antinutritional factors present in plant-derived alternate fish feed ingredients and their effects...

Ž .Aquaculture 199 2001 197–227www.elsevier.nlrlocateraqua-online

Review article

Antinutritional factors present in plant-derivedalternate fish feed ingredients and their effects

in fish

George Francis a, Harinder P.S. Makkar b, Klaus Becker a,)

a Department of Animal Nutrition and Aquaculture, Institute for Animal Production in the Tropics and( )Subtropics, UniÕersity of Hohenheim 480 , D 70593 Stuttgart, Germany

b Animal Production and Health Section, International Atomic Energy Agency, P.O. Box 100, Wagramerstr. 5,A-1400 Vienna, Austria

Abstract

The use of plant-derived materials such as legume seeds, different types of oilseed cake, leafmeals, leaf protein concentrates, and root tuber meals as fish feed ingredients is limited by thepresence of a wide variety of antinutritional substances. Important among these are proteaseinhibitors, phytates, glucosinolates, saponins tannins, lectins, oligosaccharides and non-starchpolysaccharides, phytoestrogens, alkaloids, antigenic compounds, gossypols, cyanogens, mimo-sine, cyclopropenoid fatty acids, canavanine, antivitamins, and phorbol esters. The effects of thesesubstances on finfish are reviewed. Evidently, little unanimity exists between the results ofdifferent studies as to the specific effects of antinutrients, since most studies have been conductedusing an ingredient rich in one particular factor and the observed effects have been attributed tothis factor without considering other antinutrients present in the ingredient, or interactions betweenthem. Tentatively, protease inhibitors, phytates, antigenic compounds, and alkaloids, at levelsusually present in fish diets containing commercially available plant-derived protein sources, areunlikely to affect fish growth performance. In contrast, glucosinolates, saponins, tannins, solublenon-starch polysaccharides, gossypol, and phorbol esters, are more important from a practicalpoint of view. The effectiveness of common processing techniques such as dry and wet heating,solvent extraction and enzyme treatment in removing the deleterious effects of antinutrients fromfeed materials is discussed. More insights into the nutritional, physiological and ecological effectsof antinutrients on fish need to be accumulated through studies using purified individualantinutrients and their mixtures in proportions similar to those in alternative nutritional sources infish feeds. Such studies would provide data useful for designing optimum inclusion levels of

) Corresponding author. Tel.: q49-711-4593158; fax: q49-711-4593702.Ž .E-mail address: [email protected] K. Becker .

0044-8486r01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 01 00526-9

( )G. Francis et al.rAquaculture 199 2001 197–227198

plant-derived materials and treatment methods that would neutralise the negative effects of theantinutritional factors. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Aquaculture; Plant-derived nutritional sources; Antinutrients

1. Introduction

As estimated by FAO, the human demand for food fish will climb from the currentŽ .level of consumption of about 90 million metric tonnes Mtn to about 110 Mtn by the

year 2010. The share of aquaculture in the total world food fish production is set toincrease from 29% in 1996 to 38% by the year 2010. In fact, aquaculture has becomethe fastest growing food production sector of the world, with an average annual increaseof about 10% since 1984 as compared to 3% increase for livestock meat and 1.6%

Ž .increase for capture fisheries FAO, 1997 . To sustain such high rates of increase inaquaculture production, a matching increase in the levels of production of fish feeds isrequired. Aqua-feed production is currently one of the fastest expanding agriculturalindustries of the world, with annual growth rates in excess of 30% per year. Estimates ofthe global levels of aqua-feed production for the year 2000 vary widely from 16.8 to 4.5

Ž .Mtn FAO, 1997 . Almost one third of the 122 Mtn of fish harvested in the year 1997was converted into fishmeal or fish oil to be used in producing animal feed, includingaquaculture feed. Of the total global production of fishmeal in 1996, 2 Mtn was used infish farming, with farming of shrimp accounting for 20.3%, of salmon 18.8%, carp18.3%, marine fish 13.9%, trout 10.9%, eel 10.7%, milkfish 1.6% and catfish farming

Ž . Ž .1.3% of that tonnage Sargent and Tacon, 1999 . FAO 1999 estimates that about 40%of the total aqua-feed production was for carnivorous finfish species, 35% for non-carnivorous species, and 25% for shrimp. Thus, although the bulk of fishmeal is used insalmon, trout and marine fish farming in western countries, freshwater fish farming,largely that of carp, also consumes a substantial proportion. Given the current very rapidincrease in the intensification of fresh-water farming in Asia, particularly in China,intense future competition for limited global supplies of fishmeal and fish oil are likelyŽ .Sargent and Tacon, 1999; Naylor et al., 2000 . This predicted strong demand from Asiafor available feed resources will have a considerable impact on the world commodity

Ž .markets and feed prices in general FAO, 1999 . Fishmeal production is also ratherlocalised in some regions of the world, as a result of which it is becoming moreexpensive and difficult to obtain in many countries practising aquaculture. The need foralternative protein sources to replace fishmeal in aqua-feeds is therefore obvious andwas strongly recommended by the Second International Symposium on Sustainable

Ž .Aquaculture 1998 in Oslo, Norway. The very sustainability of the growing aquacultureŽindustry depends on the progressive reduction of wild fish inputs into fish feed Naylor

.et al., 2000 .

2. Antinutritional factors

Most of the potential, alternative, plant-derived nutrient sources are known to containŽ .a wide variety of antinutritional substances Table 1 . Hundreds of studies have been

( )G. Francis et al.rAquaculture 199 2001 197–227 199

Table 1Important antinutrients present in some commonly used alternative fish feed ingredients

Plant-derived nutrient source Antinutrients present

Soybean meal Protease inhibitors, lectins, phytic acid, saponins,phytoestrogens, antivitamins, allergens

Rapeseed meal Protease inhibitors, glucosinolates, phytic acid, tanninsLupin seed meal Protease inhibitors, saponins, phytoestrogens, alkaloidsPea seed meal Protease inhibitors, lectins, tannins, cyanogens, phytic acid,

saponins, antivitaminsSunflower oil cake Protease inhibitors, saponins, arginase inhibitorCottonseed meal Phytic acid, phytoestrogens, gossypol, antivitamins,

cyclopropenoic acidLeucaena leaf meal MimosineAlfalfa leaf meal Protease inhibitors, saponins, phytoestrogens, antivitaminsMustard oil cake Glucosinolates, tanninsSesame meal Phytic acid, protease inhibitors

published where the effects of inclusion of these materials in the diets of commonculture fish are reported. A few studies in which plant-derived materials containingantinutrients have been included in fish diets and the effect of this inclusion on fishgrowth are summarised in Table 2. The main observations from various experiments thatstudied the effects of purified antinutritional factors in fish are listed in Table 3.

Antinutrients have been defined as substances which by themselves, or through theirmetabolic products arising in living systems, interfere with food utilisation and affect the

Ž .health and production of animals Makkar, 1993 . They could be broadly divided intoŽ .four groups: 1 factors affecting protein utilisation and digestion, such as protease

Ž .inhibitors, tannins, lectins; 2 factors affecting mineral utilisation, which includeŽ . Ž .phytates, gossypol pigments, oxalates, glucosinolates; 3 antivitamins; 4 miscella-

neous substances such as mycotoxins, mimosine, cyanogens, nitrate, alkaloids, photo-sensitizing agents, phytooestrogens and saponins. Antinutrients may also be classifiedaccording to their ability to withstand thermal processing, the most commonly employedtreatment for destroying them. Heat labile factors include protease inhibitors, phytates,lectins, goitrogens and antivitamins, whereas heat stable factors are represented bysaponins, non-starch polysaccharides, antigenic proteins, estrogens and some phenolic

Ž .compounds van der Peol, 1989; Rumsey et al., 1993 . Oligosaccharides becomesomewhat more digestible after heat treatment, whereas the effect of thermal treatmenton substances such as tannins remains uncertain. An overview on the effects of some

Ž .antinutrients present in oilseeds and pulses on fish has been presented by Tacon 1997 .The present review takes into account additional information provided by experimentsconducted in recent years, particularly those in which the effects of purified antinutrientssuch as saponins, tannins and phorbol esters have been studied on different species offish. Our endeavour is to provide information on the nature, possible mode of action andeffects of antinutrients, and known methods to eliminate potent antinutrients in conven-tional and unconventional plant resources. It is hoped that the information collated in

