JOURNAL OF THE Vol. 40, No. 6WORLD AQUACULTURE SOCIETY December, 2009

Estimating Amino Acid Requirement of Brazilian Freshwater Fishfrom Muscle Amino Acid Profile

Alvaro Jose de Almeida BicudoEscola Superior de Agricultura “Luiz de Queiroz,” Universidade de Sao Paulo-Programa de

Pos-Graduacao em Ciencia Animal e Pastagem. CP 09-13418-900-Piracicaba, SP-Brazil

Jose Eurico Possebon Cyrino1,2

Escola Superior de Agricultura “Luiz de Queiroz,” Universidade de Sao Paulo-Departamentode Zootecnia/Setor de Piscicultura. CP 09-13418-900-Piracicaba, SP-Brazil

Abstract

Information on nutritional requirement of some Brazilian farmed fish species, especially essentialamino acids (EAA) requirements, is scarce. The estimation of amino acids requirements based onamino acid composition of fish is a fast and reliable alternative. Matrinxa, Brycon amazonicus, andcurimbata, Prochilodus lineatus, are two important Brazilian fish with potential for aquaculture. Theobjective of the present study was to estimate amino acid requirements of these species and analyzesimilarities among amino acid composition of different fish species by cluster analysis. To estimateamino acid requirement, the following formula was used: amino acid requirement = [(amount of anindividual amino acid in fish muscle tissue) × (average totalEAA requirement among channel catfish,Ictalurus punctatus, Nile tilapia, Oreochromis niloticus, and common carp, Cyprinus carpio)]/(averagefish muscle totalEAA). Most values found lie within the range of requirements determined for otheromnivorous fish species, in exception of leucine requirement estimated for both species, and argininerequirement estimated for matrinxa alone. Rather than writing off the need for regular dose–responseassays under the ideal protein concept to determine EAA requirements of curimbata and matrinxa,results set solid base for the study of tropical species dietary amino acids requirements.

Formulation complete, balanced aquafeedsrequire complete knowledge of the nutritionalrequirements of the farmed species. The majorconstraints in the formulating and processingof cost-effective diets are a lack of informationon nutritional requirements of tropical, novelaquaculture species and the digestibility ofsuitable feed ingredients. The survival, health,growth, and efficient feed utilization of fishdepend on the nutritional value of feedstuff.

Fish, like any other animal, do not have nutri-tional requirement of protein per se but havea requirement of a well-balanced mixture ofindispensable or essential amino acids (EAA)and dispensable or nonessential amino acids.Usually, dose–response trials are used to deter-mine amino acid requirement in fish. This is a

1 Corresponding author.2 Present address: J. E. P. Cyrino is a scholar (1-B)

of Conselho Nacional de Desenvolvimento Cientıfico eTecnologico (Brazil’s National Council for Technologicaland Scientific Development–CNPq).

costly and time-consuming method, especiallywhen aimed at determining requirements forall EAA (Akiyama et al. 1997). Data on com-plete EAA requirements are available only fora small number of fish, such as Nile tilapia,Oreochromis niloticus, common carp, Cypri-nus carpio, channel catfish, Ictalurus punctatus,Chinook salmon, Oncorhynchus tshawytscha,chum salmon, Oncorhynchus keta, and Japaneseeel, Anguilla japonica (NRC 1993). However,several species had one or more amino acidsrequirement determined.

Studying the amino acids requirement forswine, Cole (1978) introduced the “ideal pro-tein” concept in the nutrition of monogastricanimals—the body amino acid profile of swinewould reflect the amino acid requirement of theanimals. Historically, the concept was adaptedfor and first used in fish nutrition studies byOgino (1980). The EAA requirements weresoon shown to correlate well with the EAAprofile of the whole-body tissue of rainbow

© Copyright by the World Aquaculture Society 2009

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AMINO ACID REQUIREMENT ESTIMATION FROM MUSCLE AMINO ACID PROFILE 819

trout and Atlantic salmon by Wilson and Cowey(1985).

Mambrini and Kaushik (1995) postulated thatcarcass amino acid profile best reflects the idealpattern of a reference protein, and that it canbe used as a guideline for formulating feedsor for studying EAA requirements. Accord-ing to Ngamsnae et al. (1999) there is a highcorrelation between phenylalanine and argi-nine requirements experimentally determinedand the value estimated using the A/E ratiofor silver perch, Bidyanus bidyanus. As a mat-ter of fact, Montes-Girao and Fracalossi (2006)determined lysine requirement of jundia, Rham-dia quelen, by dose–response experiment in5.1% of dietary protein. This value is very sim-ilar to the requirement in lysine −5.8% dietaryprotein—previously estimated for the samespecies by Meyer and Fracalossi (2005) basedon the species’ muscle amino acid composi-tion. Therefore, it seems reasonable to suggestthat information on EAA contents of fish wholebody may be useful in designing test diets forfish when their amino acid requirements havenot been established yet (Wilson 2002).

