3e epitope. These lymphocytes were not reactive
to an antibody developed against salmonid IgM as
would be the case if they were B-cells (also lymphocytes). Real-time polymerase chain reaction with primers developed for salmon CD3 polypeptide, as well
as CD4 and CD8b, also revealed signi¢cantly increased expression (Po0.05) in the distal intestinal
tissue of SBM-fed ¢sh compared with ¢sh meal-fed
reference ¢sh. These results indicate that a mix of
putative T-cell subtypes was involved in the in£ammatory response after 3 weeks of dietary exposure
to SBM. More speci¢c investigations are needed to
reveal which subtypes of T cells are present. In any

r 2010 The Authors

Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344


Aquaculture Research, 2010, 41, 333^344

An update on antinutrients in aquaculture feeds — Krogdahl et al.

case a T-cell-mediated response appears to be involved in this example of a food-sensitive enteropathy
(Bakke-McKellep et al. 2007).

Proteinase-activated receptors (PARs)
The PARs play important roles in response to tissue
injury, notably in the process of in£ammation and repair. They show increased activation in a range of human in£ammatory diseases, including in£ammatory
responses in the GI tract (Cenac, Coelho, Nguyen,
Compton, Andrade-Gordon, MacNaughton, Wallace,
Hollenberg, Bunnett, Garcia-Villar, Bueno & Vergnolle 2002; Schmidlin, Amadesi, Dabbagh, Lewis,
Knott, Bunnett, Gater, Geppetti, Bertrand & Stevens
2002; Cenac, Garcia-Villar, Ferrier, Larauche, Vergnolle, Bunnett, Coelho, Fioramonti & Bueno 2003;
Kim, Choi, Yun, Kim, Han, Seo, Yeom, Kim, Nah &
Lee 2003). Our hypothesis was that the PARs are present in salmon and are involved in soybean enteritis.
Cloning and characterization of the full-length sequence of Atlantic salmon PAR-2 was successfully
conducted and made it possible to investigate the expression in both early and chronic stages of SBMinduced enteropathy (Thorsen, Lilleeng, Valen &
Krogdahl 2008). Two full-length versions of PAR-2
cDNA were identi¢ed and designated PAR-2a and
PAR-2b. Expressions of the two PAR-2 transcripts
were investigated in 18 tissues and the highest expressions were detected in the intestine and gills. A
signi¢cant up-regulation in the distal intestine was
observed for the PAR-2a transcript after 1 day of exposure to diets containing SBM. After 3 weeks of
feeding, PAR-2a was down-regulated compared with
the ¢sh fed control diets. These ¢ndings may indicate

that PAR-2a participates in in£ammatory responses
in both the early and the later stages of the SBM enteropathy. In the chronic stages of the enteropathy,
down-regulation of PAR-2a may indicate desensitization of the PAR-2a receptor. Expression of the PAR-2b
gene was not altered in the ¢rst 7 days of SBM feeding, but a signi¢cant up-regulation was observed
after 3 weeks, suggesting a putative role in the
chronic stages of SBM-induced enteritis. The expression di¡erences of the two PAR-2 transcripts in the
feeding trials may indicate that they play di¡erent
roles in SBM-induced enteritis.
Elucidating the mechanisms of in£ammation and
pathogenesis of SBM enteropathy not only improves
our basic understanding of immune system function
in salmonids, it also provides potential targets for

modi¢cation to abrogate or regulate these responses.
While speculative, using SBM enteropathy in Atlantic salmon as a model for diseases in other animals,
including humans, may help identify points or components in the disease process that represent potential targets for therapeutic agents or preventive
measures. PAR-2 antagonism has been shown to reduce joint in£ammation in mice when used before
the onset of in£ammation (Kelso, Lockhart, Hembrough, Dunning, Plevin, Hollenberg, Sommerho¡,
McLean & Ferrell 2006), but its e¡ect on existing
in£ammation has not been investigated. Obviously,
several obstacles need to be overcome before any
such treatment modality could be introduced in
large-scale commercial aquaculture. However, other
downstream components of the in£ammatory process may provide more suitable targets. Further characterization of immune responses may also identify
suitable markers for monitoring the health and
welfare status of animals in large-scale production
systems.