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Table 2An overview of the results of selected studies where plant-derived ingredients were used to replace fishmeal in fish diets

Plant material Fish Inclusion Crude Percentage Biological effects ReferencesŽ .species rate % protein of fishmeal

Ž .level % replaced

Commercial soybean meal Grass carp 40 to 60 29 to 46 100 Growth performance lower than fish fed Dabrowski andfry fishmeal based diet and it declined with Kozack, 1979

increasing soybean content.Thermally and Carp 50 44 to 50 50 Fishmeal based control fish showed Abel et al., 1984hydrothermally treated best performance, among the soybeanfull-fat soybean meal meals the hydrothermally treated and

the intensely thermally treated mealssupported better growth performance

ŽCommercial hexane Tilapia O. 17 or 23 24 or 32 30 At 24% protein level inclusion of Shiau et al., 1987extracted soybean meal niloticus= soybean meal did no affect growth, but

.O. aureus at the 32% level groth was significantlyreduced if methionine was not suppliedadditionally

Ž . Ž . Ž .Raw R or boiled B or Nile tilapia 55 R or 56 30 83 Fish fed diet with boiled or defatted Wee and Shu,Ž . Ž .mixture of R and B RqB or to 58 RqB soybean diet grew better than control 1989

Ž . Ž .defatted D soybean meal or 58 B or while increasing levels of raw mealŽ .52 D progressively depressed growth

Commercial soybean meal Rainbow 13, 25 and 38 25 to 100 The 13% group showed performance as Dabrowski et al.,trout 50 control, depression of growth in 25% 1989

group; growth arrestment and highmortality in the 50% group; decreasein amino acid absorption in all soyfed groups

Ž . Ž .Full-fat soybean meal SBM Atlantic 30 SBM or 43 30 or 48 Abnormal intestinal morphology in van den InghŽ .or soy protein concentrate salmon 28 SPC SBM fed fish; no abnormality in the et al., 1991

Ž .SPC SPC group

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Solvent extracted soybean Fingerling 71, 53, 35.5, 34 25 to 100 Supression of feed intake, growth and Reigh and Ellis,meal red drum 18 high mortality at the highest level; 1992

lower feed consumption, higher feedefficiency and lower growth at theinclusion level of 53.2%; no significantdifference between the other two groupsand the control fed a diet based onfishmeal alone.

Ž .Solvent extracted 1 , special Rainbow 33 to 44 50 70 Growth of group 2 was similar to Rumsey et al.,processed solvent extracted trout control; other groups had lower growth; 1993Ž .2 , enzyme-treated solvent protein utilisation was comparable in all

Ž .extracted 3 , ethanol-treated groups except 3, which had significantlyŽ .solvent extracted 4 and lower values for protein utilisation and

Ž .alkali-treated 5 solvent carcass proteinextracted soybean meal

Ž .Full-fat FF , full-fat Rainbow 21 to 30 40 30 Growth rates of soybean fed groups Oliva-TelesŽ .extruded FFE , solvent trout except FFE better than control; FFE et al., 1994Ž .extracted FFSE , and group had significantly lower growth

solvent extracted andinfrared radiation-treatedŽ .FFR soybean mealSoybean meal Rainbow 32 41 40 No differences in weight gain and Sanz et al.,

trout growth rate, feed intake was higher 1994in fish fed thesoybean meal diet

Ž .Methionine supplemented Hybrid 15.7, 34 or 35 24 to 64 With small fish 5 g there were no Gallagher,soybean meal striped 44 difference in performance at the lowest and 1994

bass highest levels but growth was significantlylower at the 34% level; in bigger fishthere was no differences in growth

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Ž .Soy protein concentrate Rainbow 22 to 62 SPC 46 33 to 100 or No significant difference in growth Kaushik et al., 1995Ž . Ž .SPC or commercial soy trout or 24 to 42 SF 25 to 50 performances between the different groups,

Ž .flour SF an insignificant decline in growth whensoy flour was used, significant reductionin protein retention when soy flourreplaced fishmeal

Soybean meal Gilthead 10, 20 or 55 to 57 10 to 30 Growth performance similar to control; Robaina et al., 1995seabream 30 each trypsin activity retarded at 30% levels;

deposition of lipid in the liver at highsoybean level

Commercial soy concentrate Atlantic 48 43 to 45.7 75 Growth performance similar to control; Storebakken et al.,Ž .SC or phytase-treated SC salmon treatment of SC with phytase further 1998

improved protein utilisationA 1:4 mixture of defatted Rainbow 44 35 73 Fish on the test diet grew significantly Vielma et al., 2000soybean meal and soy trout better than control; supplementation byprotein concentrate phytase did not affect weight gain of fishRaw or heated lupin Rainbow 10, 20, 30 44 to 46 10 to 40 Performance similar to control up to a de la Higuera et al.,seed meal trout or 40 level of 30% inclusion; heating did 1988

not improve performanceLupin seed meal Gilthead 10, 20 or 53 to 56 10 to 30 Growth performance similar to control; Robaina et al., 1995

seabream 30 each trypsin activity retarded at 30% levelExtruded lupin flour Rainbow 30, 50 40 to 46 33 to 88 Growth performance similar to control Burel et al., 1998

trout or 70 up to the 50% level, lipid depositionin the liver; significantly lowerperformance at 70% level of inclusion

ŽColzapro coextruded Rainbow 8, 16, 24 34 to 42 5 to 48 Growth slightly better than control at Gomes et al., 1993.rapeseed and peas trout or 45 the first 3 levels and significantly lower

growth rate but higher protein efficiencyratio and feed to body mass gain ratioat 45% level.

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Undephytinized rapeseed Juvenile 12, 24, 36 42 to 45 33 to 100 Growth was depressed only when Teskeredzic et al.,protein concentrate rainbow fishmeal was totally replaced, feed 1995

trout consumption was highest at this levelDephytinized rapeseed Juvenile 13, 26 42 to 45 33 to 100 Growth was depressed only when Teskeredzic et al.,protein concentrate rainbow or 39 fishmeal was totally replaced, feed 1995

trout consumption was highest at this level,dephytinisation lowered protein quality.

Low and high glucosinolate Rainbow 30 or 50 40 18 and 33 Significant decrease in growth Burel et al., 2000bŽ .rapeseed RM meal trout each performance in all RM based diets

Ž . Ž .Raw R , commercial C , Rainbow 25 each 50 26 to 40 of Growth depression in the R and Herman, 1970Ž .low heated LH , trout casein was LH groups, LH being the worst

Ž .heated H or replaced hereŽ .glandless G

cottonseed mealCottonseed cake Tilapia 16.4 to 19.4 18 to 32 100 Growth depression at all levels Ofojekwu and

Ejike, 1984Ž .Decorticated DC or Nile tilapia 65 or 80 30 100 or 86 Significant depression in growth rate and El-Sayed, 1990

Ž .corticated CC other food utilisation parameters atcottonseed meal both levels; DC was better than CC

as far as growth was concerned;supplementation with L-lysine did notimprove the nutritiponal quality ofcottonseed meal

Sunflower meal Rainbow 42.5 41 40 No differences in weight gain and Sanz et al., 1994trout growth rate

Cassava or rice meal Carp 15 to 45 30 15 to 45 The 45% carbohydrate diets led to the Ufodike andgreatest weight increases, best food Matty, 1983utilisation and protein sparing

Autoclaved or roasted African 50 40 80 Performance not significantly different Fagbenro, 1999winged bean catfish from control

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Ž .Table 2 continued



Soaked, sundried or Nile tilapia 31 to 90 21 to 30 26 to 100 Significant decline in growth at all levels Wee and Wang,commercial leucaena of inclusion; the soaked leaf meal performed 1987leaf meal better than the other leaf meal dietsLeucaena leaf meal Nile tilapia 20 to 80 20 19 to 78 Up to 40% leucaena meal was tolerated Santiago et al.,

without negative effects on growth by males; 1988but growth was affected at all levelsfor females; fry production significantlyreduced beyond the 40% level

Soaked sun dried Nile tilapia 20 to 100 30 20 to 100 Cassava leaf meal significantly reduced Ng and Wee,or sun dried growth performance with growth reduction 1989cassava leaf meal increasing linearly with increasing leaf

meal inclusion; the treatment of the cassavaleaf meal did not improve fish performance.

