Aquaculture research, tập 41, số 3, 2010

E-mail: kaushik@st-pee.inra.fr Abstract Optimising the amino acid supply in tune with the requirements and improving protein utilization for body protein growth with limited impacts on t

The special issue of Aquaculture Research is

com-prised 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

ex-cellence in ¢sh nutrition research and the

opportu-nity for communication among researchers New

knowledge in ¢sh nutrition research plays an

impor-tant role in the development of global aquaculture as

well as allows for the production of safe and healthy

food for human consumption The widespread

inter-est 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

en-compassed a workshop on ‘Sustainable aquafeeds for

the third millennium’, followed by 296 oral and

pos-ter contributions in the following eight sessions:

Protein; Nutrient Requirement and Availability;

Nu-trition and Gene Expression; NuNu-trition and Health;

Environmental Quality and Feeding Strategies; Fish

Quality and Food Safety; Feed Ingredients and FeedProcessing; and Broodstock and Larvae Seven in-vited lecturers, and four review papers from thesepresentations are published in this special issue Se-lected contributions were submitted for peer reviewand the resulting manuscripts are published here.The participants earned our appreciation, espe-cially those who gave oral presentations, posters andinvited papers The planning and organizing of thissymposium was a considerable undertaking forwhich scienti¢c and local organizing committeesmet with enthusiasm, energy and strong commit-ment Our special thanks are due to all the anon-ymous reviewers for the assistance in reviewing allthe papers for this proceeding We hope the articlespresented in this special issue will contribute to-wards the advancement of our knowledge in the fas-cinating ¢eld of ¢sh nutrition

De¤bora M Fracalossi, Organizing

Committee ChairSantosh P Lall, Scienti¢c Committee Chair

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: kaushik@st-pee.inra.fr

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

pro-vision 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

pro-tein 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

par-ticularly 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

or-der 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

the developing world as well as in the emerging mies of the world At a global level, about 45% of all ¢shconsumed by humans, totalling about 48 millionstonnes is farm raised (FAO 2007) Aquaculture thusplays a vital role in supplying products known to have

econo-a high biologicecono-al vecono-alue to humecono-ans (Bender &econo-amp; Hecono-aizelden1957) besides providing healthy long-chain w3 polyun-saturated fatty acids (Sargent 1997)

From a quantitative point of view, e⁄ciency of tein utilization and muscle protein growth are the mostcrucial issues Although ¢sh are generally consideredbetter converters of dietary protein, compared with ter-restrial vertebrates, given the global context of rapid de-velopment of aquaculture and the increasing costs anddearth of protein-rich feedstu¡s, there is an impendingnecessity for improvements in dietary protein utiliza-tion, achievable only by optimising dietary supply intune with the di¡erent physiological needs of organ-isms This then necessitates a full understanding of thephysiological basis for the requirements and e⁄cientexploitation of available sources to meet such needs

pro-Protein/energy nutrition of fish: generalconsiderations

Critical assessment of protein requirements havealready been made by a number of authors over thepast two decades (Cowey & Luquet 1983; Bowen 1987;Cowey 1994, 1995) The general observation is that

¢sh require a higher level of dietary protein than restrial farmed vertebrates The general contentionswhich have very often been put forward with regard

ter-to this high protein requirement of farmed ¢sh are asfollows: (i) ¢sh have high ß apparent  protein needs,the basal energy needs of ¢sh are lower than those of

r 2010 The Authors

terrestrial animals, due to the aquatic mode of life,

poikilothermy and ammoniotelism Based on

com-parisons of protein e⁄ciency ratios in a number of

farmed animals, it becomes clear that ¢sh and

terres-trial animals di¡er only in relative protein

concentra-tion 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

pro-tein 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

consid-ered high Much progress has however been achieved

through optimising the digestible protein (DP) to

di-gestible 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

di-gestive tract level and at a post-absorptive level

(Sargent,Tocher & Bell 2002) whereas dietary

carbo-hydrates require heat treatment to improve its

digest-ibility and supply of DE (Bergot & Breque 1983;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

uti-lization 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

dif-ferences between species (Furuichi & Yone1982;

Pan-serat, 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 bolism as a¡ected by dietary protein levels is indeedconsidered to be one major reason for the high pro-tein requirements of ¢sh (Cowey & Walton1989) This

cata-is somewhat comparable to what cata-is found in the nivorous cat, where the high protein requirement isconsidered to be a consequence of the high obligatorynitrogen losses incurred in the conversion of nitro-gen from indispensable amino acids (IAA) to dispen-sable amino acids (DAA) in the liver and to a slow rate

car-of catabolism car-of IAA (Taylor, Morris, Kass & Rogers1998)

IAA requirementsQuantitative data on amino acid requirements for all

10 IAA are available only for a limited number of cies (National Research Council 1993; Wilson 2002;Lall & Anderson 2005; Tibaldi & Kaushik 2005).Given the large number of species of farmed ¢n¢shand shrimp, we should admit that it is indeed di⁄cult

spe-to establish the quantitative requirements for all the

10 IAA for each of the species concerned Measuredamino acid requirements of di¡erent species,expressed as a proportion of the diet, show also anapparently high degree of variation (Cowey 1994;Mambrini & Kaushik 1995b;Wilson 2002) One majorexplanation for this apparent variability in IAA re-quirement data are linked to methodology issues(Cowey 1995): (i) the mode of expression of data (rela-tive 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 andwhether the ¢sh were able to reach their near maxi-mum growth potential, (iii) the criterion used for theestimation of requirement and (iv) the statisticalmethod used for analysing numerical data ondose^response Besides conventional dose^responsecurves using di¡erent response criteria such asgrowth, nitrogen utilization, direct or indirect meth-ods of measurement of amino acid oxidation, meta-bolic responses, new approaches such as singleamino acid deletion or reduction (Fournier, GouillouCoustans, Metailler,Vachot, Guedes,Tulli, Oliva-Teles,Tibaldi & Kaushik 2002; Green & Hardy 2002; Rollin,Mambrini, Abboudi, Larondelle & Kaushik 2003) ordiet-dilution techniques (Liebert & Benkendor¡2007) have also been attempted in ¢sh with resultscon¢rming data obtained by conventional methods

A close analysis of reliable data available howeverpoints towards some degree of homogeneity between

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-para-meter nutrition kinetics analysis (Mercer 1982) was

used Based on data from several studies on

require-ments of di¡erent species for lysine and sulphur

amino acids (methionie1cystine) the di¡erent

para-meters 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/ami-no 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

stu-dies, it appears that the ideal protein would be the one

that re£ects the whole body IAA pro¢le of the

corre-sponding species The whole body protein bound

amino acid pro¢les are however very much similar

between di¡erent species and the amino acids

depos-ited during growth are also similar between di¡erentteleosts as well as crustaceans (Table 1) It has clearlybeen shown that there is possibly more apparentvariability in AA requirement pro¢les than in thewhole bodyAA pro¢les of di¡erent teleosts (Akiyama,Oohara & Yamamoto 1997) It is however reassuringthat a study by Green and Hardy (2002) con¢rmedthat the requirement pro¢le as proposed by National

Met, %CP

Figure 1 Analysis of erature data on lysineand methionine require-ments of di¡erent species

lit-of ¢sh and shrimp

Table 1 Whole body amino acid composition of di¡erent

¢n¢sh and crustaceans (expressed as g16gN 1 )

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

r 2010 The Authors

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

regres-sion 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,

Calduch-Giner, 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

(Rode-hutscord, Becker, Pack & Pfe¡er 1997) Some other

studies have estimated the maintenance needs for

in-dividual 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

pro-tein-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¡er-ences in endogenous losses in amino acids between

rainbow trout and turbot (Fig 2), strongly suggesting

di¡erences in protein degradation rates and tissues

involved

From needs to feeds: developing low or

non-fish meal diets

One of the major issues a¡ecting the aquaculture

in-dustry is the availability of protein-rich feedstu¡s

Under intensive ¢sh farming conditions, ¢sh meal

and ¢sh oil are the most common feedstu¡s ing the essential nutrients (amino acids, fatty acids,minerals and trace elements) vital for growth, health,reproduction and physiological well-being of farmed

supply-¢sh.While the marine capture ¢sheries remains stant, the demand for such feedstu¡s derived fromcapture ¢sheries is on the increase and the costs arerocketing In this context, replacement of ¢sh meal byalternative protein sources remains a major thrustarea of research and much has been accomplished

con-in reduccon-ing the level of ¢sh meal con-in all species (Gatlcon-in,Barrows, Brown, Dabrowski, Gaylord, Hardy, Her-man, 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 tein having an ideal IAA pro¢le for ¢sh and shrimp

pro-An example of data on lysine and sulphur amino acidcontents of di¡erent plant protein sources in compar-ison with that of the requirements of ¢sh is illustrated

in Fig 3 Fish meal is also a good source of essentialfatty acids, minerals and trace elements In choosingalternatives to ¢sh meal, it is then necessary to look