Research perspectives for the future
We are only in the initial stages of understanding the

e¡ects of ANFs in ¢sh. Strengthening of the knowledge base is urgently needed to understand the negative e¡ects and to ¢nd means of overcoming them.
Interactions between the e¡ects of ANFs seem to be
very important. The picture is complicated as the
gut microbiota may modify the antinutrients and
hence their interactions and biological e¡ects. Moreover, not only salmonids show soybean-induced enteritis and plant protein sources other than
soybeans contain important ANFs. With strengthened knowledge, we can develop better diets for improvement of nutrition, health and economy in
aquaculture.

Acknowledgments
Many of the recent ¢ndings from our group reported
herein were supported by a Norwegian Research
Council grant (no. 145949/120). We are grateful to
the Research Council of Norway for establishing the
Aquaculture Protein Centre as a Norwegian Centre of
Excellence, and committing to co-funding the centre
for 10 years. This has allowed focused basic research
on nutrition and health in ¢sh to improve our understanding of responses to feedstu¡s and ANFs.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344

341


An update on antinutrients in aquaculture feeds — Krogdahl et al.

References
Anderson R.L. & Wolf W.J. (1995) Compositional changes in
trypsin inhibitors, phytic acid, saponins and iso£avones
related to soybean processing. Journal of Nutrition 125,

S581^S588.
Baeverfjord G. & Krogdahl A. (1996) Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L, distal intestine: a comparison with
the intestines of fasted ¢sh. Journal of Fish Diseases 19,
375^387.
Bakke-McKellep A.M., Press C., Baeverfjord G., Krogdahl —.
& Landsverk T. (2000) Changes in immune and enzyme
histochemical phenotypes of cells in the intestinal mucosa of Atlantic salmon, Salmo salar L., with soybean mealinduced enteritis. Journal of Fish Diseases 23, 115^127.
Bakke-McKellep A.M., Froystad M.K., Lilleeng E., Dapra F.,
Refstie S., Krogdahl A. & Landsverk T. (2007) Response to
soy: T-cell-like reactivity in the intestine of Atlantic salmon, Salmo salar L. Journal of Fish Diseases 30,13^25.
Bardocz S., Grant G., Ewen S.W.B., Pryme I.F. & Pusztai A.
(1998) E¡ects of Antinutrients on the NutritionalValue of Legumes, Vol. 5. O⁄ce for O⁄cial Publications of the European Communities, Luxembourg.
Belitz H.-D. & Grosch W. (1999) Legumes. In: Food Chemistry
(ed. by H.-D. Belitz & W. Grosch), pp. 693^715. SpringerVerlag, Berlin, Germany.
Berg-Lea T., BrattÔs L.-E. & Krogdahl A. (1989) Soybean proteinase inhibitors a¡ect nutrient digestion in rainbow
trout. In: Recent Advances of Research in Antinutritional
Factors in Legume Seeds (ed. by J. Huisman, T.F.B. van der
Pool & I. Liener), pp. 99^102. Pudoc,Wageningen.
Bhat P.G., Jacob R.T. & Pattabiraman T.N. (1981) Enzyme-inhibitors from plants ^ enterokinase inhibitors in tubers
and seeds. Journal of Biosciences 3, 371^378.
Buttle L.G., Burrells A.C., Good J.E.,Williams P.D., Southgate
P.J. & Burrells C. (2001) The binding of soybean agglutinin
(SBA) to the intestinal epithelium of Atlantic salmon, Salmo salar and Rainbow trout, Oncorhynchus mykiss, fed
high levels of soybean meal. Veterinary Immunology and
Immunopathology 80, 237^244.
Cellini F., Chesson A., Colquhoun I., Constable A., Davies
H.V., Engel K.H., Gatehouse A.M.R., Karenlampi S., Kok
E.J. & Leguay J.-J. (2004) Unintended e¡ects and their detection in genetically modi¢ed crops. Food and Chemical
Toxicology 42,1089^1125.
Cenac N., Coelho A.M., Nguyen C., Compton S., AndradeGordon P., MacNaughton W.K., Wallace J.L., Hollenberg