Ž . Ž Ž .Chloroplastic CH and Tilapia O. 11 to 41 CH 40 15 to 55 Fish fed lowest level of 1 and up to 20% Olvera-Novoa et al.,Ž . . .cytoplastic CY alfalfa mossambicus or 9 to 32 CY of 2 showed significant improvement 1990

leaf protein in growth from control; others showednegative effects

Ž . Ž .Dehydrated alfalfa DA Tilapia 6 to 34 DA 40 5 to 30 Negative effects on growth at all Yousif et al.,Ž . Ž . Ž .or saltbush Artiplex O. aureus L. or 6 to 38 SB levels of inclusion and these 1994

Ž .SB leaves increased with increasing levelsŽ . Ž .Autoclaved A or Tilapia 13 to 47 A 39 10 to 35 Depressed feed intake and increased Martinez-Palacios

Ž .otherwise treated, OT, or 33 OT or 26 mortality with increased use of autoclaved et al., 1988Žchopped soaked and jack bean seed meal; the treatmentsboiled; chopped and resulted removal of non-themolabile toxinsautoclaved; chopped so that replacement of 26% of fishmealand soaked in ethanol– protein by treated meal did not reduce

Ž .sulphuric acid 1:1 growth performance.and autoclaved

jack bean meal

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Ž . Ž .Autoclaved A Tilapia 9 to 33 A 38 10 to 36 Depressed feed intake with increased Olvera-Novoa et al.,Ž . Ž .or raw R or or 24 R mortality with increased use of sesbania 1988

Ž .differently treated or 25 DT seed meal; fish fed the water-treatedŽ .with water DT meal performed better but still was not

Sesbania grandiflora as good as controlseed mealPea seed meal Juvenile European 20 or 40 48 9 to 18 No significant differences in performance Gouveia and

sea bass from control Davies, 1998Unheated or heat-treated Common carp 23 35 46 Growth lower than control, heat treatment Makkar andJ. curcas meal had no significant effect Becker, 1999

ŽCopra, groundnut, soy, Tilapia S. 17.4 to 69.4 30 25, 50 Leucaena meal at 25% level; copra, Jackson et al.,.sunflower, rapeseed, Mossambicus or 75 groundnut and soy at 50% levels produced 1982

cottonseed or poor growth, sunflower produced comparableleucaena meal and rapeseed and cottonseed better growth

than control at the 50% level; at 75%level all induced growth depression

Mustard oil cake, Common carp 70 to 88 30 100 All ingredients had significantly Hossain andlinseed meal or lower nutrient digestibility coefficients Jauncey, 1989asesame meal than control

Ž . Ž .Mustard oil cake MC , Common carp 30 or 61 MC , 40 25 to 75 Growth significantly lower than control Hossain andŽ .linseed meal LM or 30 or 59 or in all cases, among the various treatments Jauncey, 1989bŽ . Ž .sesame meal SM 23 LM , 25% MC and 25% LM gave best results

Ž .47 or 70 SM for growthŽ . Ž .Linseed meal LM or Common carp 30 LM or 40 25 Growth performance was significantly Hossain andŽ . Ž .sesame meal SM 27 SM better in the control fish, aqueous Jauncey, 1990

extraction and autoclaving improved thenutritional value of the meals to carp butnot to the level of fishmeal

Commercial soybean meal, Tilapia 21 to 34 28.5 32 Fish fed the sunflower meal and cottonseed Sintayehu et al.,cottonseed meal or meal showed slightly better performance than 1996sunflower seed meal control, but the soybean meal fed fish had

inferior growth performance and feed conversion

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Table 3Effects of purified antinutritional chemicals occuring in plant material on fish

Ž .Substance tested Fish species Inclusion rate % Biological effect References

Gossypol acetate Fingerling trout 0.025, 0.1 or 0.2 At the first two levels, fish accepted the feeds Roehm et al., 1967well but accumulated gossypol in thevascular tissues; complete suppressionof feed intake at 2000 ppm level

Gossypol acetate Rainbow trout 0.1 Growth depression and high mortality; Herman, 1970thickening of the glomerular basement membraneand fatty degeneration of the liver

Gossypol acetate Fingerling tilapia 0.1 or 0.2 No significant differences in growth or feed Robinson et al., 1984conversion

Sterculic acid Rainbow trout 0.01 or 0.02 Growth inhibition; alterations in liver fatty Roehm et al., 1970acid composition and abnormal glycogendeposition in liver

Phytic acid Rainbow trout 0.5 Depression in growth and food conversion efficiency Spinelli et al., 1983Sodium phytate Juvenile chinook 0.16, 0.65 or 2.58 The 25.8 grkg group showed depressed growth, Richardson et al., 1985

salmon food and protein conversion and thyroid function.Sodium phytate Common carp 5 or 10 mg per g Depression of growth, food utilization and protein Hossain and Jauncey, 1990

dogestibility; effect was exacerbated bysimultaneous increases in dietary calciumand magnesium levels. Intestinal epitheliumshowed abnormalities.

Phytoestrogens-formononetin, Yearling Siberian Administered at rates Except formononetin, all induced hepatic synthesis Pelissero et al., 1991adaidezin, genistein and equol sturgeon of 0.05 to 0.5 mg of vitellogenin

per g body weight

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Soybean protease inhibitors Rainbow trout 0.37, 0.74, 1.11 Increased trypsin inhibition with increased inclusion; Krogdahl et al., 1994or 1.48 this was partly compensated by increased enzyme

secretion and absorption by the intestine;the compensation was complete at lowerlevels of the inhibitor

Quillaja bark saponin Chinook salmon 0.15 or 0.30 Growth depression only at the higher level; Bureau et al., 1998abnormal intestinal morphology at both levels

Quillaja bark saponin Rainbow trout 0.15 or 0.30 Growth depression only at the higher level; Bureau et al., 1998abnormal intestinal morphology at both levels

Purified alcohol extract of Chinook salmon 1 or 0.3 Suppression of feed intake and growth in Bureau et al., 1998Ž .soybean meal PAES or both cases

Ž .soy protein isolate SPIactive principle beingsoy saponins

Ž . Ž .PAES 1 or SPI 2 , active Rainbow trout 1 or 0.3 Depression in growth and moderate intestinal Bureau et al., 1998principle being soy saponins damage when fed 1; No significant effect

Ž .when fed 2 only 2 weeks durationPhorbol esters Common carp 03.75 to 1000 ppm Feed rejection, faecal mucus production, significant Becker and Makkar, 1998

suppression in growth starting from 31 ppm onwardsŽ .Tannic acid hydrolysable tannin Common carp 2 Condensed tannin did not affect the performance Becker and Makkar, 1999

and quebracho tannin of fish but hydrolysable tannin had adverseŽ .condensed tannin effects after 28 days when it completely

supressed feeding


this review will provide an impetus to further research in this area, enabling betterutilisation of plant resources as sources of protein and carbohydrate for sustaining thecurrent growth rate of aquaculture production.

3. Protease inhibitors

Protease inhibitors are widespread antinutrient substances in many plant-derivedŽnutritional ingredients that could be used in fish feed, particularly the legumes Norton,

.1991 . Their potency depends on their origin and the target enzyme. In soybean, forexample, there are two groups of protease inhibitors: the Kunitz soybean trypsininhibitor that is relatively heat- and acid-sensitive, and the more stable Bowman–Birkprotease inhibitor. One molecule of the former blocks either one trypsin or onechymotrypsin molecule, while one molecule of the latter blocks either two trypsin orchymotrypsin molecule or one trypsin and one chymotrypsin molecule at the same timeŽ .Norton, 1991 .