050100150200

Figure 3 Lysine and sulphur amino acid contents of lected protein sources compared with the requirementsfor these amino acids by ¢sh

se-at the amino acid pro¢le, but also se-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

re-sort 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

quanti-tative data on amino acid availability and the

biologi-cal 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,

Faucon-neau & Laroche 1995) Non-¢shmeal diets

incorporat-ing 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

gilt-head seabream (Benedito-Palos, Saera-Vila,

Calduch-Giner, 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

le-vels 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

de-monstrated with postprandial blood free amino acid

peaks appearing later for protein-bound amino acids

(Cowey & Walton 1988) They also showed that

incor-poration of labelled carbon residues into glycogen and

lipid from an amino acid diet was greater than from a

whole protein diet, whereas incorporation of

radioac-tivity 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

dietary pH improves the utilization of diets withhigh levels of synthetic amino acids Increasingthe number of meals was proposed to improveamino acid utilization (Yamada, Tanaka & Katayama1981) in carp In order to reduce absorption of freeamino acids while digestion of intact proteinoccurs, coating amino acids with agar for instancehas been found to be e⁄cient resulting in improvednitrogen utilization (Cho, Kaushik & Woodward1992; Fournier et al 2002) These necessary precau-tions de¢nitely improves utilization of amino acidssupplied in the free form even at very high levels.There are indeed several reports showing thatcrystalline amino acids are well utilized both withsemi-puri¢ed (Cho et al.1992; Rodehutscord, Mandel,Pack, Jacobs & Pfe¡er 1995; Fournier et al 2002) andpractical 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 uti-lized as well as protein bound amino acids even at a10% incorporation level

Amino acid utilizationRegulation of feed intake by dietary amino acid bal-ance has been little studied.Whether a dietary aminoacid de¢ciency or excess leads to increases in volun-tary feed intake over a long term has not been ana-lysed in depth A preliminary analysis shows thatsingle amino acid de¢ciencies (arginine, leucine, luy-sine, methionine) lead to decreased feed intake (de laHiguera 2001) In European seabass, responsesappear to di¡er depending on the amino acid, trypto-phan de¢ciency exerting the most signi¢cant depres-sion in voluntary feed intake (Tibaldi & Kaushik 2005).Further insight on the consequences of marginal ami-

no acid de¢ciencies linked with dietary DP levels onshort or long-term feed intake is needed to optimizedietary amino acid supply and utilization

Nutritional regulation of amino acid metabolismhas already been dealt with in detail in a number ofin-depth reviews (Walton 1985; Cowey & Walton1989; Dabrowski & Guderley 2002) At the hepaticlevel, dietary protein levels appear to exert little e¡ect

on amino acid catabolism whereas there is a tively good response of enzymes of amino acid meta-bolism to the corresponding amino acid intakes Thelack of control by dietary protein levels on amino acidProtein and amino acid nutrition and metabolism in ¢sh S J Kaushik and I Seiliez Aquaculture Research, 2010, 41, 322^332

rela-r 2010 The Authors

oxidation in ¢sh contrasts with what is generally

seen in mammals and this is considered to be the

ma-jor reason explaining teleosts’adaptation to high

diet-ary protein levels

Somatic growth involves irreversible

transforma-tion of dietary substrates and tissue energy stores

into tissue and organs There is de¢nitely a good

co-rrelation between somatic growth rate and

instanta-neous protein synthesis rates (Haschemeyer & Smith

1979; Smith 1981; Fauconneau 1985; Carter &

Houli-han 2001) Protein synthesis rates di¡er between

tis-sues 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

deposi-tion of synthesized protein is however high in the

muscle of ¢sh (Fauconneau 1985; Peragon,

Ortega-garcia, 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

ac-counts 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¡er-ent species (Young 1991) Inclusion of data from

rain-bow 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

consid-ered to be several fold higher than in mammals

(Dab-rowski & 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

this metabolic expenditure are areas worth furtherinvestigation

Fasting followed by re-feeding induces an increase

in protein turnover (Smith 1981; McMillan & han 1989a) The role of insulin, and insulin-likegrowth factor 1 (IGF-1) as mediators of the anabolicdrive is well described in mammals (Millward 1989,1995) Current knowledge in higher animals showsthat this is accomplished by stimulation of the mam-malian target of rapamycin (mTOR), a cell signallingpathway involved in the regulation of initiation ofmRNA translation (Garami, Zwartkruis, Nobukuni,Joaquin, Roccio, Stocker, Kozma, Hafen, Bos &

Fed Fasted Re-fed

Figure 4 Fractional rates of protein synthesis (%/day) in

various tissues of rainbow trout (redrawn from McMillan

& Houlihan 1989b)

Adipose 3%

Fins 1%

Skin 11%

Gills 3%

Head 13%

Viscera 11%

Blood 2%

Liver 1%

Muscle 55%

Liver 1% Dig.Tract 7%

Other

50%

Muscle 22% Other

25%

Dig.Tract 39%

Liver 14%

(a)

Figure 5 Relative importance of tissue size (a) comparedwith protein content (b) and protein synthesis rates in dif-ferent tissues (c) in rainbow trout

Chickens Rat

Mouse

0100200300400500600700800

Protein turnover, g/kg/d

Metabolic rate,kJ/kg/d

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

meta-Thomas 2003; Tee, Manning, Roux, Cantley & Blenis

2003; Zhang, Cicchetti, Onda, Koon, Asrican,

Bajras-zewski, 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,

Pan-serat, Skiba-Cassy, Fricot, Vachot, Kaushik &

Tesser-aud 2008b), opening new research perspectives

on the molecular bases of amino acid utilization in

teleosts

Given the dynamic status of protein turnover

im-plying 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

degra-dation pathways In ¢sh, we do not yet have a clear

idea of the relative importance of the three major

pro-teolytic systems operating in vivo (lysosome, Ca21

de-pendent and ubiquitin^proteasome dede-pendent) 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

pep-tides of 7^9 amino acid residues (Voges, Zwickl &

Baumeister 1999) In mammals, the ATP-dependent

ubiquitin^proteasome proteolytic pathway is

consid-ered to be the major route of protein degradation

in-volved 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

rain-bow trout (Oncorhynchus mykiss), the activity of the

proteasome in muscle does not change during

star-vation-induced muscle degradation (Martin, Blaney,

Bowman & Houlihan 2002) Furthermore,

microar-ray gene expression analysis in atrophying rainbow

trout showed that mRNA levels for the subunits of

the proteasome were either not a¡ected or

down-regulated (Salem, Kenney, Rexroad III & Yao 2006),

leading to the suggestion that degradation of muscle

proteins in trout occurs by a route distinct from the

one observed in mammals (Mommsen 2004) But,our own recent data show that, in the muscle of rain-bow trout, the polyubiquitination step of the ubiqui-tin^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 ideathat we have to reconsider the role of this proteolyticroute in muscle protein degradation and its nutri-tional regulation

Future researchGiven the general context of aquaculture and the im-portance of dietary protein/amino acids in aqua-feeds, a number of areas need our attention.We need

to strengthen our understanding of the quences of marginal amino acid imbalances underminimal dietary DP levels capable of maximumgrowth and physiological well being Given the rela-tively high contribution of excess amino acids for en-ergy, we need to gain knowledge on the relativecontributions or preferential utilization of individualamino acid oxidation to overall metabolic demands.Similarly, understanding the role of individual aminoacids directly or indirectly through hormonal factors

conse-in elicitconse-ing the anabolic drive conse-in the regulation ofmuscle growth is necessary.While we know that sig-ni¢cant ‘protein-sparing’ and reduction in nitrogen-ous losses are achieved by decreasing the DP/DEratios, we need to get more insight on the underlyingmechanisms especially as a¡ected by dietary factors.Comprehensive data should bring forth more simila-rities than di¡erences between terrestrial animalsand aquatic organisms in their nitrogen metabolismand utilization

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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, Stle 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, Sunndalsra, Norway

Correspondence: — Krogdahl, Norwegian School of Veterinary Science, PO Box 8146 Dep., NO-0033 Oslo, Norway E-mail: Ashild.Krogdahl@veths.no

Abstract

This review presents an overview of antinutritive

fac-tors (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

ad-dressing the involvement of T cells and

proteinase-activated 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;

interac-tions 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

soy-beans 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

apparent function of protecting them from becomingfood for microbes, insects and higher animals Con-sequently, many of these compounds may exertharmful e¡ects when ingested by humans and ani-mals Such substances are often called antinutritivefactors (ANFs), although they may also have bene¢-cial e¡ects, such as being antioxidative, immunosti-mulatory or prebiotic, depending on the amountingested Possible harmful e¡ects include reduced pa-latability, less e⁄cient utilization of feed nutrients forgrowth, altered nutrient balances of the diets, inhibi-tion of growth, intestinal dysfunction, altered gutmicro£ora, immune modulation, goitrogenesis, pan-creatic hypertrophy, hypoglycaemia or liver damage.The species of animal, its age, size, gender, state ofhealth and plane of nutrition and any stress factorsmay modify these responses

In his textbook on toxic constituents of plant foods,Liener (1980) wrote the following: ‘What has only re-cently been realized is that although there might not

be an immediate violent reaction to a certain foodcomponent there might still be a slow cumulative ad-verse e¡ect resulting in overt disease or less than op-timal health This poses a great challenge, sinceknowledge of these e¡ects is gained slowly and withdi⁄culty, particularly if the causative principles re-main unidenti¢ed’ Liener’s statement is still validand relevant During the last 10^15 years, inclusion

of plant feedstu¡s in ¢sh feed has increased markedlyand consequently so has exposure to ANFs Antinu-tritive factors are novel to most cultivated ¢sh species,particularly carnivorous species Fish nutritionistsshould keep in mind Liener’s warning that ‘there

may be slow cumulative adverse e¡ects resulting in

overt disease or less than optimal health’ It is not

un-likely that ANFs are involved in the aetiology of

dis-eases that are emerging in the aquaculture industry

today related to gut function and the immune

appara-tus, for example enteritis, low protein and lipid

digest-ibility, diarrhoea and neoplasia

Important antinutrients

Antinutrients are de¢ned as endogenous compounds

in feedstu¡s that may reduce feed intake, growth,

nu-trient 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-galacto-side 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

infor-mative overviews of the general biological e¡ects of

ANFs and summarized the state of knowledge at

their respective times of publication Among the

rele-vant 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;