M.D., Bunnett N.W., Garcia-Villar R., Bueno L. & Vergnolle
N. (2002) Induction of intestinal in£ammation in mouse
by activation of proteinase-activated receptor-2. American
Journal of Pathology 161, 1903^1915.
Cenac N., Garcia-Villar R., Ferrier L., Larauche M.,Vergnolle
N., Bunnett N.W., Coelho A.M., Fioramonti J. & Bueno L.
(2003) Proteinase-activated receptor-2-induced colonic
in£ammation in mice: possible involvement of a¡erent

342

Aquaculture Research, 2010, 41, 333^344

neurons, nitric oxide, and paracellular permeability. Journal of Immunology 170, 4296^4300.
Cleland J.L., Kensil C.R., Lim A., Jacobsen N.E., Basa L., Spellman M.,Wheeler D.A.,Wu J.Y. & Powell M.F. (1996) Isomerization and formulation stability of the vaccine adjuvant
QS-21. Journal of Pharmaceutical Sciences 85, 22^28.
Cuadrado C., Ayet G., Burbano C., Muzquiz M., Camacho L.,
Cavieres E., Lovon M., Osagie A. & Price K.R. (1995) Occurrence of saponins and sapogenols in Andean crops.
Journal of the Science of Food and Agriculture 67,169^172.
Cummings J.H., Englyst H.N. & Wiggins H.S. (1986) The role
of carbohydrates in lower gut function. Nutrition Reviews
44, 50^54.
Eeckhout W. & Depaepe M. (1994) Total phosphorus, phytate
phosphorus and phytase activity in plant feedstu¡s. Animal Feed Science andTechnology 47,19^29.
Francis G., Makkar H.P.S. & Becker K. (2001) Antinutritional
factors present in plant-derived alternate ¢sh feed ingredients and their e¡ects in ¢sh. Aquaculture 199, 197^227.
Francis G., Makkar H.P.S. & Becker K. (2005) Quillaja saponins ^ a natural growth promoter for ¢sh. Animal Feed
Science and Technology 121,147^157.
Gatlin D.M., Barrows F.T., Brown P., Dabrowski K., Gaylord
T.G., Hardy R.W., Herman E., Hu G.S., Krogdahl A., Nelson

R., Overturf K., Rust M., Sealey W., Skonberg D., Souza
E.J., Stone D., Wilson R. & Wurtele E. (2007) Expanding
the utilization of sustainable plant products in aquafeeds:
a review. Aquaculture Research 38, 551^579.
Gee J.M., Wortley G.M., Johnson I.T., Price K.R., Rutten
A.A.J.J., Houben G.F. & Penninks A.H. (1996) E¡ects of Saponins and Glycoalkaloids on the permeability and viability of mammalian intestinal cells and on the integrity of
tissue preparations in vitro. Toxicology InVitro 10,117^128.
Gestetner B., Birk Y. & Tencer Y. (1968) Soybean saponins ^
fate of ingested soybean saponins and physiological aspect of their hemolytic activity. Journal of Agricultural and
Food Chemistry 16, 1031^1035.
Grant G. (1991) Lectins. In: Toxic Substances in Crop Plants
(ed. by J.P.F. D’Mello, C.M. Du¡us & J.H. Du¡us), pp. 49^67.
The Royal Society for Chemistry, Cambridge, UK.
Hemre G.-I., Amlund H., Aursand M., Bakke A.M., Olsen
R.E., RingÖ E. & Svihus B. (2009) Criteria for safe use of
plant ingredients in diets for aquacultured ¢sh. Opinion of
the Panel of Animal Feed of the Norwegian Scienti¢c
Committee for Food Safety.
Hendriks H.G.C.J., van den Ingh T.S.G.A., Krogdahl A., Olli J.
& Koninkx J.F.J.G. (1990) Binding of soybean agglutinin
to small intestinal brush-border membranes and brushborder membrane enzyme-activities in Atlantic Salmon
(Salmo-Salar). Aquaculture 91, 163^170.
Iwashita Y., Yamamoto T., Furuita H., Sugita T. & Suzuki N.
(2008) In£uence of certain soybean antinutritional factors supplemented to a casein-based semipuri¢ed diet on
intestinal and liver morphology in ¢ngerling rainbow
trout Oncorhynchus mykiss. Fisheries Science 74, 1075^
1082.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344

Aquaculture Research, 2010, 41, 333^344

An update on antinutrients in aquaculture feeds — Krogdahl et al.