Ž .Commercial soybean products mostly show trypsin inhibitors TI in the range of 2–6Ž .mgrg, averaging 4 mgrg Synder and Kwon, 1987 . TI have a wide distribution in the

plant kingdom and is present in most legume seeds and cereals. The common cultureŽfish species differ in their ability to tolerate dietary TI. Tilapia Jackson et al., 1982;

. ŽShiau et al., 1987, 1989; Wee and Shu, 1989 , carp Abel et al., 1984; Makkar and. ŽBecker, 1999 , rainbow trout Dabrowski et al., 1989; Rumsey et al., 1993; Krogdahl et

. Ž . Žal., 1994 , channel catfish Wilson and Poe, 1985 , salmon Higgs et al., 1982; Olli et. Ž .al., 1994a and seabream Robaina et al., 1995 are able to maintain growth rates

Ž .comparable to fish-meal based controls see Table 2 after the inclusion of varyinglevels of soybean meal, lupin seed meal, rapeseed meal and Jatropha seed meal, all ofwhich are known to contain TI. TI levels of 1.6 mgrg or higher in the diet retarded Niletilapia growth, but the fish tolerated and grew well at dietary levels of 0.6 mg TIrg dietŽ . Ž .Wee and Shu, 1989 . Makkar and Becker 1999 found that carp fed diets containingmeal of J. curcas seeds of non-toxic provenance, with 24.8 mg TIrg and heat-treatedmeal with 1.3 to 8.3 mg TIrg, showed no differences in growth performance implyingthat the fish were able to tolerate the high levels of TI. The reduction in growth of carps

Ž .and tilapia Oreochromis aureus=O. niloticus fed diets containing TI as compared toŽthe controls Dabrowski and Kozak, 1979; Viola et al., 1983; Abel et al., 1984; Shiau et

.al., 1987 may have been caused by amino acid imbalance in the soybean of experimen-tal diets. Rainbow trout has been found to be highly sensitive to the soybean proteaseinhibitors and a direct relation was observed between the amount of TI in the diet and

Žthe availability of protein and energy for trout Sandholm et al., 1976; Krogdahl et al.,. Ž .1994 . Dabrowski et al. 1989 suggested that the impairment of chymotrypsin secretion

in rainbow trout fed soybean-supplemented diets is caused by the suppression of thepancreatic feedback mechanism. Trypsin production in Atlantic salmon peaked when the

Ž .TI level in the diet was around 4.8 mgrg Olli et al., 1994a . Juvenile channel catfishshowed the best growth performance in this trial when the TI level was 2.2 mgrg of the

Ž .diet Wilson and Poe, 1985 . The negative effect of TI on growth in fingerling channel


catfish at higher levels in this experiment was less pronounced when dietary protein wasŽ .also high. Krogdahl et al. 1994 observed that rainbow trout were able to partly

compensate for TI action by increased enzyme secretion and enhanced absorption ofŽ .protein in the distal parts of the intestine. Rumsey 1991 found little effect on growth or

feed intake at TI levels below 5 mgrg, when feeding trout at levels of TI ranging from2.6 to 51.0 mgrg.

It seems that below the 5 mgrg level, most cultured fish are able to compensate forthe presence of TI by increasing trypsin production. At the usual levels at which thematerials containing protease inhibitors such as commercially available soybean mealare included in fish diets, other antinutritional factors or interactions between them may

Žbe more important. For other materials, moist heat treatment autoclaving for 15–30.min; Norton, 1991 is recommended as a means of reducing the amount of trypsin

inhibitors below the critical levels. This heating process should be carefully regulated tominimise the loss of nutritional quality of the feed material, such as the loss ofavailability of amino acids like lysine and the decrease in protein degradability due toexcessive heat denaturation.

4. Phytates

Ž .Phytate hexaphosphates of myo-inositol is common in plant seeds. They can chelatewith di- and trivalent mineral ions such as Ca2q, Mg2q, Zn2q, Cu3q and Fe3q resulting

Ž .in these ions becoming unavailable for consumers Duffus and Duffus, 1991 . Sincephytates cannot be broken down by non-ruminants, their occurrence in feed reduces the

Ž .availability of phosphorus to these animals Liener, 1989 . Phytates also form sparinglydigestible phytate–protein complexes, thus reducing the availability of dietary proteinŽ .Richardson et al., 1985 . Commonly used and potentially usable plant-derived fish feedingredients such as soybean meal, rapeseed meal, and sesame meal contain 10–15,50–75 and 24 grkg phytate, respectively. Growth in commonly cultured fish species,such as carp, tilapia, trout and salmon, is negatively affected by inclusion of phytate

Ž .containing ingredients in the diet see Table 2 . The involvement of phytates in inducingthe negative effects has been corroborated by feeding studies where synthetic phytate

Ž .was added to fish diets. Spinelli et al. 1983 observed decreased growth rates inrainbow trout fed a diet containing 5 grkg synthetic phytic acid. Formation of sparinglydigestible phytic acid–protein complexes was found to be the main reason for growth

Ž .depression in this study. High dietary phytic acid synthetic, 25.8 grkg dramaticallyŽ .depressed the rate of growth in Chinook salmon Richardson et al., 1985 . The lowered

feed and protein conversion ratios observed here were partly due to diminished zincŽbio-availability, as supplementation of the high phytate diets with zinc 0.35 to 0.4

.grkg partially improved the food and protein conversion when dietary calcium wasalso high. The authors also found abnormalities in thyroid, kidney and alimentary tractmorphology of the fish. Furthermore, the pyloric caecae were found to be abnormallyhypertrophied and showing cytoplasmic vacuolation. Phytate obviously had a toxiceffect on the epithelial layer of the pyloric caecae. In juvenile Chinook salmon fed high


phytate and low zinc diets, the same authors observed incidence of cataract, indicatingŽ .mineral-chelating action of phytic acid. Pure synthetic phytic acid 5 and 10 grkg feed

Ž .resulted in lower growth performance in common carp Hossain and Jauncey, 1993 .The effects of phytic acid were exacerbated by the simultaneous addition of calcium andmagnesium to the diet. Dietary phytate in this experiment caused hypertrophy andvacuolization of the cytoplasm of the intestinal epithelium. A significant depression ofzinc levels in the carcass was observed in juvenile rainbow trout fed a diet containing

Ž .undephytinised rapeseed protein concentrate Teskeredzic et al., 1995 . The rapeseedprotein concentrate used in this study contained 53 to 75 grkg phytic acid. Feeding

Ž .trials by Sugiura et al. 1999 demonstrated that apparent availability of phosphorus wassignificantly higher when mutant varieties of corn and barley, low in phytic acid content,were used in rainbow trout diets.

Supplementation of phytate-containing diets with the enzyme phytase neutralised thenegative effects of phytate. True P availability to rainbow trout from various plantfoodstuffs, which ranged between 9.7% and 48.4%, significantly increased to 46.2% to

Ž .75.6% on supplementation with phytase Riche and Brown, 1996 . Soy concentrate,previously incubated with phytase, when included in the diet, improved protein utilisa-tion parameters, apparent digestibility coefficients, and body levels of Ca, Mg and Zn

Ž .and retention of P in Atlantic salmon Storebakken et al., 1998; Vielma et al., 1998 .The lowered growth performance of fish fed high phytate diets can be attributed to

various factors, namely reduced bio-availability of minerals, impaired protein digestibil-ity caused by formation of phytic acid–protein complexes, and depressed absorption ofnutrients due to damage to the pyloric cecal region of the intestine. Phytates, particularlyin cereals, are concentrated in the outer endosperm. Milling to remove the outer layer ofseeds therefore reduces the phytate content of the seed considerably. Fermentation hasalso been shown to reduce the phytic acid content of grains because of the action of

Žphytases produced by yeast or lactic acid bacteria Duffus and Duffus, 1991; Mukhopad-. Ž .hyay and Ray, 1999a . Heat treatment autoclaving was also found to reduce phytic

Žacid in linseed and sesame meals by up to 72% and 74%, respectively Hossain and.Jauncey, 1990 . Salmonids seem to be able to tolerate dietary levels of phytate in the

range of 5–6 grkg, while carp appears to be sensitive to these levels. It seems to beadvisable to maintain the level of phytates below 5 grkg in fish feeds. The addition ofminerals such as Zn has been shown to be only partially capable of counteracting thenegative effects of dietary phytate.

5. Glucosinolates

Glucosinolates are thioglucosides commonly found in plants belonging to the familyCruciferae. They are always accompanied by thioglucosidase enzymes in plants but thetwo are kept separated in different cell compartments. When the contents of these twocomponents come together by cellular damage, breakdown products like isothiocyanatesand nitriles, capable of causing potentially harmful effects to animals, are releasedŽ .Duncan, 1991 . The presence of intact glucosinolates has been correlated with the


occurrence of liver haemorrhage in laying hens while nitriles have been shown to causetissue damage in liver and kidney and increased organ weights and isothiocyanates

Ž .affect thyroid function see Duncan, 1991 . Glucosinolates are the main antinutrientsŽ . Ž .present in rapeseed Brassica spp. meal and mustard oil cake see Duncan, 1991

which are potentially attractive protein sources in fish feeds. Plant geneticists havedeveloped improved varieties of both of the common species, Brassica napus and B.