Krog-dahl & Holm1981; Berg-Lea, Bratts & KrogKrog-dahl1989;

Thompson1993); lectins, which bind to gut cell

recep-tors, possibly stimulating intestinal growth, make the

gut more permeable for increased in£ux of

macromo-lecules and bacteria, stimulate insulin production

and alter metabolism (Grant 1991); saponins, which

interfere with lipid and protein digestion and may

in-crease 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

the diet is increased accordingly (Liener1980); estrogens (iso£avons/coumestan), which may interferewith the e¡ects of endogenous oestrogen (Mazur &Adlercreutz 1998); phytosterols, which may interferewith cholesterol absorption and metabolism (Os-tlund, Racette & Stenson 2003); quinolizidine alka-loids, such as lupanin, which may cause nervoussystem symptoms and intestinal disorders (Wink,Schmeller & Latz-Bruning 1998); or oligosaccharides,which may alter microbiota of the intestinal tractand increase osmotic pressure in the intestine if notmetabolized by the microbiota (Wiggins 1984; Cum-mings, Englyst & Wiggins 1986)

phyto-Some ANFs are easy to eliminate by processing,and others are more di⁄cult to eliminate There arealso examples for which common processing steps,such as heat treatment, may activate the antinutri-tional e¡ects For all ANFs, fermentation or enzymetreatments directly focusing on inactivation of a spe-ci¢c ANF may reduce content or activity in the feed-stu¡ Selective breeding and genetic modi¢cationmay also alter the content E¡orts are being made inseveral laboratories to modify the ANF contents ofimportant crops such as soybeans and rapeseeds(Shewry, Tatham & Halford 2001) However, geneticmodi¢cation may also result in unintended altera-tions in the contents of ANFs (Cellini, Chesson,Colquhoun, Constable, Davies, Engel, Gatehouse,Karenlampi, Kok & Leguay 2004) Table 1lists the ma-jor ANFs present in a variety of feedstu¡s and treat-ments that may reduce biological activity either byelimination or by inactivation

Proteinase inhibitorsInhibitors of proteinases, i.e of trypsin, chymotryp-sin, elastases and carboxypeptidases, are proteinsthat form stoichiometric complexes with the respec-tive enzymes and inhibit their activity in the gastro-intestinal (GI) tract Proteinase inhibitors are found

in most plants (Liener 1980) In addition, nase inhibitors have been described (Bhat, Jacob &Pattabiraman 1981) The molecular weight of protei-nase inhibitors is found in the range between 6000and 50 000 kDa and their speci¢cities vary consider-ably Some inhibit only one type of enzyme, e.g tryp-sin or chymotrypsin, and others inhibit two or three.Some inhibit one enzyme molecule per inhibitormolecule, and others inhibit two or more An over-view of plants with endogenous proteinase inhibitorscommonly used as food has been provided by Belitz

enteroki-An update on antinutrients in aquaculture feeds — Krogdahl et al Aquaculture Research, 2010, 41, 333^344

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& 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

inhi-bitor has a molecular weight of about 8000 kDa and

is characterized by the seven disulphide bridges that

stabilize the molecule and make it relatively stable toproteolytic breakdown, acid denaturation as well asheat This inhibitor may bind one trypsin and onechymotrypsin simultaneously Inhibitors with a si-milar structure can be found in several legumes Thelarger (21000 kDa) Kunitz inhibitor with a speci¢csite for trypsin may also bind chymotrypsin but in aless stable complex and only one enzyme molecule isinhibited at a time

The e¡ects of proteinase inhibitors have been died thoroughly in mammals and birds since theirdiscovery in 1947 (Kunitz 1947) Based on these stu-dies, an understanding of their actions has been de-veloped (Liener 1980) In the intestine, inhibitors (e.g

stu-a trypsin inhibitor) ¢rst form stu-a rstu-ather ststu-able complexwith trypsin, thereby reducing trypsin activity This

in turn stimulates the secretion of pancreozymin^cholecystokinin from the gut wall This hormonestimulates the secretion of trypsin from pancreatictissue and stimulates the gall bladder to empty itscontents into the intestine Trypsin synthesis in thepancreas is stimulated, resulting in an increased re-quirement 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 inhibitorshave been found to reduce the apparent digestibility

of both protein and lipid (Berg-Lea et al 1989; dahl, Berg Lea & Olli 1994; Olli & Krogdahl 1994; Olli,Krogdahl, van den Ingh & Bratts 1994) Among thefatty acids, the longer saturated and monounsatu-rated were most a¡ected The e¡ects on the digestibil-ities correlated with a decrease in trypsin activity and

Krog-Table 1 Antinutrients in common plant ingredients and

treatments that will eliminate or reduce biological activity

Antinutrient Sources Type of treatment

Proteinase

inhibitors

Legumes Heat, methionine

supplementation Amylase inhibitors Peas Heat

Lipase inhibitor Beans Heat

Lectins All plants

seeds

Heat, supplementation with specific carbohydrates Phytic acid All plants Mineral supplementation

Fibre All plants Dehulling

beans

Dehulling, restriction of heat treatment

Saponins Legumes Alcohol extraction

Sterols Legumes Alcohol/non-polar

extraction, cholesterol supplementation Oestrogens Beans Alcohol/non-polar extraction

Gossypol Cotton seed Non-polar extraction, iron

supplementation Oligosaccharides Legumes Alcohol/aqueous extraction

Quinolozidine

alkaloids

Lupins Aqueous extraction

Goitrogens Rape seed Iodine supplementation

For all antinutrients, levels may be reduced by fermentation,

treat-ment with enzymes that speci¢cally inactivate the compound,

selective breeding and genetic modi¢cation.

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)

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)

presumably also in chymotrypsin, both of which are

inhibited by soybean proteinase inhibitors (Fig 2)

The results presented in Fig 3 indicate that the

pro-teinase inhibitors stimulated pancreatic enzyme

se-cretion, causing the enzyme protein level of the

intestinal content (trypsin protein) to increase

How-ever, the activity in the intestinal content was not

in-creased 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

en-zyme activity by increased secretion Enen-zyme

pro-duction appeared not to keep up with the increased

demand

Work by Krogdahl et al (1994) investigated the

ef-fects of increasing levels of dietary proteinase

inhibi-tors 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

in-creased the secretion of cysteine-rich pancreatic

en-zymes 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

as-sumed to enter the GI tract, yielding a negative

appar-ent digestibility in the pyloric region of the intestine

Many ¢sh studies have been performed with

feed-stu¡s showing varying proteinase inhibitor activities

(1^30 g kg 1) From these studies, it is apparent that

there are considerable di¡erences in sensitivity

be-tween ¢sh species Salmonids and Nile tilapia may

be particularly sensitive (Francis et al 2001) Most ¢sh

species seem to be able to compensate when

protei-nase inhibitor activities are below 5 g kg 1(Olli &

Krogdahl 1994; Olli et al 1994; Francis et al 2001)

LectinsLectins, also called haemagglutinins, are sugar bind-ing proteins apparently present in all organisms withmetabolic and protective functions Lectins are pro-duced in most plant feedstu¡s, particularly in le-gumes and cereals (1^20 g kg 1) They aremonomers, dimers or polymers of the same peptidechain, or more complex aggregates of di¡erent pep-tides (Fig 4) Lectins di¡er in speci¢city to which type

of carbohydrate they bind Their mode of action ismediated through binding to glycated cell receptors,which may cause receptor activation Lectins withtwo or more binding sites will agglutinate cells withreceptors of similar glycation, e.g blood cells and en-terocytes Because various receptors di¡er in glyca-

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 thedistal intestine (Olli et al 1994)

Figure 4 The agglutinating (clumping) e¡ect of a modellectin on cells A lectin is a protein with speci¢c a⁄nity forcarbohydrate moieties for example on cell receptors Eachmolecule may bind to more than one carbohydrate moietyand therefore can bind more than one cell and agglutinatethe cells The lectin e¡ects depend on the function of thereceptors

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tion, their susceptibility to a particular lectin may

dif-fer Hendriks, van den Ingh, Krogdahl, Olli and

Ko-ninkx (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

re-lease, 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

muco-sa 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

poten-tially lead to sepsis and death Germ-free animals are

therefore not as a¡ected by these lectins as

conven-tionally 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,

Wil-liams, Southgate & Burrells 2001;

Iwashita,Yamamo-to, Furuita, Sugita & Suzuki 2008) Buttle and

colleagues’study was conducted with soybean lectin

included in a diet (35 g kg 1) fed to rainbow trout for

53 days The authors concluded that soybean lectin

binds to the distal intestine in particular and

contri-butes to the pathology observed in soybean-fed

sal-monids ¢rst described by van den Ingh & Krogdahl

(1990) and van den Ingh et al (1991) However, the

ef-fects 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

prolifera-tion of ¢brous connective tissue in the lamina propria

of the distal intestine Supplied alone in the

semi-syn-thetic 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, andthe authors did not discuss it

The basis for answering the question of whetherlectins are involved in enteritis seen in soybean-fedsalmonids is weak and further investigation regard-ing interaction with other ANFs is necessary How-ever, from the existing knowledge, it appears thatlectins play a minor, if any, role