Johnson I.T., Gee J.M., Price K., Curl C. & Fenwick G.R. (1986)
In£uence of saponins on gut permeability and active nutrient transport in vitro. Journal of Nutrition 116, 2270^
2277.
Kelso E.B., Lockhart J.C., Hembrough T., Dunning L., Plevin
R., Hollenberg M.D., Sommerho¡ C.P., McLean J.S. &
Ferrell W.R. (2006) Therapeutic promise of proteinase-activated receptor-2 antagonism in joint in£ammation.
Journal of Pharmacology and Experimental Therapeutics
316, 1017^1024.
Kim J.A., Choi S.C., Yun K.J., Kim D.K., Han M.K., Seo G.S.,
Yeom J.J., Kim T.H., NahY.H. & LeeY.M. (2003) Expression
of protease-activated receptor 2 in ulcerative colitis. In£ammatory Bowel Diseases 9, 224^229.
Knudsen D., Jutfelt F., Sundh H., Sundell K., Koppe W. &
Frokiaer H. (2008) Dietary soya saponins increase gut
permeability and play a key role in the onset of soyabean-induced enteritis in Atlantic salmon (Salmo salar
L.). BritishJournal of Nutrition 100, 120^129.
Krogdahl —. & Holm H. (1979) Inhibition of human and rat
pancreatic proteinases by crude and puri¢ed soybean
proteinase inhibitors. Journal of Nutrition 109, 551^558.
Krogdahl —. & Holm H. (1981) Soybean proteinase inhibitors
and human proteolytic enzymes: selective inactivation of
inhibitors by treatment with human gastric juice. Journal
of Nutrition 111, 2045^2051.
Krogdahl —., Berg Lea T. & Olli J.J. (1994) Soybean proteinase
inhibitors a¡ect intestinal trypsin activities and amino

acid digestibilities in rainbow trout (Oncorhyncus mykiss).
Comparative Biochemistry and Physiology A, Comparative
Physiology 107A, 215^219.
Krogdahl —., Roem A. & Baeverfjord G. (1995) E¡ects of soybean saponin, ra⁄nose and soybean alcohol extract on
nutrient digestibilities, growth and intestinal morphology in Atlantic salmon. In: Quality in Aquaculture. Proceedings of the International Conference of Aquaculture ’95
and the Satellite Meeting Nutrition and Feeding of ColdWater
Species, Special Publication No. 23, Trondheim, Norway,
August 9^12, pp. 118–119. European Aquaculture
Society, Gent, Belgium.
Krogdahl —., Bakke-McKellep A.M. & Baeverfjord G. (2003)
E¡ects of graded levels of standard soybean meal on intestinal structure, mucosal enzyme activities, and pancreatic response in Atlantic salmon (Salmo salar L.).
Aquaculture Nutrition 9, 361^371.
Kunitz M. (1947) Crystalline soybean trypsin inhibitor. Journal of General Physiology 30, 311^320.
Liener I. (1980) Toxic Constituents of Plant Foodstu¡s. Academic Press, NewYork.
Malinow M.R., Mclaughlin P., Papworth L., Sta¡ord C., Kohler G.O., Livingston A.L. & Cheeke P.R. (1977) E¡ect of Alfalfa saponins on intestinal cholesterol absorption in rats.
American Journal of Clinical Nutrition 30, 2061^2067.
Mazur W. & Adlercreutz H. (1998) Naturally occurring oestrogens in food. Pure and Applied Chemistry 70, 1759^1776.

Nordrum S., Bakke-McKellep A.M., Krogdahl A. & Buddington R.K. (2000) E¡ects of soybean meal and salinity on
intestinal transport of nutrients in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss).
Comparative Biochemistry and Physiology B ^ Biochemistry
and Molecular Biology 125, 317^335.
Oda K., Matsuda H., Murakami T., Katayama S., Ohgitani T.
& Yoshikawa M. (2000) Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food
plants. Biological Chemistry 381, 67^74.
Olli J.J. & Krogdahl —. (1994) Nutritive value of four soybean
products as protein sources in diets for Rainbow trout
(Oncorhyncus mykiss, Walbaum) reared in fresh water.
Acta Agricultura Scandinavica 44, 185^192.
Olli J.J., Krogdahl —., van den Ingh T.S.G.A. & BrattÔs L.E.