Ž .campestris, with low glucosinolate content less than 3 mgrg in the seed; such varietiesŽ . Ž .are known as canola. Higgs et al. 1982 found that dietary rapeseed meal 16%

affected the thyroid structure in juvenile Chinook salmon whereas dietary canola mealdid not. Growth performance was comparable to control in both cases. The thyroids ofthe affected fish were characterised by the presence of clear hyperplasia and follicularhypertrophy, indicating higher than normal thyroid activity. The thyroid follicles in thesefish had little colloid, numerous mitotic figures and significantly taller epithelial cellswhen compared to control fish; symptoms of an obvious effort by the fish to maintainblood thyroid hormone levels through increased thyroid gland activity. Similar thyroid

Ž .abnormalities were found in carp Cyprinus carpio fed a diet containing 3.3 g purifiedŽ . Ž .glucosinolaterkg Hossain and Jauncey, 1989b and tilapia O. mossambicus fed a diet

Ž .containing 2.5 g glucosinolaterkg, Davies et al., 1990 . Increased thyroid activity wasobserved in juvenile rainbow trout fed undephytinised rapeseed protein concentrate

Ž . Ž .containing glucosinolates Teskeredzic et al., 1995 . Trout and turbot Psetta maximawere found to compensate for decreased thyroid functioning caused by dietary glucosi-nolates through higher activity of the deiodinases, which convert thyroxine into the

Ž .active component, triiodothyronine Burel et al., 1998, 2000a,c . It is interesting thatŽ .turbot fed a high glucosinolate 11.6 mmolrg , rapeseed meal-based diet had normal

thyroxine and triiodotyronine contents in plasma even though higher deiodinase activityŽ .was observed Burel et al., 2000c . This might have been due to lack of breakdown of

glucosinolates into toxic by-products in untreated rapeseed meal. The ingestion of veryŽ .low amounts of glucosinolates 1.4 mmolrg diet led to a decrease of both growth rate

and feed efficiency in rainbow trout, but this effect was not exacerbated when dietaryglucosinolate was increased to 11.6 mmolrg. Stronger depression of growth wasobserved when the level of glucosinolates went up to 19.3 mmolrg in the diet. Thyroid

Žactivity in trout was already affected at the lowest level of glucosinolate Burel et al.,.2000b .

ŽHeat treatment is effective in reducing the glucosinolate content of feed materials in.rapeseed meal from 40 to 26 mmolrg after wet pressure-cooking; Burel et al., 2000a .

Extracting with water was found to be a cost-effective method of removing glucosino-Ž .lates from full-fat and fat-free Moringa oleifera kernels Makkar and Becker, 1997 .

‘Colzapro’, a co-extruded product of rapeseed and peas, did not affect thyroid morphol-ogy in rainbow trout, indicating the effectiveness of this treatment in minimising the

Ž .negative effects of glucosinolates Gomes et al., 1993 .Over the long term, thyroid dysfunction induced by continuous consumption of

glucosinolate containing feed is certain to affect metabolism and growth in fish. Settinga threshold level of glucosinolates in fish diets is, however, difficult because ofinadequacies of data available on the quantities of its toxic derivatives, such asisothiocyanates and nitriles, which are mainly responsible for the negative effects.


6. Saponins

Saponins are steroid or triterpenoid glycosides found in many of the potential,Žalternate plant-derived feed ingredients for fish, like legumes ranging between 18 and

41 mgrkg in various legume seeds; 67 mgrkg in defatted roasted soybean flour;.Fenwick et al., 1991 . When added to water, they are highly toxic to fish because of the

damage caused to the respiratory epithelium of the gills by the detergent action of theŽ .saponins. Tea Camellia sinensis seed cake, containing about 7–8% saponins, when

Žadded to water at a dose of 100 ppm resulted in the death of tilapia within 5 to 6 h De.et al., 1987 . They are also considered to be the active components of many traditionally

Ž .used fish poisons, like mahua oil cake. Saponins in lupin seed meal 1.1% and alfalfaŽMedicago satiÕa, -0.30% in low saponin varieties to )1.5% in high saponin

.varieties could have been important contributing factors for the lower growth perfor-Ž . Žmance of rainbow trout de la Higuera et al., 1988 , and tilapia Olvera-Novoa et al.,.1990; Yousif et al., 1994 fed diets containing high levels of these ingredients. Krogdahl

Ž .et al. 1995 however did not find any negative effects of saponins included in the dietof Atlantic salmon at levels similar to that expected to be found in a soybean mealŽ .30–40% based diet. In the same study, an alcohol extract of soybean meal causedgrowth retardation, altered intestinal morphology and depressed mucosal enzyme activ-

Ž .ity in the distal intestine. Bureau et al. 1998 observed that rainbow trout was moreŽtolerant to inclusion of purified alcohol extracts of soybean meal extracted to isolate

.soy saponins in the diet than Chinook salmon. Chinook salmon fed this diet had anintestinal morphology resembling that of a fasting fish, probably caused by the deterrentaction of the saponin on feeding. Extensive damage to the intestinal mucosa wasobserved in both fish species at a dietary level of 1.5 grkg Quillaja bark saponin. Thecondition of the intestine of these fish was similar to that of fish fed the raw soybeanmeal diet, indicating the role of the saponins in causing the damage. The negative effectsof saponins could be caused by the well-known effects of these surface-active compo-nents on biological membranes. Ingested soy saponins do not seem to be absorbed either

Žas saponins or even as constituent sapogenins in chickens, rats and mice Gestener et al.,.1968 . In these animals, hind gut microorganisms break down the saponins into

sapogenins and sugars. The fate of saponins in the alimentary tract of fish has not beenstudied. Some saponins readily increase the permeability of small intestinal mucosalcells and inhibit active nutrient transport, although different saponins might differ ineffectiveness. For example, soy saponin was less effective compared to Gypsophyllasaponins in reducing transmural potential difference, the main driving force for activehexose transport across the brush border membrane in the small intestine of ratsŽ .Johnson et al., 1986 . Other properties of saponins may also play a role in its growthdepressing action. Endogenous saponins have been shown to reduce the protein di-

Ž .gestibility of soybean by chymotrypsin Shimoyamada et al., 1998 , probably by theŽ .formation of sparingly digestible saponin–protein complexes Potter et al., 1993 .

Complex formation between saponins and other antinutrients could, however, lead toŽ .the inactivation of the toxic effects of both the substances Makkar et al., 1995a .

Simultaneous consumption of saponin and tannin resulted in the loss of their individual


Ž .toxicity to rats Freeland et al., 1985 . This is considered to be due to chemical reactionsbetween them, leading to the formation of tannin–saponin complexes, inactivating thebiological activity of both tannins and saponins. Soy saponins have been found toactually stimulate feeding in oriental clouded yellow larva, Coliaserate poliographusŽ .Matsuda et al., 1998 . Saponins might also increase the digestibility of carbohydrate-richfoods because of their detergent-like activity, which reduces viscosity and thus preventsthe normal obstructing action of such foods against movement of digesta in the intestine.

Because of the high solubility of most saponins in water, aqueous extraction wouldremove most saponins from feed ingredients and this could be recommended forremoving saponins provided it does not otherwise affect the nutritional quality of thematerial. Levels below 1 grkg of diet are unlikely to affect growth performance ofcommon culture fish. More investigations are required on the effects and tolerance limitsof this widely present factor in plants to fish.

7. Tannins

Tannins are secondary compounds of various chemical structures widely occurring inplant kingdom and are generally divided into hydrolysable and condensed tannins. Theirantinutritional effects include interference with the digestive processes either by binding

Ž .the enzymes or by binding to feed components like proteins or minerals Liener, 1989 .Tannins also reduce the absorption of vitamin B . Some materials tested as alternative12

Ž .nutrient sources for fish do contain this substance Table 1 . Common carp has beenŽshown to be able to tolerate a 2% addition of quebracho tannin powder a condensed

. Žtannin without any effect on growth while a similar level of hydrolysable tannin tannic. Ž .acid induced feed rejection after 28 days of feeding Becker and Makkar, 1999 .