Present knowledge regarding the e¡ects of ent plant lectins in ¢sh is far from su⁄cient for anysuggestion of upper limits As various lectins fromdi¡erent sources are highly speci¢c in their binding,upper levels must be determined for each individuallectin

di¡er-SaponinsSaponins are glycosides produced by more than 100families of plants such as soy, pea, sun£ower and lu-pin Soybeans generally contain saponins in therange of 1^5 g kg 1, and the level in soybean is gen-erally higher than in other common feedstu¡s (An-derson & Wolf 1995) Saponins are amphipathicmolecules, containing a hydrophobic steroidal or tri-terpenoid aglycone to which one or more hydrophilicsugar chains are attached Glycation varies consider-ably among saponins and may include glucose, ga-lactose, glucuronic acid, xylose or rhamnose.The amphipathic nature of saponins is directly re-lated to many of their biological activities Saponinsform micelles and can intercalate into cholesterol-containing membranes, forming holes Saponins alsoa¡ect the functions of mammalian intestinal epithe-lia by increasing the permeability of intestinal muco-sal cells, inhibiting active mucosal transport andfacilitating uptake of substances that are normallynot absorbed (Johnson, Gee, Price, Curl & Fenwick1986), such as allergens (Gee,Wortley, Johnson, Price,Rutten, Houben & Penninks 1996) Orally adminis-tered saponins that are incorporated into cell mem-branes will eventually be lost in the normal process

of intestinal epithelial replacement (Sjolander & Cox1998) They can be degraded by acid and alkalinehydrolysis (Cleland, Kensil, Lim, Jacobsen, Basa,Spellman, Wheeler, Wu & Powell 1996) and glucosi-dases of bacterial origin (Gestetner, Birk & Tencer1968) Saponins are also lost due to binding with cho-lesterol, forming an insoluble complex that cannot beabsorbed (Malinow, Mclaughlin, Papworth, Sta¡ord,Kohler, Livingston & Cheeke 1977) It seems that

saponins, at least in some species, may stimulate feed

intake Several papers, summarized by Francis,

Mak-kar 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

ques-tioned (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,

Ka-tayama, Ohgitani & Yoshikawa 2000) Considering

the wide range of dietary habits, it is not

unreason-able 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

enter-itis 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,

Jut-felt, Sundh, Sundell, Koppe & Frokiaer 2008)

Supple-mentation of a semi-puri¢ed soy saponin product in a

¢sh meal-based diet (1.7 and 2.6 g kg 1diet) did not

alter the intestinal morphology When the diet

con-tained lupine kernel meal, the saponin

supplementa-tion 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-meability The authors concluded that soybean nins increased the intestinal epithelial permeabilitybut did not, per se, induce enteritis In the work ofIwashita et al (2008), saponin supplementation(3.8 g kg 1) in a semi-synthetic diet (casein, gelatine,gelatinized starch and dextrin, pollock oil, vitaminsand minerals) fed to rainbow trout induced histologi-cal alterations similar to those observed in soybean-fed salmonids Figure 5 shows the results of a recentstudy in our laboratory comparing the characteris-tics of the distal intestine of Atlantic salmon fedeither a reference diet (Ref.), a reference diet with pur-i¢ed soybean saponins added (Saponin) or a diet with30% extracted SBM In this experiment, ¢sh fed thereference diet showed normal characteristics; thetreatment with saponin caused slight histologicalchanges and also seemed to reduce the activity ofbrushborder membrane enzymes The saponinsalone, however, did not increase chyme trypsin activ-ity as seen in the SBM-fed ¢sh From these studies, weconclude that soy saponins play a key role in soy-bean-induced enteritis, but the pure compound willnot induce enteritis unless some other plant compo-nent is also present in the diet

sapo-Our knowledge of the role and e¡ects of saponins

in ¢sh diets requires strengthening, particularlyregarding interactions with other feed components,possible growth-promoting potentials and especiallyregarding e¡ects in species other than salmonids.Current knowledge does not allow an accurate uppersafe 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 ofleucine 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 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 com-pared to those seen in the soybean fed ¢sh, and also seemed to reduce the activity of brushborder enzymes but did notincrease the chyme trypsin activity Results within each parameter marked with di¡erent letters were signi¢cantly di¡er-ent (Po0.05)

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ferent sources, but levels up to 1g kg 1appear 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 of 15^80 g kg 1

(Saini 1989) (see Fig 6) These oligosaccharides are

not hydrolysed by endogenous enzymes in

monogas-trics 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

oli-gosaccharides 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

short-term study with ra⁄nose and soybean molasses in

diets for Atlantic salmon gave no indication of

nega-tive 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

oligosac-charides disappear from the intestinal contents in

¢sh (Refstie, Sahlstrom, Brathen, Baeverfjord &

Kro-gedal 2005) Thus, it is likely that an increase in

diet-ary 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 gosaccharides, may alter the micro£ora If so, thismay explain why results on enteritis di¡er somewhatbetween experiments and points to a possible role ofoligosaccharides as prebiotics

oli-Update on recent findings on immuneresponses involved in soybean-inducedenteritis

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 pri-mary and secondary mucosal folds and a widening ofthe lamina propria, which is in¢ltrated by a mixedpopulation of in£ammatory cells identi¢ed as lym-phocytes, macrophages, eosinophilic and neutrophi-lic granular cells, and di¡use immunoglobulin

M (IgM) (Baeverfjord & Krogdahl 1996; lep, Press, Bverfjord, Krogdahl & Landsverk 2000).The functional alterations include a reduction inbrushborder enzyme activities (Bakke-McKellep

Bakke-McKel-et al 2000; Krogdahl, Bakke-McKellep & Baeverfjord2003), reduced uptake of macromolecules (Uran,Aydin, Schrama, Verreth & Rombout 2008) and in-creased permeability (Nordrum, Bakke-McKellep,Krogdahl & Buddington 2000; Knudsen et al 2008).The e¡ects seem to be dose dependent both regardinghistological and functional characteristics (Krogdahl

et al 2003) At dietary levels below 10% inclusion ofSBM, the e¡ects vary from absent to very distinct Arecent Norwegian risk evaluation of plant ingredients

in ¢sh feed suggests 5% as an upper limit (Hemre,Amlund, Aursand, Bakke, Olsen, Ring & Svihus2009)

The work of Uran, Goncalves, Taverne-Thiele,Schrama,Verreth and Rombout (2008) indicates that

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

salmonids are not the only species that develop

enter-itis when fed diets with SBM The common carp seem

to respond with similar changes In the carp,

how-ever, 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

en-teropathies Our recent studies have therefore

fo-cused 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),

cyto-toxic (TC), memory (TM), regulatory (Treg) and natural

killer (NKT) T cells characterized in mammals,

mod-ulate the immune response depending on the

situa-tion The di¡erent types of T cells di¡er somewhat in

their protein expression of the TCR complex, ing a basis for characterization of various T-cell popu-lations Whether the cells in¢ltrating the laminapropria of the distal intestine in soybean fed salmo-nids comprise T cells has remained an unansweredquestion due to lack of appropriate markers However,recently, we were able to develop a protocol for thedetection of T-cell-like cells in Atlantic salmon usinghuman antibodies developed against a conserved epi-tope of the CD3e protein, which is part of the TCRcomplex of all known T cells (Bakke-McKellep, Froys-tad, Lilleeng, Dapra, Refstie, Krogdahl & Landsverk2007) The results showed that during the SBM-in-duced enteropathy, many of the cells in¢ltrating thelamina propria of the distal intestine were lympho-cytes with reactivity to the antibody against theCD3e epitope These lymphocytes were not reactive

provid-to an antibody developed against salmonid IgM aswould be the case if they were B-cells (also lympho-cytes) Real-time polymerase chain reaction with pri-mers developed for salmon CD3 polypeptide, as well

as CD4 and CD8b, also revealed signi¢cantly creased expression (Po0.05) in the distal intestinaltissue of SBM-fed ¢sh compared with ¢sh meal-fedreference ¢sh These results indicate that a mix ofputative T-cell subtypes was involved in the in£am-matory response after 3 weeks of dietary exposure

in-to SBM More speci¢c investigations are needed in-toreveal which subtypes of T cells are present In any

Figure 7 Histological characteristics of distal intestinal mucosa from Atlantic salmon fed a reference diet based on ¢shmeal (left) and a diet with410% standard soybean meal (right) The soybean response is characterized by a shortening ofthe 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¡useimmunoglobulin

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case a T-cell-mediated response appears to be

in-volved 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

re-pair They show increased activation in a range of

hu-man in£ammatory diseases, including in£ammatory

responses in the GI tract (Cenac, Coelho, Nguyen,

Compton, Andrade-Gordon, MacNaughton, Wallace,

Hollenberg, Bunnett, Garcia-Villar, Bueno &

Verg-nolle 2002; Schmidlin, Amadesi, Dabbagh, Lewis,

Knott, Bunnett, Gater, Geppetti, Bertrand & Stevens

2002; Cenac, Garcia-Villar, Ferrier, Larauche,

Verg-nolle, Bunnett, Coelho, Fioramonti & Bueno 2003;

Kim, Choi, Yun, Kim, Han, Seo, Yeom, Kim, Nah &

Lee 2003) Our hypothesis was that the PARs are

pre-sent in salmon and are involved in soybean enteritis

Cloning and characterization of the full-length

se-quence of Atlantic salmon PAR-2 was successfully

conducted and made it possible to investigate the

ex-pression in both early and chronic stages of

SBM-induced 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

ex-pressions 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

ex-posure 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

en-teropathy In the chronic stages of the enteropathy,

down-regulation of PAR-2a may indicate

desensitiza-tion of the PAR-2a receptor Expression of the PAR-2b

gene was not altered in the ¢rst 7 days of SBM

feed-ing, but a signi¢cant up-regulation was observed

after 3 weeks, suggesting a putative role in the

chronic stages of SBM-induced enteritis The

expres-sion 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 Atlan-tic salmon as a model for diseases in other animals,including humans, may help identify points or com-ponents in the disease process that represent poten-tial targets for therapeutic agents or preventivemeasures PAR-2 antagonism has been shown to re-duce joint in£ammation in mice when used beforethe onset of in£ammation (Kelso, Lockhart, Hem-brough, Dunning, Plevin, Hollenberg, Sommerho¡,McLean & Ferrell 2006), but its e¡ect on existingin£ammation has not been investigated Obviously,several obstacles need to be overcome before anysuch treatment modality could be introduced inlarge-scale commercial aquaculture However, otherdownstream components of the in£ammatory pro-cess may provide more suitable targets Further char-acterization of immune responses may also identifysuitable markers for monitoring the health andwelfare status of animals in large-scale productionsystems