Matrinxa, Brycon amazonicus (=cephalus),is an Amazonian, omnivorous Characin, andcurimbata, Prochilodus lineatus (=scrofa), isa bottom-feeder Characin from the Parana andParaguay river basins, usually farmed in Brazil.The objective of this study was to estimate theamino acid requirements of these two speciesbased on their body amino acid profile.

Material and Methods

Data on body amino acid contents from thestudied species were obtained from Maia andRodriguez-Amaya (1983) for P. lineatus andCyrino (1984) for B. amazonicus. The aminoacid requirement estimate was calculated withthe aid of the equation used by Meyer andFracalossi (2005): amino acid requirement =[(amount of an individual amino acid in fishmuscle tissue) × (average TEAA requirementamong channel catfish, Nile tilapia, and com-mon carp)]/(average fish muscle TEAA).

The A/E ratio (essential to total aminoacids ratio) was calculated using the formula

suggested by Kaushik (1998) to compare mus-cle tissue amino acids of species studied tothose from other 16 fish species registered inliterature by cluster analysis:

A/Eratio = essential amino acid

total of essential amino acids× 1000

The matrix of the amino acids profile of fishused for cluster analyses is listed in Table 1.Cluster analysis is a multivariate analysis tech-nique that organizes information about variablesin a way that relatively homogeneous groups, or“clusters,” can be formed. The clustering pro-cess involves three steps: data standardization;assessment of a dissimilarity measure amongsamples; and the use of a grouping technique.In the present study, data standardization wasnot necessary because the AA were expressedin a uniform unit (A/E ratio). The Euclideandistance was used as the dissimilarity distance(Sneath and Sokal 1973) and Ward’s methodwas used as the grouping technique (Ward1963). Cluster analyses were run in the softwareStatistica® (version 7.0, Statsoft, Inc., Tulsa,OK, USA).

Results and Discussion

The results of cluster analyses are repre-sented in a dendogram built-up from the matrixof AA profile of fish dissimilarities (Fig. 1).Considering a break point at the linkage dis-tance around 48, three fish groups with sim-ilar AA profile were identified. No similari-ties were detected between B. amazonicus andPseudoplatystoma corruscans AA profiles andany other fish analyzed. Species were randomlydistributed in the dendogram, and there was noevidence of phyletic relationships influencingspecies distribution.

Similar results were found by Akiyama et al.(1997) in a study which involved 12 differ-ent fish species. According to Fu et al. (2000),quantitative differences between some AA maybe related to changes in specific genetic infor-mation in individual fish, on the basis of whichmuscle proteins are synthesized.

The AA requirements estimated for P. line-atus and B. amazonicus are shown in Table 2.Usually, the amino acid requirements of fish,

820 BICUDO AND CYRINO

Table 1. Essential to total amino acid ratio (A/E) of different freshwater and marine fish.

Arg His Ile Leu Lys Met+Cys Phe+Tyr Thr Trp Val

Brycon amazonicusa,n 90.05 35.52 95.55 166.83 153.33 71.29 165.83 87.04 31.52 103.05Catostomus commersoni b,l,m 124.53 44.76 76.88 166.39 175.30 55.33 152.09 81.64 20.93 102.15Hippoglossus hippoglossusc,m 129.64 54.50 82.51 147.99 167.49 70.02 140.99 87.43 20.25 99.17Ictalurus punctatusd,m 131.87 42.90 84.82 146.30 168.25 74.73 146.70 87.19 15.42 101.82Lota lotab,l,m 129.84 48.26 82.75 165.50 161.63 62.21 151.55 80.23 19.38 98.64Micropterus salmoidese,n 131.96 44.78 75.43 150.87 177.83 79.13 148.04 90.87 17.83 83.26Oncorhynchus kisutch f,m 115.24 57.52 71.18 144.09 166.22 91.57 145.83 98.31 26.93 83.11Oncorhynchus masoug,m 119.97 46.02 76.26 145.20 169.65 86.27 158.10 89.16 15.98 93.39Oncorhynchus mykissh,n 123.25 56.91 83.45 145.93 163.24 70.76 149.20 91.52 17.88 97.87Oreochromis niloticuse,n 142.88 44.23 94.18 160.97 161.15 66.43 129.99 89.88 18.44 91.85Paralichthys olivaceusc,m 130.74 45.71 75.73 147.01 177.22 75.34 152.24 86.96 20.53 88.51Perca flavescensb,l,m 123.92 51.00 84.50 168.02 149.30 60.61 156.44 78.59 26.11 101.50Pleuronectes ferrugineac,m 133.48 48.16 80.79 148.81 168.27 68.80 127.97 87.08 25.95 110.67Pomoxis nigromaculatusb,l,m 117.65 44.61 83.82 169.85 170.10 55.64 157.11 80.39 24.51 96.32Prochilodus lineatus i,m,n 122.97 46.94 90.72 152.84 180.18 50.58 141.77 85.98 20.86 107.16Pseudoplatystoma corruscans j,m,n 135.73 47.72 85.38 99.39 199.65 84.50 153.46 66.99 22.55 104.64Pseudoplatystoma fasciatume,n 133.02 58.67 82.50 154.00 166.23 69.26 139.95 91.67 18.13 86.58Rhamdia quelenk,n 115.41 40.78 78.75 156.00 180.13 96.33 148.71 93.15 8.42 82.30Salmo salarh,m 125.55 57.36 83.76 146.63 176.26 52.80 149.29 94.02 17.66 96.68Stizostedion vitreumb,l,m 122.71 49.42 83.65 163.45 168.76 60.75 152.36 84.38 18.32 96.19