Contrary to condensed tannins, the hydrolysable tannins are easily degraded in biologi-cal systems, forming smaller compounds that can enter the blood stream and over a

Ž .period of time cause toxicity to the organs e.g., liver and kidney . How far the purifiedcommercially available tannins simulate those naturally occurring in plant productsneeds to be investigated. Condensed tannins present in copra at a level of approximately

Ž .2.4% could have been the cause of growth depression in tilapia and rohu Labeo rohitaŽfingerlings even at such low levels of inclusion as 25% or 20% Jackson et al., 1982;

.Mukhopadhyay and Ray, 1999b . Other tannin containing feeds, like rapeseed and peaseed meal, have reportedly been tolerated by different fish species at moderate to high

Ž . Ž .levels of inclusion see Table 2 . Broad bean Vicia faba meal, with a high condensedtannin content, had lower protein digestibility than soybean in in vitro experimentsŽ .Grabner and Hofer, 1985 . The differences in digestibility were more pronounced afterdigestion under the simulated conditions of the carp gut than those of the rainbow troutgut. These differences probably indicate the differences in tolerances of different fishspecies and differences in the structure of the tannins or their interactions with othercomponents in the diet. Tannins are also known to interact with other antinutrients. Forexample, interaction between tannins and lectins removed the inhibitory action of

Ž .tannins on amylase Fish and Thompson, 1991 , and interactions between tannins and


Žcyanogenic glycosides reduced the deleterious effects of the latter Goldstein and.Spencer, 1985 .

Recommended methods for the removal of condensed tannins include de-hulling theseeds to remove the tannin rich outer layer, autoclaving or treatment with alkaliŽ . Ž .Griffiths, 1991 . Mukhopadhyay and Ray 1999a observed a reduction in the tannincontent of sesame seed meal from 20 to 10 grkg after fermentation with lactic acidbacteria. The treatment of tannin-containing feeds with oxidising agents and supplemen-tation with a tannin-complexing agent, polyethylene glycol, could mitigate their negative

Ž .effects on animals Makkar et al., 1995b; Makkar and Becker, 1996 . Limited literatureon the effects of purified tannins on fish suggest that fish are sensitive to tannins andcaution should be exercised in incorporating seeds and agroindustrial byproducts con-taining high amounts of tannins.

8. Lectins

Plant lectins or phytohaemagglutinins are found in many legume seeds and are able tobind reversibly to carbohydrate moieties of complex glyco-conjugates present onmembranes. Even though they are proteins, they are at least partially resistant toproteolytic degradation in the intestine. Their common biological effects include disrup-

Žtion of the small intestinal metabolism and morphological damage to the villi Grant,.1991 .

Ž .Soybean lectins 60 haemagglutinating unitsrmg protein , otherwise known asŽ .soybean agglutinin SBA , have been shown to be able to bind extensively to the brush

border surfaces, particularly in the distal part of the small intestine of Atlantic salmonand may contribute to the toxic effect of full-fat soybeans and soybean products in diets

Ž . Ž .for salmonids Hendriks et al., 1990 . van der Ingh et al. 1991 observed distinct effectsŽ .of full-fat soybean meal FFSB on the mucosa of the distal intestine in the Atlantic

Ž .salmon as compared to a standard herring meal diet HM fed control. In the FFSBgroup, the epithelium had an increased density of goblet cells and a marked decrease orabsence of absorptive vacuoles; the microvilli of the enterocytes were shortened withincreased microvillar vesicle formation. These effects are similar to those observed after

Ž .SBA was given to other animals see Grant, 1991 . The increase in goblet cell densitymay have been the result of hypertrophic mucus production in the intestine whensubjected to irritation by lectins. Carp growth was similar when fed diets containing highŽ . Ž .51 haemagglutination units or low lectin -1.2 haemagglutination units activityŽ .Makkar and Becker, 1999 . Other effects of lectins, such as muscle wastage and

Ž .depletion of lipids in the adipose tissue and liver enlargement Grant, 1991 , have notbeen observed in fish so far.

Ž .Lectins can be removed by aqueous heat treatment 1008C for 10 min or autoclavingŽ . Ž .Grant, 1991 . Aregheore et al. 1998 reduced the lectin content in Jatropha seed mealfrom 102 to 1.17 haemagglutination units by moist heating at 1008C for 10 min.Irritation caused by lectins to the intestinal membrane resulting in over secretion of


mucus may impair the enzymatic and absorptive capacity of the intestinal wall. Theirdeleterious effect may be more potent when present along with other antinutrients.

9. Oligosaccharides and non-starch polysaccharides

Ž .Oligosaccharides of the raffinose family and non-starch polysaccharides NSPs areŽ .important constituents of a wide variety of grain legumes and cereals Saini, 1989 . In

fish, their negative effects may be either due to binding to bile acids or obstructingaction against action of digestive enzymes and movement of substrates in the intestineŽ . Ž .Storebakken et al., 1998 . Arnesen et al. 1989 found a decrease in nutrient utilisationcaused by soybean carbohydrates in Atlantic salmon. They, however, found little effectof soy oligosaccharides on protein utilisation in rainbow trout. The main oligosaccha-

Ž . Ž .rides of defatted soybean meal are sucrose 6–7% , raffinose 1–2% and stachyoseŽ .5–6% , accounting for a total soluble carbohydrate content of 12–15%. Decreased feed

Ž . Žintake in hybrid striped bass Gallagher, 1994 and rainbow trout de la Higuera et al.,. Ž .1988 , and lower feed digestibility in trout Sanz et al., 1994 fed diets containing

soybean, lupin, and sunflower meal, were attributed to the presence of high proportionsof NSPs such as pectins, galactans, cellulose and lignin. High fecal water content,probably induced by the presence of osmotically active NSPs, was observed when soy

Žproducts were included in diets of salmonid fish Olli and Krogdahl, 1994; Olli et al.,.1994b; Refstie et al., 1997 . NSPs such as arabinan, arabinogalactan and acid poly-

saccharides, which form about 14% to 18% of the total carbohydrate content of defattedsoybean meal might also bind minerals in the intestine and reduce the digestibility of fatŽ .Storebakken et al., 1998 . The addition of soybean oligosacccharides to a fishmeal

Žbased diet did not cause morphological changes in the intestinal tract van der Ingh et. Ž .al., 1991 or affect nutrient digestibility or growth Krogdahl et al., 1995 in Atlantic

salmon. It must be added that trout have been shown to be able to satisfactorily utiliseŽ .diets containing sunflower meal Tacon et al., 1984; Sanz et al., 1994 and soybean meal

Ž .Rumsey et al., 1993; Sanz et al., 1994; Kaushik et al., 1995 . Feed containing highŽlevels of carbohydrates was also shown to be well tolerated by tilapia Jackson et al.,

. Ž .1982 and carp Ufodike and Matty, 1983 .Ž .Erfanullah and Jafri 1998 observed that steam cooking improved the digestibility

coefficients of yellow corn and potato in these fish species. However, heat-treated lupinseed meal, abundant in galactans that were made more digestible by the treatment,

Žreduced the feed intake of rainbow trout, probably by inducing hyperglycaemia de la.Higuera et al., 1988 . Extrusion at high temperature has the potential to improve the

Ž .carbohydrate digestibility in legume seeds Bangoula et al., 1993; Burel et al., 2000abecause of a higher break up of cell walls andror a partial degradation of a-galacto-sides. There are discrepancies among different studies as to the level of tolerance andutilisation of feed carbohydrates by fish species such as trout. This could be because ofthe different origins of the carbohydrates and the different treatment methods employedin different studies. It could be concluded that NSPs, particularly the soluble NSPs, are

Ž .more detrimental to growth of fish than the oligosaccharides Refstie et al., 1999 . Theytrap water and form gum-like masses in the intestine, increase the viscosity of intestinal


contents, and obstruct the digestive enzyme activity, thus exerting potent antinutritionalactivity when present in fish diets.