Research perspectives for the future

We are only in the initial stages of understanding thee¡ects of ANFs in ¢sh Strengthening of the knowl-edge base is urgently needed to understand the nega-tive e¡ects and to ¢nd means of overcoming them.Interactions between the e¡ects of ANFs seem to bevery important The picture is complicated as thegut microbiota may modify the antinutrients andhence their interactions and biological e¡ects More-over, not only salmonids show soybean-induced en-teritis and plant protein sources other thansoybeans contain important ANFs With strength-ened knowledge, we can develop better diets for im-provement of nutrition, health and economy inaquaculture

AcknowledgmentsMany of the recent ¢ndings from our group reportedherein were supported by a Norwegian ResearchCouncil grant (no 145949/120) We are grateful tothe Research Council of Norway for establishing theAquaculture Protein Centre as a Norwegian Centre ofExcellence, and committing to co-funding the centrefor 10 years This has allowed focused basic research

on nutrition and health in ¢sh to improve our standing of responses to feedstu¡s and ANFs

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ac-An update on antinutrients in aquaculture feeds — Krogdahl et al Aquaculture Research, 2010, 41, 333^344

r 2010 The Authors

1 Alltech Aqua, Cephalonia, Greece

2 Grupo de Investigacion en Acuicultura (GIA), ICCM & ULPCG, Telde, Canary Islands, Spain

3 Aquaculture and Fish Nutrition Research Group, School of Biological Sciences, University of Plymouth, Plymouth, UK

Correspondence: J W Sweetman, Samoli, Livadi, 28200 Lixouri, Cephalonia, Greece E-mail: jsweetman@alltech.com

Abstract

The promotion of nutritional strategies that optimize

natural defence mechanisms in ¢sh is of critical

importance in producing robust juveniles and adult

¢sh These animals are more capable of minimizing

the impact of opportunistic pathogen attack, thus

improving liveability and performance characteristics

The importance of the piscine gastrointestinal tract as

a major endocrine and osmoregulatory organ is well

reported as is its function as a defensive barrier to

pathogen attack Investigations using the inclusion of

a speci¢c structural form of mannan oligosaccharide

have been shown to improve the performance

para-meters, immune status, and gut morphology and

improve an important aspect of barrier protection

through the enhancement of mucal production in a

number of aquaculture species The selenium status

of an animal is pivotal in determining the success

of the innate and adaptive immune response of the

animal, and the use of an organic selenium source, in

the form of a selenoyeast, has been shown to improve

enzyme function and tissue uptake The antioxidant

role of many of the selenoptroteins and the role of

selenium in the glutathione peroxidase enzyme

path-ways involved in the control of oxidative stress is

criti-cal if oxidative damage and cell membrane lipid

peroxidation are to be prevented The use of these

com-pounds as feed additives has important implications

for health management in commercial aquaculture

facilities Further research is needed to evaluate the

bene¢ts o¡ered by a range of commercial products

Keywords: natural defence, gastrointestinal tract,mannan oligosaccharide, Bio-Moss, organic sele-nium, aquaculture species

IntroductionThe successful commercial production of an aqua-culture species requires the optimization of a num-ber of operational parameters so that the productmay be delivered to its market place in the most cost-e¡ective manner, thus maximizing the potential forpro¢t within that activity Critically, on the produc-tion side, optimal growth, performance and productquality play a crucial role in the overall cost-e¡ective-ness and ultimate success of the business Often inindustrial-scale farms, unfavourable environmentalconditions (oxygen levels, pH, water quality andtemperature £uctuations), sub-optimal growth con-ditions (inadequate nutrition, overcrowding andoverfeeding) and the reality of practical husbandrypractices combine resulting in the development ofstress situations that ultimately express themselves

in poor performance, suppressed immune defencemechanisms and variable product quality (Wede-meyer 1997; Pickering 1998) This makes farmed ¢shmore vulnerable to ubiquitous opportunistic bacter-ial, viral and parasitic infections, further impactingpro¢tability negatively (Hansen & Olafsen 1999;Verschuere, Rombaur, Sorgeloos & Verstraete 2000).The promotion of nutritional strategies that en-hance the natural defence mechanisms of ¢sh, thusenabling them to adequately combat the negative

e¡ects of stress, will help to minimize the risks

asso-ciated with intensive culture conditions The primary

defence mechanism of a ¢sh, exposed to an infectious

or damaging agent, can be considered to be the

phy-sical barrier [skin, gills and gastrointestinal (GI)

tract] and their protection mechanisms, at the point

of interaction of the environment and the physiology

of the ¢sh Both externally and internally, therefore,

the mucosal barrier and the tissue cellular

mem-branes play a vital role in this process

This paper reviews recent work carried out with a

mannan oligosaccharide (MOS) (Bio-Moss) and a

se-lenoyeast providing organic selenium (Sel-Plexs),

which have been shown to be e¡ective prophylactic

nutritional strategies helping natural barrier defence

mechanisms, including gut morphology and nutrient

uptake, mucosal development, microbial £ora balances

and improving immunocompetence and disease

resis-tance Other mannan oligosaccharides have been

shown to have similar e¡ects in terrestrial animals;

however, from the few aquaculture studies available

variable responses have been observed in di¡erent

spe-cies (Genc, Aktas, Genc & Yilmaz 2007; Genc,Yilmaz,

Genc & Aktas 2007, Yilmaz, Genc & Genc 2007) The

di¡erences observed between the di¡erent MOS’s may

also arise due to the structural di¡erences of the MOS

used, but no details of this are given in these

publica-tions Similarly, while there are several studies using

selenomethionine (SeMet) in aquaculture species, the

use of other selenoyeasts in aquaculture has only been

reported by Jovanovic, Grubor-Lajsic, Djukic,

Gardino-vacki, Matis and Spasic (1997) (of unspeci¢ed origin) in

their investigation of the e¡ect of selenium on the

anti-oxidant system of carp but selenium muscle deposition

was not investigated

The function of the GI tract

The digestive tract of ¢sh has been described by

Ringo, Myklebust, Mayhew and Olsen (2007) as a

‘muscular tube lined by a mucous membrane of

columnar epithelial cells that exhibit regional

varia-tions in structure and function’ The major function

of the GI tract is to process the ingested feed material

and to digest and degrade this into a form that can be

easily absorbed and assimilated by the animal and

thereby supplies dietary nutrients to the body tissues

Digestion by its very nature is a complex process

in-volving enzyme and £uid secretions, motility,

absorp-tion and ultimately evacuaabsorp-tion

The GI tract has long been recognized as one of themajor routes of infection in ¢sh, and the histologicalstudy of the intestine has been important in estab-lishing the status of structural integrity; it acts as atool that helps improve our understanding of dietaryin£uences that can either positively improve thestructure or factors such as infectious diseases oranti-nutritional components that may ultimatelycause physical damage (Grisez, Chair, Sorgeloos &Ollevier 1996; Acosta, Real, Caballero, Sieiro, Fernan-dez & Rodriguez 2002; Bakken 2002; Ringo, Jutfelt,Kanapathippillai, Bakken, Sundell, Glette, Mayhew,Myklebust & Olsen 2004)

An array of protection systems have evolved to tect the GI tract from the risk of damage Mucosa iscritical in digestion, absorption and metabolicprocesses and acts as a barrier to pathogenic infec-tions, preventing both viable and non-viable bacteriaand their products from migrating from the intestinallumen through the epithileal mucosa to infect other-wise sterile tissues In addition, mucosa plays a role inthe electrolyte balance, immune response and endo-crine functions Mucins and glycoproteins associatedwith the intestinal brush border serve as importantbarriers protecting the absorptive surface from feed-stu¡s, bacteria colonization and toxins In addition,endogenous acids, digestive enzymes and bile reducebacterial growth while digestive £ow and peristalticmovements transport the digesta through the tract,limiting bacterial development

pro-Stress has been shown to induce severe cell mage on the intestinal and enterocyte function incarp, eel and cat¢sh (Szakolczai 1997) and also Atlan-tic salmon and rainbow trout (Olsen, Sundell, Han-sen, Hemre, Myklebust, Mayhew & Ringo 2003;Olsen, Sundell, Mayhew, Myklebust & Ringo 2005).The loss of intestinal integrity resulting in enhancedepithelial permeability may lead to the enhanced up-take of macromolecules, bacterial translocation andantigens across the epithelium Enteritis and poorgut morphology may lead to ine⁄cient feed conver-sion, and the repair of damaged enterocytes is anenergy-consuming activity, which in turn directsvaluable resources from growth to the more immedi-ate urgency of tissue repair and maintenance

da-Role of MOS

In the last 10 years, more has been discovered aboutthe complex carbohydrate structures of the yeast cellwall and how di¡erent strains of yeast, di¡erent fer-Enhancing natural defences in aquaculture species J W Sweetman et al Aquaculture Research, 2010, 41, 345^355

r 2010 The Authors

mentation conditions and di¡erent processing

meth-ods used in the manufacture of MOS can a¡ect the

characteristics and the function of the MOS

(New-man 2007) In the work reported here, a speci¢c

MOS, Bio-Moss, Alltech, Lexington, KY, USA, has

been used This MOS can be brie£y described as being

composed of yeast cell wall mannoproteins from

Sac-charomyces cerevisiae, which are highly glycosated

polypeptides, often 50^95% carbohydrate by weight,

that form radially extending ¢brillae at the outside of

the cell wall (Lipke & Ovalle 1998; Kapteyn, Van Den

Ende & Klis 1999) Many mannoproteins carry

N-linked glycans with a core structure of Man10^

14GlcNAc2^Asn structures very similar to

mamma-lian high-mannose N-glycan chains ‘Outer chains’

present on N-glycans consist of 50^200 additional

a-linked mannose units, with a longa-1,6-linked

back-bone decorated with shorta-1,2 and a-1,3-linked side

chains Alltech has pioneered industrial extraction

procedures that are currently used to produce

func-tional mannan oligosaccharide extracts from

Sacchar-omyces cerevisiae cell wall and marketed under the

Bio-Mossbrand name

Mannan oligosaccharides has been shown to a¡ect

gut health by pathogen adsorption and modulation

of both humoral and cellular immune function

These e¡ects have been well documented in terrestrial

animals where the incorporation of MOS in poultry

and swine diets has led to increased body weight and

survival (Spais, Giannenas, Florou ^ Paneri, Christaki

& Botsoglou 2003; Miguel, Rodriguez-Zas & Pettigrew

2004) and improved feed conversion ratios (Fritts

& Waldroup 2003; Hooge, Sims, Sefton, Connolly &

Spring 2003; Waldroup, Oviedo-Rondon & Fritts 2003;