(1994) Nutritive value of four soybean products in diets
for Atlantic salmon (Salmo salar, L.). Acta Agricultura Scandinavica 44, 50^60.
Ostlund R.E., Racette S.B. & StensonW.F. (2003) Inhibition of
cholesterol absorption by phytosterol-replete wheat germ
compared with phytosterol-depleted wheat germ. AmericanJournal of Clinical Nutrition 77, 1385^1389.
Refstie S., Sahlstrom S., Brathen E., Baeverfjord G. & Krogedal P. (2005) Lactic acid fermentation eliminates indigestible carbohydrates and antinutritional factors in
soybean meal for Atlantic salmon (Salmo salar). Aquaculture 246, 331^345.
Saini H.S. (1989) Legume seed oligosaccharides. In: Recent
Advances of Research in Antinutritional Factors in Legume
Seeds (ed. by J. Huisman, A.F.B. van der Pool & I. Liener),
pp. 329^341. Pudoc,Wageningen.
Schmidlin F., Amadesi S., Dabbagh K., Lewis D.E., Knott P.,
Bunnett N.W., Gater P.R., Geppetti P., Bertrand C. & Stevens M.E. (2002) Protease-activated receptor 2 mediates
eosinophil in¢ltration and hyperreactivity in allergic in£ammation of the airway. Journal of Immunology 169,
5315^5321.
Shewry P.R., Tatham A.S. & Halford N.G. (2001) Genetic
modi¢cation and plant food allergens: risks and bene¢ts.
Journal of Chromatography B ^ AnalyticalTechnologies in the
Biomedical and Life Sciences 756, 327^335.
Sjolander A. & Cox J.C. (1998) Uptake and adjuvant activity of
orally delivered saponin and ISCOM (TM) vaccines. Advanced Drug Delivery Reviews 34, 321^338.
Thompson L.U. (1993) Potential health bene¢ts and problems associated with antinutrients in foods. Food Research International 26, 131^149.
Thorsen J., Lilleeng E.,Valen E.C. & Krogdahl A. (2008) Proteinase-activated receptor-2: two potential inflammatory
mediators of the gastrointestinal tract in Atlantic
salmon. Journal of In£ammation (London) 5, 18.
Uran P.A., Aydin R., Schrama J.W., Verreth J.A.J. & Rombout
J.H.W.M. (2008) Soybean meal-induced uptake block in
Atlantic salmon Salmo salar distal enterocytes. Journal of
Fish Biology 73, 2571^2579.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344

343


An update on antinutrients in aquaculture feeds — Krogdahl et al.

Uran P.A., Goncalves A.A.,Taverne-Thiele J.J., Schrama J.W.,
Verreth J.A.J. & Rombout J.H.W.M. (2008) Soybean
meal induces intestinal in£ammation in common carp
(Cyprinus carpio L.). Fish and Shell¢sh Immunology 25,
751^760.
van den Ingh T.S., Krogdahl A. (1990) Negative e¡ects of
anti-nutritional factors from soybeans in Salmonidae.
[article in Dutch]. Tijdschrift voor Diergeneeskunde 15,
935–938.
van den Ingh T.S.G.A.M., Krogdahl —., Olli J.J., Hendrix
H.G.C.J.M. & Koninkx J.G.J.F. (1991) E¡ects of soybean containing diets on the proximal and distal intestine in

344

Aquaculture Research, 2010, 41, 333^344

Atlantic salmon Salmo salar: a morphological study.
Aquaculture 94, 297–305.
van der Kamp J.W., Asp N.-G., Miller Jones J. & Schrama J.W.
(2004) Dietary Fibre: Bioactive Carbohydrates for Food and
Feed.Wageningen Academic Publishers,Wageningen.
Wiggins H.S. (1984) Nutritional-value of sugars and relatedcompounds undigested in the small gut. Proceedings of the

Nutrition Society 43, 69^75.
Wink M., Schmeller T. & Latz-Bruning B. (1998) Modes of action of allelochemical alkaloids: interaction with neuroreceptors, DNA, and other molecular targets. Journal of
Chemical Ecology 24,1881^1937.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 333^344