10. Phytoestrogens

Non-steroidal estrogenic substances are widely distributed among potential plant-de-Ž .rived feeds Farnsworth et al., 1975a,b . Estrogenic activity has been reported to be

present in soybeans, cottonseed, linseed, safflower etc. Chemically, plant estrogens aremostly isoflavones that occur in the form of glycosides. The sugar moiety is attached toone or more of the hydroxyl groups located at various positions of the isoflavone

Ž .nucleus Liener, 1980 . For example, soybean contains estrogenic isoflavones andderivatives such as coumestrol, formononetin, diadzein, biochanin A, genistein and

Ž .equol about 0.25% in defatted meal; Pelissero et al., 1991a . Studies with sturgeonindicated that dietary phytoestrogens were estrogenic in fish and induced vitellogenesisŽ .Pelissero et al., 1991b . Coumsterol, and isoflavonic compounds such as genistein anddaidzein had estrogenic properties when administered intraperitoneally in the pure formto the same fish species. These compounds act either by binding direct to oestrogenreceptors or getting converted into compounds that have estrogenic effects, like equol.Dietary estrogenic effects of plant-derived materials can have wide ranging conse-quences, as oestrogen is known to have wide ranging effects on various physiologicalprocesses in animals. Siberian sturgeon fed a commercial trout diet containing estradiolalso had hypertrophic and hyperlipidemic livers, features which disappeared when fish

Ž . Ž .were fed a diet free of any steroids Pelissero et al., 1991a . Kaushik et al. 1995detected daidzein and genistein in the bile of trout fed soy flour rich diets and attributedthe slightly poor growth rates to these substances. Plasma vitellogenin levels of thetreated group in this study tended to be higher compared to the levels in the control

Ž .group. Mambrini et al. 1999 postulates that the presence of isoflavones could only beconsidered as one of the reasons for reduced growth of trout fed a soy proteinconcentrate diet.

Additional fish studies are required to evaluate the significance of observed effects ofŽ .phytoestrogens Mambrini et al., 1999 . The presence of these substances, however,

Ž .need to be kept in mind when fish diets are formulated Pelissero and Sumpter, 1992

11. Alkaloids

Alkaloids are secondary metabolites widely found in plants. With few exceptions,true alkaloids are basic, contain nitrogen in the heterocyclic ring, and are derived from

Ž .amino acid precursors Petterson et al., 1991 . Alkaloid-containing grain legumes, suchŽ .as lupins Lupinus albinus , are otherwise ideally suited as feed ingredients in aquacul-

Ž .ture because of their high digestible protein content 30–50% . The presence ofquinolizadine alkaloids, which interfere with nerve functioning, makes them unsuitablefor human consumption.


The organoleptic properties of alkaloid-containing diets may lower the feed intake inŽ . Ž .rainbow trout de la Higuera et al., 1988 . According to Bangoula et al. 1993 , rainbow

trout fed a diet containing crude lupin seed meal showed lower feed efficiency. In morerecent studies, rainbow trout and turbot were found to be tolerant to high inclusion levelsof lupin seed meal in their feed, probably because extruded meal of a variety with low

Ž . Ž .alkaloid content -20 mgrkg was used Burel et al., 1998, 2000a .Breeding for low alkaloid varieties has been successful in the case of lupin and is the

obvious solution to reduce the alkaloid content in alternative nutritional sources.Ž .Aqueous extraction also removes alkaloids from some materials. Bangoula et al. 1993

observed that extrusion at 1458C led to higher digestive utilisation, and higher growthperformance in trout than the same lupin seed meal extruded at 1208C. Few studies areavailable on the effects of alkaloid-containing feeds in other species of fish. Themechanism of action of alkaloids in fish and their metabolic fate in the fish body are notproperly understood. It is reasonable to assume that fish would be capable of toleratinglupin meal with a low alkaloid content at moderate levels of dietary inclusion.

12. Antigenic compounds

A few protein components of some legume seeds and cereals elicit antigenic effectsin animals; these compounds are capable of inducing intestinal mucosal lesions,abnormalities in the villi, specific and non-specific immune responses and abnormal

Ž .movement of digesta through the gut D’Mello, 1991a . Soybean protein containsŽ . Ž .compounds such as glycinin G and beta conglycinin bC , which act as allergens to

Ž .several animals. Rumsey et al. 1993 reported that high levels of immunologicallyactive G and bC in different soy preparations seemed to negatively affect growthperformance in rainbow trout. They assumed that the comparatively under-investigatedeffects of allergens may provide answers to why conventionally processed soybean, inwhich the protease inhibitors and lectins have been largely inactivated, results in poor

Ž .growth of salmonid fish. Haemagglutination inhibition assays HIA by the same authorsshowed that normal processing measures like toasting and de-fatting did not signifi-

Ž .cantly reduce antigenicity levels in soybean meal. Krogdahl et al. 2000 observedŽ .enteritis-like changes in the distal intestine of Atlantic salmon Salmo salar L. fed diets

containing solvent-extracted soybean meal or an alcohol extract of soybean meal.The antigenic compounds present in feed may trigger a variety of non-specific and

specific immune responses in the fish body and this might lead to a reduction in growth.Their presence in common plant-derived feed ingredients, however, remains a matter of

Ž .controversy. Kaushik et al. 1995 failed to detect any antigenic proteins in the soyprotein concentrate, nor was there any agglutinating activity against soybean protein inthe sera of rainbow trout fed with diets containing soy protein concentrate. It may besafe to conclude that at those levels at which they are likely to be present in practicalfish feeds, the allergen content does not cause any serious growth reduction in culturedfish species.


13. Gossypols

Gossypols are polyphenols, contained in the pigment glands of plants of the genusGossypium and in certain other members of the order Malvales. Feeding diets containinggossypols cause negative effects such as growth depression and intestinal and other

Ž .internal organ abnormalities Berardi and Goldblatt, 1980 . The formation of indigestiblegossypol–protein complexes may produce deficiencies of some amino acids, such as

Ž .methionine, which are essential for the normal fat metabolism Herman, 1970 . Com-mercially available cottonseed meal has gossypol levels ranging from -0.01% in‘glandless’ cottonseed meals to an average of about 1.3% in ‘glanded’ varieties, levels

Žthat may cause toxicity in fish. Raw cottonseed meal supplementation in the diet the.diet contained 0.029% free gossypol caused a reduction in growth and tissue pathology

Ž .in rainbow trout Herman, 1970 . Necrotic changes in the liver cells, thickening of theglomerular basement membrane, and accumulation of ceroid pigment granules in theliver in this study were all attributed to the presence of gossypol. A dietary level of 0.1%gossypol resulted in quick development of severe focal fatty degeneration in the liverand extensive kidney damage in the same study. At the end of this study, an extremely

Ž .unbalanced sex ratio 1 male:4 female in fish fed a diet containing a moderate levelŽ .0.0531% of gossypol was also observed. Gossypol is known to cause problems to thereproductive system in mammals by affecting the reproductive tissues directly or

Ž .pituitary and gonadal hormone secretion Randel et al., 1992 . Cottonseed meal at levelsabove 8% in the diet resulted in alterations in spermatic activity, increase in abnormal

Ž .spermatozoids, and abnormalities in the histology of the testes Salaro et al., 2000 .Ž .Yellow perch Perca flaÕescens sperm cells were immobilised in vitro by a level of

Ž .200 mM gossypol Ciereszko and Dabrowski, 2000 .The growth rate of fingerling carp was depressed to half of that of the control on a

Ž .1% dietary inclusion of gossypol–acetate Roehm et al., 1967 . A 2% gossypol levelresulted in feed rejection in the same study. Significant amounts of gossypol becamebound to liver, kidney and spleen tissue. This bound gossypol remained in the liverŽ .which is the main organ responsible for metabolising these compounds even after fishwere fed a gossypol-free control diet for 10 weeks.

Ž .Tilapia O. aureus growth was affected at a dietary free gossypol content of 0.012%Ž .Robinson et al., 1984 . Tilapia and catfish fed diets containing full-fat cottonseed mealhad higher levels of palmitic acid and linoleic acid, and lower levels of oleic acid intheir carcass. O. niloticus niloticus exhibited a poor growth response when fed cotton-seed meal based diets, even when the crude protein level of these diets was higher than

Ž .that of the fishmeal based control diet Ofojekwu and Ejike, 1984 . Lysine unavailabilitycaused by gossypol cannot be the only reason for the negative effect of cottonseed meal,

Žas lysine supplementation failed to improve the growth performance of tilapia O.. Ž .niloticus fed diets containing cottonseed meal El-Sayed, 1990 . It is, however, notable

that the use of cottonseed meal was calculated to be more profitable even with lowergrowth rates in this study. It is not evident from these studies whether gossypol toxicitywas the only reason for reduced fish growth even though toxicity of this compound to

Ž .fish seems to be high even at low levels ;0.05% . High levels of inclusion ofcottonseed meal resulted in a growth performance comparable to a control fed a


Ž . Žfishmeal diet in channel catfish Reigh, 1999 and tilapia Jackson et al., 1982;.Sintayehu et al., 1996; Mbahinzireki et al., 2000 . Different treatments undergone by the

cottonseed meal used in the different trials may have been the reason for the erraticgrowth performance of the fish in different studies. Pre-pressed and solvent extracted‘glandless’ cottonseed meal should be able to support good growth even at high

Ž .inclusion levels in the range of 50% in most cultured fish species, if supplementedwith the deficient amino acids such as cystine, lysine and methionine.