Sims, Dawson, Newman, Spring & Hooger 2004)

Similar results have been observed in several

aqua-culture species.When MOS was included in the diet of

Jian carp higher weight gains, improved feed

conver-sion ratio (FCR) and immune parameters were

observed (Zhou & Li 2004) Similar e¡ects of MOS with

improved growth, lower FCR, mortalities and

improved immune parameters have been reported in

carp (Staykov, Denev & Spring 2005; Culjak, Bogut,

Has-Schon, Milakovic & Canecki 2006), rainbow trout

(Staykov, Spring, Denev & Sweetman 2007) and cat¢sh

(Bogut, Milakovic, Pavlicevic & Petrovic 2006) Daniels

(2005) reported decreased mortalities and improved

survivals in lobster larvae fed MOS to stage IV

In sea bass juveniles, Torrecillas, Makol, Caballero,

Montero, Robaina, Real, Sweetman,Tort and

Izquier-do (2007), Torrecillas, Caballero, Sweetman, Makol

and Izquierdo (2007) have reported that the dietary

incorporation of MOS signi¢cantly increases growth,

by approximately 10%, and produces a better speci¢cgrowth rate at low ¢sh densities; however, when ¢share stocked at higher densities the FCR is enhanced.The mucus layer in the GI tract acts as a ¢rstdefence barrier between the intestinal surface andthe intestinal luminal content and is a component ofthe innate host immune response Nutritionalfactors, such as high dietary ¢bre or protein content,have been shown to a¡ect mucin synthesis andsecretion in the small intestine of several terrestrialanimals and this may in£uence the content andthickness of the mucous layer (Sharma, Fernandez,Hinton & Schumacher 1997; Montagne, Piel & Lalles2004; Piel, Montagne, Seve & Lalles 2005; Morita,Tanabe, Ito, Yuto, Matsubara, Masuda, Sugiyama &Kiriyama 2006) Uni (2007) showed that dietarysupplementation with MOS in broiler diets resulted

in increased production of mucin, as indicated byincreased MUC 2 mRNA expression, increased thethickness of the mucin adherent layer and increasedthe size of the goblet cells in the small intestine whencompared with control diets Torrecillas, Makol,Caballero, Montero, Sweetman and Izquierdo (2008)reported that in sea bass fed 2 months of MOS supple-mentation, the number of cells secreting acid mucins

in the posterior gut was signi¢cantly increased Theincrease in mucus secretion with its antiadhesiveproperties could be directly related to the decrease

in the number of infected ¢sh in disease challengetrials reported previously (Torrecillas, Makol, et al.2007; Torrecillas, Caballero et al 2007)

Increased skin mucous secretion has also beenrecorded in salmon (C Wallace and J Johansen,pers comm.) fed diets supplemented with 0.4% MOS

in comparison with control diets Trial work withMOS at Gildeskal Forskningsstasjon AS in Norwayhas shown signi¢cantly reduced lice counts After 7weeks of feeding juvenile salmon sea lice counts un-

Table 1 The number of sea lice found to be present on sampled juvenile salmon

Control cases MOS cages

1 2 3 4 Average 1 2 3 4 Average t-test

dertaken (Table 1) showed that ¢sh fed with the

addi-tion of MOS in the feed had signi¢cantly lower overall

numbers of sea lice present compared with ¢sh fed

the control diet (P 5 0.044) No signi¢cant di¡erences

were found between the diets for the di¡erent stages

of Lepeophtheirus salmonis or for Caligus elongates

even though ¢sh fed with the addition of MOS had

lower numbers present for each of the di¡erent stages

as well as for C elongates

One of the key bene¢ts of this speci¢c MOS is its

ability to bind or agglutinate a number of strains of

bacteria known to cause disease in shrimp and ¢sh,

thereby preventing colonization of the gut and

subse-quent infection The presence of Escherichia coli in the

intestinal digesta of Jian carp was signi¢cantly

decreased while signi¢cant increases were observed

in bi¢dobacterium and lactobacillus when 0.24% of

MOS was included in the diet (Zhou & Li 2004)

Dimi-troglou, Davies, Moate, Spring and Sweetman (2007)

demonstrated that MOS signi¢cantly reduced the

bacterial load in the gut of both rainbow trout and

sea bream by reducing the total aerobically cultivated

bacteria In the case of rainbow trout, the MOS

fed ¢sh had reduced numbers of Micrococcus spp.,

Staphylococcus spp., Aeromonas/Vibrio spp and other

unidenti¢ed Gram1 bacteria and increased

Acineto-bacteria spp., Pseudomonads spp and EnteroAcineto-bacteria

spp This indicated that the MOS promoted the

coloni-zation of bene¢cial bacteria associated with

the natural gut £ora of the rainbow trout when

healthy

The immune function was improved with the

in-clusion of MOS in sea bass juveniles (Torrecillas,

Makol, et al 2007; Torrecillas, Caballero et al 2007)

The immune parameters, phagocytic activity of

leucocytes and the bacterial activity of the sera in

the MOS fed groups showed a statistically signi¢cant

improved dose response when compared with thecontrol group (Figs 1 and 2)

Disease resistance to bacterial infection, both bycohabitative challenge and by direct inoculation

in the gut, was enhanced when MOS was rated into the diets In cohabitation trials, the pre-sence of Vibrio alginolyticus on the head kidney ofsea bass was 33% for the control group and 8%and 0%, respectively for the 0.2% and 0.4% MOSfed groups

incorpo-The incorporation of MOS also resulted in an provement in the hepatocyte morphology, with moreregularly shaped hepatocytes and less hepatocyteswith displaced nuclei to the cellular periphery Theactivity of lipogenic enzymes in the liver was signi¢-cantly reduced at the di¡erent incorporation levels ofMOS (Table 2)

im-This development work has shown interestingnew trends indicating the possibility of interactionwith nutrient uptake mechanisms as indicated bythe reduced liver fat deposition and the improvedhepatic composition that may be an indicator ofbetter utilization of dietary nutrients

Gut morphologyThe e¡ect of MOS on the GI morphology of several spe-cies, rainbow trout, salmon, sole and sea bream, hasbeen examined using optical, scanning and transmis-sion electronic microscopy (Dimitroglou et al 2007)

In adult rainbow trout and sole MOS had a cant e¡ect on the external and internal perimeterratio both in the anterior and in the posterior gut re-gions, indicating a more complicated architecturalgut structure with longer villi, and hence a large sur-

b

a

b a

c

a

a

b c

Figure 1 In£uence of mannan oligosaccharide (MOS) on

the total phagocytic activity of head kidney leukocytes in

European sea bass in Experiment I at 30, 45 and 60 days of

supplementation (mean SD; n 515) Di¡erent letters

within a line denote signi¢cant di¡erences (Po0.05)

0.00.10.20.30.40.50.60.7

Enhancing natural defences in aquaculture species J W Sweetman et al Aquaculture Research, 2010, 41, 345^355

r 2010 The Authors

face area for nutrient absorption than the non-MOScontrol diet (Fig 3).

The inclusion of MOS improved the microvilli sity only in the posterior gut region of the freshwater

den-¢sh, rainbow trout However, in marine ¢sh species,salmon, sole and sea bream MOS produced a morepronounced integral e¡ect by increasing the micro-villi density in both the anterior and the posteriorgut regions The microvilli length was also signi-

¢cantly increased in all the adult ¢sh examined

in both the anterior and posterior gut regions Injuvenile rainbow trout, the e¡ect of MOS on themicrovilli length was restricted to the anterior part

IU/mg protein 0.0116a  0014 0.0082b  0.0004 0.0079b  0.0013 0.0094ab  0.0003

Di¡erent letters within a line denotes signi¢cant di¡erences (P o0.05) Control 5 0% MOS; BM2 52% MOS; BM4 54% MOS; BM6 56% MOS Values expressed in mean  SD (n 5 9).

MOS, mannan oligosaccharide.

Figure 3 Comparative light microscopy micrographs of

the gut transect of rainbow trout fed either a control diet

or mannan oligosaccharide (MOS) Note the more

com-plex villi structures in the Bio-Mos fed ¢sh

SEM and TEM - microvilli structures

Control

Control

Bio-Mos

Bio-MosMicrovilli

height 1.28

Microvilliheight 1.55

Microvillidensity 2.10 Microvillidensity 9.75

Figure 4 Comparative

scan-ning electron microscopic

(SEM) and transmission

elec-tron microscopic (TEM)

mi-crographs of the microvilli

structures in the

gastroin-testinal tract of salmon fed

Bio-Mos and their control

groups without Bio-Mos in

their diets

Figure 4 shows the e¡ect of MOS supplementation

combined with dietary organic minerals on the

mi-crovilli structures of salmon, where the mimi-crovilli

density of the MOS fed groups was 4.62 times greater

than that of the control groups of salmon, and the

mi-crovilli length was increased by 21.1% in the MOS fed

groups when compared with the control groups The

microvilli density photographs also indicate that

there are considerably less damaged areas in the

MOS fed groups Similar results have been recorded

when only MOS supplementation was included in

the salmon diets (A Dimitroglou, unpub data)

It is clear from these results that MOS can

have a signi¢cant e¡ect on gut morphology The

changes observed, increased perimeter ratio,

increased villi and microvilli density and length,

indicate that the absorptive surface of the gut has

been improved and hence a better absorptive

capa-bility appears to be possible Increased microvilli

density without signs of damage or irritation will

also lead to an increase in the absorptive capability

of the enterocytes

In the case of sole, MOS supplementation was made

in the feed in order to improve the condition of

vacci-nated and non-vaccivacci-nated ¢sh In both cases, MOS

improved the villi structure and increased the

micro-villi density A disease outbreak in these ¢sh and the

overall mortalities were reduced in the MOS fed

groups when compared with the control groups

(Dimitroglou, Janssens & Davies 2006)

The role of selenium and

organoselenium in antioxidant

protection and immune function

One of the major determinants of the physical

proper-ties of cell membranes is the degree of unsaturation

of the fatty acyl components of the membrane

phos-pholipids Polyunsaturated fatty acids (PUFA) are

essential in ¢sh development and DHA, the w3

polyunsaturate with six double bonds, is one of the

most highly unsaturated fatty acids to be found in

cellular lipids and is therefore very vulnerable to

at-tack by free radicals Oxidative metabolism in aerobic

tissues results in the continuous production of

super-oxide radicals and hydrogen persuper-oxide.When the

gen-eration of reactive oxygen species (ROS) exceeds their

normal removal, such as in times of stress conditions,

oxidative stress occurs Under conditions of oxidative

stress induced by ROS, the lipid peroxidation process

starts, which can lead to peroxidation of susceptible

DHA and lead to membrane damage Cell membranesare therefore very dependent on antioxidants for pro-tection and therefore many of the biological func-tions of the PUFAs and selenium are interlinked.Selenium is incorporated, instead of sulphur, intothe protein amino acids: methionine and cysteine toform SeMet and selenocysteine (SeCys), in yeastgrown in the presence of inorganic selenium In ¢sh,SeCys is at the active site in all active selenoenzymesinvolved in redox reactions (Arthur 2000)

The role of the antioxidant selenoenzymes in theprotection of phagocytic cells of the innate immunesystem is one of the best-characterized aspects of theessentiality of selenium in disease defence (Arthur,McKenzie & Beckett 2003) Phagocytic cells, neutro-phils and macrophages, ingest and destroy pathogensusing enzymatic processes or by the generation oftoxic oxygen radicals Preventing oxidative damage tothe phagocytic cells is a balance between producingenough radicals to kill the pathogens and the activity

of the radical-neutralizing enzymes The antioxidantselenoproteins, particularly glutathione peroxidase(GSH-Px), must act quickly to neutralize the oxygenradicals to protect the immune cellular machineryand its ‘killing ability’

Phagocytic cells from selenium-de¢cient animalshave been shown to be less able to kill pathogens due

to the reduced activity of GSH-Px (Arthur et al 2003).Selenium de¢ciency has also been shown, by impair-ing the leukotriene B4 synthesis, to slow the chemo-taxis of phagocytes to sites of injury and infection(McKenzie, Beckett & Arthur 2006)

Fish under a state of stress may draw on tissuereserves of selenium to prevent oxidative damageand membrane lipid peroxidation through theGSH-Px pathways Tissue selenium loss has beendemonstrated after transport stress in Chinooksalmon when moving juvenile ¢sh between sites

on the Columbia River (Halver, Felton & Zbanyszek2004)

The selenium form in the diet has a very huge pact on its ability to ful¢l its role in immune function

im-In its puri¢ed form, SeMet is unstable and easilyoxidized Moreno, Quijamo, Gutierrez, Perez-Condeand Camara (2002) demonstrated that in freeze-driedsamples of oyster total Se and the Se species evalu-ated were stable for at least 12 months under all thetested conditions However, if the Se species are pur-i¢ed in the enzymatic extracts, including SeMet, theyare only stable for 10 days if stored at 4 1C SeMet isquite stable in yeast and Block, Glass, Jacobsen, John-son, Kahakachchi, Kaminski, Skowronska, BoakyeEnhancing natural defences in aquaculture species J W Sweetman et al Aquaculture Research, 2010, 41, 345^355

r 2010 The Authors

Tyson and Uden (2004) showed that SeMet was the

major Se product in a high-Se yeast stored at room

temperature for410 years Selenoyeasts are

there-fore advantageous as a selenium source in the

com-mercial production of aquaculture feeds Selenium in

this form is incorporated into proteins that are

pre-sent in the yeast cell wall as glucomanno-protein

complexes and in the intracellular space as O- and

N-glucosylated proteins The presence of the

carbo-hydrate component increases Se protein stability

at elevated temperatures and pressures during the

feed extrusion process, similar to the formation of

complexes between yeast proteins and trehalose in

yeast exposed to heat stress Substituting an organic

selenium yeast source for the inorganic sodium

selenite has been demonstrated to reduce the impact

of disease challenge with lower mortality and less

pathology (tissue damage), and thereby improve

e⁄ciency, uniformity and product quality The

reason for this is that the organoselenium

com-pounds formed by yeast are more easily metabolized

and can be taken up into tissue proteins such as

muscle non-speci¢cally in place of methionine,

which provides tissue reserves of this critical trace

element Normal cell and protein turnover releases

the required selenium, a process that escalates

during periods of increased demand such as immune

challenge

Increasing the selenium status of

farmed aqua species: response to

organic selenium

When considering the selenium requirements of

commercial aquaculture species, it has been reported

that selenium in whole livers of coho salmon was

twice that of commercially reared species (Felton, Ji

& Mathews 1990), and 10-fold di¡erences were

observed in liver selenium levels when comparing

wild and farmed Atlantic salmon (Maage, Julshamn

& Ulgenes 1991) Felton, Landolt, Grace and

Palmisa-no (1996) reported that it was very di⁄cult to obtain

tissue selenium concentrations similar to wild ¢sh

when inorganic selenium sources were used in the

diets Rider and Davies (2007, 2008) reported that

in experiments with rainbow trout (Oncorhynchus

mykiss) at the University of Plymouth in the United

Kingdom, trout given dietary organic selenium

(Sel-Plexs, Alltech) retained more selenium in muscle

than trout given selenite (Fig 5) This is in agreement

with other studies that demonstrated that organic

but not inorganic selenium is deposited in these sues due to the non-speci¢c incorporation of SeMet(Lorentzen, Maage & Julshamn 1994; Wang & Lovell1997; Cotter, Craig & McLean 2008)

tis-Improving selenium status through attention tolevel and form has been shown to boost cell-mediatedimmune parameters and defence against viral infec-tion in many species, from humans to invertebrates.The sensitivity of immune defences against viruses

to selenium status is also a function of the impact ofviral infections on oxidative stress Oxidative stress is

a hallmark of viral infection and is known to play arole in the progression of several viral diseasesincluding HIV and hepatitis B in humans (Stehbens2004) Radical oxygen species are released from in-fected host cells and activated phagocytes In addition,viral ‘hijacking’ of host synthetic processes disturbsthe normal function of the endoplasmic reticulumand mitochondria, which increases ROS production

Muscular tissue Se

0.21

0.23

00.050.10.150.20.250.30.350.4

012345678910

Experimental week

Basal feed Inorganic Organic

∗ Means differ, P>0.05

Figure 6 E¡ect of selenium source (added at 0.3 ppm) onthe growth of Paci¢c white shrimp (Penaeus vannamei)

and depletes cellular antioxidant components

includ-ing micronutrients and glutathione (Allard, Aghdassi,

Chau, Salit & Walmsley 1998; Schwarz 1996)

The practical impact of improved Se status

on growth and health has been illustrated in a

recent work by Sritunyalucksana, Intaraprasong,

Sa-Nguanrat, Filer and Fegan (2008) with Paci¢c

white shrimp (Penaeus vannamei),which

demon-strated that the addition of organic selenium to the

diet resulted in better growth (Fig 6) More

impor-tantly, higher survival after challenge with the

Taura syndrome virus (TSV) was reported (Fig 7)