14. Miscellaneous antinutrient substances

Cyanogens are compounds found in high concentrations in a number of pulses, rootcrops, such as cassava, and some oil seeds, such as linseed, which have been tried asfish feed ingredients. Cyanogens, when hydrolysed, produce toxic products such ashydrogen cyanide and probably other carbonyl compounds that suppress natural respira-

Ž .tion and cause cardiac arrest Davies, 1991 . The enzyme required for this conversion isusually found in extracellular spaces in the plant tissues. Thiocyanate, a detoxicationproduct of cyanide, acts as an antithyroid agent. Fish fed cyanogen-containing feedmaterials, such as linseed and cassava, have generally shown reduced growth when

Žcompared to the respective controls Hossain and Jauncey, 1989b; Ufodike and Matty,.1983 . However, dietary cyanide did not depress growth in Nile tilapia, as fish fed diets

Ž .containing soaked sun-dried cassava leaf meal 9.9 ppm total cyanide and sun-driedŽ . Žcassava meal 71.1 ppm showed similar growth at similar levels of inclusion Ng and

.Wee, 1989 . More studies are required to determine the level of tolerance of differentspecies of cultured fish to this substance and whether there are any long-term effects atlow ingestion levels.

Mimosine is an unusual amino acid present in Leucaena leucocephala and comprisesŽ .about 3–5% of the dry weight of total protein Liener, 1980 . It has extensive

deleterious properties in animals including disruption of reproductive processes andŽ .teratogenic effects D’Mello, 1991b . Dietary Leuceana leaf meal does not seem to be

well tolerated by fish. Studies in tilapia indicate that Leucaena leaf proteins are poorlydigested and hence it is arguable whether enough mimosine enters the body to cause itsphysiological effects. Difference in the growth response of male and female tilapia hasbeen observed when fed a diet containing leucaena meal; males seemed to tolerate it

Ž .better than females Santiago et al., 1988 . Production of fry was significantly reducedbeyond the 40% inclusion level in this study. It would be interesting to see if theseeffects could be reproduced using pure mimosine, and if so, what the mechanism of itsaction is.

Cyclopropenoid fatty acids, like sterculic and malvalic acid present in cottonseed oiland meal, are known to cause abnormalities in the reproductive processes and alterationsin the lipid metabolism in mammals. These substances, together with other toxins, suchas aflatoxins, are suspected to be carcinogenic to fish. In rainbow trout, they have beenshown to interfere with the long chain fatty acid metabolism and with stearic andpalmitic dehydrogenation. Negative effects in rainbow trout recorded at 100 ppm methylsterculate in the diet did not increase when its concentration was increased to 200 ppm


Ž .Roehm et al., 1970 . Negative effects on growth were strongest initially, later the fishseemed to adapt, but the final weight after 87 days was still lower than the control.Tilapia fed a diet containing full-fat cottonseed meal tended to show lower growthcompared to those fed the diet containing the defatted flour, which indicated adverse

Ž .effects of cyclopropenoid fatty acid Robinson et al., 1984 .CanaÕanine is a thermoresistant-free amino acid present in many legume species

Ž . Ž .D’Mello, 1991b . It is an antagonist of arginine. Jack bean CanaÕalia ensiformis mealtreated for removal of canavanine was as efficient a feed ingredient for tilapia as

Ž .fishmeal Martinez-Palacios et al., 1988 . Tilapia fed diets containing toxic jack seedmeal exhibited depressed appetite, lethargic movements and subsequent high mortality.There is little information on effects in other fish.

AntiÕitamin factors. Many of the alternative protein sources tested in fish likesoybeans, alfalfa and oilseed meals are known to contain a variety of antivitamin factorsŽ .Liener, 1980 that might affect their efficiency as nutrient sources. Antivitamins areheat labile so these substances should not be of much physiological significance in fish,provided the plant-derived resources being incorporated in diets are properly heat-treated.Most commercially available seed meals are heat-treated to inactivate trypsin inhibitorsand lectins, and hence these are also free from antivitamins.

Ž .Phorbol esters, toxic substances found in J. curcas physic nut meal, act as aco-carcinogen and have a wide range of adverse biochemical and cellular effects inanimals. Common carp was found to be extremely sensitive to these compounds. At alevel of 31 ppm in feed, they induced depression in feed intake, growth and production

Ž .of faecal mucus Becker and Makkar, 1998 . Use of non-toxic varieties of J. curcas inwhich phorbol esters are absent is an obvious solution.

Oxycarotenoids such as zeaxanthin and lutein present in corn gluten meal reduce themarket acceptance of white fleshed fish in certain regions by giving the fillet a yellow

Ž . Ž .pigmentation Skonberg et al., 1998 . Park et al. 1997 showed that bleaching with soyflour was a practical method of removing carotenoids from corn gluten meal.

15. Conclusions

The fish feeding studies reviewed in our paper used plant-derived materials thatcontain more than one antinutritional substance, and because of this it is difficult topinpoint any one factor as ‘the culprit’ for the adverse effects that these feed ingredientsproduced when fed to fish. Understandably, there is little agreement among the results ofdifferent studies as to the specific effects of individual antinutrients. Most of theantinutrients, at levels present as a result of incorporating alternate protein sources infish feeds, do not lead to mortality, but could produce adverse effects and decreaseproductivity. However, it is difficult to make firm conclusions regarding specific plantsecondary metabolites causing deleterious effects and their threshold levels in fish diets.Tentatively, protease inhibitors, phytates, and antigenic compounds, at levels likely to bepresent in fish diets containing commercially available plant-derived protein sources, areunlikely to affect fish growth performance. In contrast, glucosinolates, saponins, tannins,phorbol esters, soluble non-starch polysaccharides and gossypol seem to be more


important in practical aquaculture nutrition. Information available on substances likelectins, phytoestrogens and alkaloids is too scarce to arrive at any conclusion. Moreinsights into the nutritional, physiological and ecological effects of these substances onfish need to be accumulated.

The common processing techniques, like dry and especially wet heating, extractingwith water, and addition of feed supplements have been widely and successfully used toreduce the concentration of antinutrients in plant feeds. Caution needs to be exercisedwhen resorting to treatment methods because they sometimes have unintended adverseeffects on the nutritional quality of the feed material, e.g., heat treatment reportedlyalters the chemical nature and decreases the nutritional quality of proteins and carbo-

Ž .hydrates van der Poel, 1989 . The different tolerance limits of individual fish species tothe presence of antinutrients also need to be considered before deciding on treatmentprocedures to reduce their levels. Tilapia species, for example, seem to be more tolerantthan carp to the increased presence of antinutrients in general. Feeding experimentsusing purified individual antinutrients are needed to determine the threshold limits thatwill not affect the productivity of common culture fish.

Another important factor to be considered is the interactions between variousantinutritional factors in a particular substance as these interactions in some instanceslead to a decrease in the toxic effect of the interacting antinutrients. For example,

Ž . Ž .saponin–tannin Freeland et al., 1985 , tannin–lectin Fish and Thompson, 1991 andŽ .tannin–cyanogen Goldstein and Spencer, 1985 interactions have all been shown to

result in a reduction in the individual toxicity of the antinutrients. A more detailed studyof such interactions would be particularly useful, as many of the plant-derived materialsthat have the potential to be used as fish feed ingredients contain more than one of theantinutrients. Studies are also needed to expose the effects of mixtures of antinutrients inproportions similar to those in plant-derived nutritional sources.

Acknowledgements

G. Francis is thankful to the ‘Katholischer Akademischer Auslander Dienst’, Bonn,¨for providing a stipend for doing PhD in Germany.

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antinutritional factors present in plant-derived alternate fish feed ingredients and their effects...

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