Conclusion

Mannan oligosaccharide has been shown to provide

multiple bene¢ts when incorporated into

aquacul-ture feed diets for improving the performance and

health status of a number of important commercial

species MOS diet supplementation has been shown

not only to increase skin and GI tract mucous

secre-tion therefore improving barrier funcsecre-tion and

protec-tion, but to improve the GI morphology and therefore

its function through an increased absorptive surface

and better absorptive capability MOS also interacts

with the immune system in a modulatory manner

and alters lipogenic enzyme activity promoting

bet-ter utilization of dietary nutrients, thus improving

performance characteristics and immune function

The multifactorial role of selenium in both the

in-nate and the adaptive immune system is expressed

through selenoproteins and regulatory cytokines.Many of the selenoproteins play antioxidant roles,which neutralize radicals produced by oxidativestress and the phagocytic cells of the innate response

to maintain their e¡ectiveness as the immune tem’s ‘¢rst responders’ The low activity of GSH-Pxdue to Se de¢ciency has been associated with in-creased viral virulence due to ROS-induced muta-tions of the viral genome, and this role in viralevolution has been proposed to explain why so manyemerging RNA viral diseases arise in Se-de¢cientregions of the world (Foster 2003)

sys-A large number and variety of pathogens tant in food animal agriculture or aquaculture live

impor-or replicate inside host cells The outcome of diseasescaused by these pathogens is dependant on e¡ectiveresponses by the T-cell-mediated arm of adaptiveimmunity, which targets these ‘altered self’cells Sele-nium status is pivotal to cell-mediated immunity; andimprovements due to providing this trace element in

an organic form that is easily stored and metabolizedhave been shown to boost the animal’s ability to resistthe impact of disease and reduce losses due to viraldisease in a variety of species

The dietary incorporation of a speci¢c MOS andorganic selenium sources results in bene¢ts such asimproved performance, livability and disease resis-tance and operates through enhancing the naturaldefence mechanisms of the ¢sh This therefore con-tributes to increased food safety as well as improvedproduction and farm economics of interest to thecommercial producer

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Nutrition and immunity: an update

Viviane Verlhac Trichet

Research Centre of Animal Nutrition and Health, NRD/CA ^ DSM Nutritional Products France, Saint-Louis Cedex, France

Correspondence: V Verlhac Trichet Research Centre of Animal Nutrition and Health, NRD/CA^DSM Nutritional Products France, BP

170, 68305 Saint-Louis Cedex, France E-mail: viviane.verlhac@dsm.com

Abstract

Immunity encompasses all mechanisms and

re-sponses used by the organism to defend itself against

bacteria, viruses or parasites Adequate supply and

balance of nutrients are required for proper e⁄ciency

of the host defences Research has identi¢ed dietary

factors that a¡ect human and animal immune

re-sponses like amino acids, fatty acids, minerals and

vitamins Some of these nutrients have been proven

to have speci¢c actions on immunity when provided

at pharmacological doses This paper will review

these nutrients and their current use in aquaculture

The immune system is an e⁄cient but complex

sys-tem Its complexity has made the assessment of the

e¡ects of diets di⁄cult Nevertheless, the

standardi-zation of methodology as well as the use of new

tech-niques at the cell or the gene level should help to

better understand the mechanisms of immune

mod-ulation This paper will review the major functions of

¢sh and shrimp immune system and the

methodolo-gies used Cellular and humoral functions including

cytokines will be discussed in relation to potential

means to modulate them and the underlying

me-chanism A better understanding of the mechanisms

of modulation of the immune functions should help

in the discovery of new dietary factors to improve

the immune status of the animal, leading to better

disease resistance

Keywords: aquaculture, ¢sh, shrimp, nutrition,

immunity, modulation

Introduction

The paper intends to provide an update on the

rela-tionships between nutrition and immunity of aquatic

animals and more speci¢cally of ¢sh and shrimp

Most recent reviews on the topic of nutrition and munity or, more generally, health are from Lall(2000) and Verlhac and Viswanath (2004)

im-The ¢rst section reviews ¢sh and shrimp immunity,comparing innate and adaptive immune mechan-isms, highlighting the importance of innate immu-nity in those animals The second section highlightsthe key ¢ndings in the area of nutrition and immu-nity, analysing the relationship between immuneresponses and the di¡erent classes of nutrients, ex-tending it to nutraceuticals used as feed additives.The last section will deal with the evaluation of theimmune responses of ¢sh and the methods currentlyused including cellular and molecular approaches, inline with the study of the nutritional modulation ofthe immune response Prophylaxis means and per-spectives are discussed in the conclusions

Fish and shrimp immunityBeing immune corresponds to the capacity of an or-ganism to resist infection through the recognition of

a foreign agent, the responses of the system to ¢ghtthis agent and the memory of this agent, in order toquickly respond to a second aggression In compari-son with mammals, ¢sh have a less speci¢c immunesystem with a shorter response, a limited immuno-globulin repertoire, a weak memory (which reducesthe potential for long-term protection) and a mucosalresponse (whose importance in comparison with thesystemic response is not really known) The immuneresponse of ¢sh is di¡erentiated between humoral-and cellular-mediated systems, with the same type

of immune cells such as B and T lymphocytes acting

of self- versus non-self recognition, phagocytosis and

a system of lectins that can be considered to be

anti-body-like proteins

The response of ¢sh to a foreign agent is rather

similar to that of mammals, while in shrimp, the

re-sponse is very rudimentary All ¢sh pathogens

con-tain antigens: viral particles, bacteria, fungi, toxins

and animal parasites

Distinction between innate and speci¢c

immunity

Figure 1 presents the di¡erent mechanisms involved

in the innate and speci¢c (or adaptive) immunity Fish

have an important ¢rst line of defence consisting of

epithelial barriers such as skin, scales, mucus

mem-branes (gastro-intestinal tract, secretions of mucus)

and physiological barriers like stomach pH, gut

mi-cro£ora and chemical mediators secreted by the

mu-cus (defensins, lysozyme, transferrin, complement

system, etc.) Involvement of cells like phagocytes,

natural cytotoxic cells (NCC) and in£ammatory

re-sponse through the release of chemical mediators

represents a second line of defence that is initiated if

the pathogen has been able to pass the epithelial and

physiological barriers The actors of the

in£amma-tory response are interferon (IFN), interleukins (ILs),chemokines and factors like tumour-necrosis factor(TNF-a) Pathogen-associated molecular patterns(PAMP) of recognition have also been discovered as

an important element of the innate immunity, ving di¡erent receptor types

invol-The third line of defence consists of the ment of a speci¢c immune response with either theproliferation of lymphocytes leading to the produc-tion of antibodies speci¢c to the antigen or the devel-opment of a T-cell-mediated response via cytotoxic

develop-T cells (e¡ector cells) in case of viral infection, for ample These responses are generally also mediated

ex-by cytokines, which play an important role in to-cell communication for a rapid expansion of theresponse to the di¡erent parts of the body The ulti-mate step of this speci¢c response is the development

cell-of a memory, allowing the immune system to tain a B-cell pro¢le corresponding to a speci¢c patho-gen In case of a second infection, these cells will berecognized and will proliferate quickly to ¢ght the in-fection This memory mechanism is much less devel-oped in ¢sh compared with mammals and does notexist in shrimp

main-In terms of kinetics of the responses, the innateimmunity can act between hours and days, while

Infection

Adaptive immunityInnate immunity

Epithelial barriers

Phagocytes

Natural cytotoxic cells

Inflammation: chemical mediators

the speci¢c immunity would need weeks to develop

depending mainly on the environment (Bowden

2008)

Cellular actors of innate immunity

Phagocytes and NCC are the main cellular elements

of the innate immunity As shown in Fig 2,

phago-cytes are able to develop di¡erent microbicidal

me-chanisms to try to de¢nitely eliminate pathogens

Enzymes, reactive oxygen species and nitric oxide

(NO) act in concert to provide the best chances to

limit the response to the second line of defences

de-scribed earlier The production of reactive oxygen

species, either internally or externally, constitutes

the oxidative burst mechanism The extracellular

response is more pronounced in ¢sh compared with

the intracellular one

Natural cytotoxic cells are involved in innate

anti-viral immunity Natural cytotoxic cells possess

recep-tors that recognize proteins expressed at the surface

of virus-infected cells Through a mechanism of forin granules released externally, the membrane ofthe virus-infected cells will be degraded and subse-quently subjected to an osmotic shock, leading totheir complete lysis and death (Fig 3)

per-Humoral factors of innate immunityInterferon is also involved in anti-viral innate immu-nity (Fig 4) Following viral infection, type I IFN isreleased and recognized by speci¢c receptors on themembrane of non-infected cells The binding of IFN

to membrane receptors initiates the secretion of zymes like lysozyme, which will then block the repli-cation of the virus and will induce an anti-viral state.The in£ammatory response consists of the release

en-of chemical mediators: histamine, prostaglandins(PG), complement and cytokines (IL1b, 6, 8, 10,

12, TNF-a) Interleukin 1b has been characterized inteleost ¢sh (Randelli, Buonocore & Scapigliati 2008).Interleukin 6 have been identi¢ed at the molecular

Figure 2 Microbicidal mechanisms of phagocytosis Adapted from Abbas et al (1999)

Nutrition and immunity: an update V VerlhacTrichet Aquaculture Research, 2010, 41, 356^372

r 2010 The Authors

level in sea bream by Castellana, Iliev, Sepulcre,

MacKenzie, Goetz, Mulero and Planas (2008)

Tumour necrosis factor-a is conserved in all

ver-tebrate classes and has been identi¢ed in teleost

¢sh Roca, Mulero, Lo¤pez-munoz, Sepulcre, Renshaw,

Meseguer and Mulero (2008) have demonstrated that

the main proin£ammatory e¡ects of ¢sh TNF-a are

mediated through the activation of endothelial cells,

leading to the recruitment and activation of cytes but they also found implications of TNF-a indi¡erent aspects as compared with mammals like in-creased susceptibility to viral infection and increasedvirus replication

phago-All this work on cytokines and chemokines is still,for a great part, carried out at the molecular level andtherefore there is a need to demonstrate the biological

Figure 3 Natural cytotoxicity cells in anti-viral innate immunity Adapted from Abbas et al (1999)

Virus-infected cell

IFN induces an anti-viral state:

blocking viral replication

Type I IFN

IFN receptor

IFN induces an anti-viral state:

blocking viral infection of cells

Figure 4 Interferon in anti-viral innate immunity Adapted from Abbas et al (1999)