aCyrino (1984).bMai et al. (1980).cKim and Lall (2000).dWilson and Poe (1985).ePortz (2001).fArai (1981).gOgata et al. (1983).hWilson and Cowey (1985).iMaia and Rodriguez-Amaya (1983).jFuruya and Furuya (2003).kMeyer and Fracalossi (2005).lAvailable methionine value only.mWhole-body composition.nMuscle composition.

when expressed as a percentage of diet, aremuch higher than for other animals. How-ever, the similarity between fish and otheranimals is closer when amino acid require-ments are expressed as percentages of dietaryprotein (NRC 1993). Most values found liewithin the range of requirements determined forother omnivorous fish species (Wilson 2002),in exception of leucine requirement estimatedfor both species, and arginine requirement esti-mated for matrinxa alone.

Leucine requirements estimated for bothspecies −9.67% dietary protein for matrinxaand 6.67% dietary protein for curimbata—are higher than values determined for otherspecies usually with similar feeding habit;

leucine requirements usually range from 3.3to 3.9% of dietary protein. Actually, Wilson(2002) consider that estimating amino acidsrequirements based on A/E ratios and pro-tein accretion tend to yield higher values thanthose determined by dose-response studies. Onthe other hand, arginine requirement estimatedfor matrinxa −3.9% dietary protein–is proba-bly underestimated. Arginine deficiency resultsin low growth and nitrogen retention (Tibaldiet al. 1994; Ruchimat et al. 1998); on the otherhand, the excess of this amino acid reduces thegrowth and feed efficiency in fish (Santiago andLovell 1988; Borlongan 1991).

According to Akiyama et al. (1997) theuse of amino acid profile from the whole

AMINO ACID REQUIREMENT ESTIMATION FROM MUSCLE AMINO ACID PROFILE 821

0 20 40 60 80 100 120 140

Linkage Distance

P. corruscansR. quelenO. kisutchO. masou

P. olivaceusM. salmoides

S. salarP. lineatus

P. ferrugineaO. niloticus

P. fasciatumI. punctatus

O. mykissH. hippoglossus

P. flavescensS. vitreum

L. lotaP. nigromaculatus

C. commersoniB. amazonicus

Figure 1. The dissimilarity diagrams of the A/E ratio profiles among fish using Ward’s method.

Table 2. Amino acids requirement estimated for Brazilian omnivorous fish matrinxa (Brycon amazonicus) andbottom-feeder curimbata (Prochilodus lineatus) based on muscle amino acids profile.

Amino acids requirement (% protein)Essential amino acids

composition (% protein)Estimation aminoacids requirement

Ictaluruspunctatus

Oreochromisniloticus

Cyprinuscarpio P. lineatus B. amazonicus P. lineatus B. amazonicus

Arg 4.30 4.20 4.30 7.78 3.60 3.96 2.90His 1.50 1.72 2.10 2.97 1.42 1.51 1.14Ile 2.60 3.11 2.50 5.74 3.82 2.92 3.08Leu 3.50 3.39 3.30 9.67 6.67 4.92 5.37Lys 5.10 5.12 5.70 11.40 6.13 5.80 4.94Met+Cys 2.30 3.22 2.10 3.20 2.85 1.63 2.30Phe+Tyr 5.30 5.54 4.40 8.97 6.63 4.57 5.34Thr 2.00 3.75 3.90 5.44 3.48 2.77 2.80Trp 0.50 1.00 3.60 1.32 1.26 0.67 1.02Val 3.00 2.80 0.80 6.78 4.12 3.45 3.32TIAA 30.10 33.85 32.70 63.27 39.98 32.22 32.22

body is recommended for feed formulationonly when dietary requirement data deter-mined by dose–response and growth studiesare not available. However, the efficiency ofutilization of some amino acids can be influ-enced by several factors. Abboudi et al. (2006)did not recorded influence of dietary proteincontents on lysine requirement in Atlanticsalmon, although increasing protein levels at

similar digestible energy concentration resultedin a better efficiency of lysine utilizationby rainbow trout (Rodehutscord et al. 2000).Digestible energy did not significantly affectlysine requirements of rainbow trout, but higherenergy levels decreased the use of lysine onwhole protein deposition (Encarnacao et al.2004), and energy source affected lysine uti-lization (Encarnacao et al. 2006) in the same

822 BICUDO AND CYRINO

species. In some diet formulations, amino acidscan be supplemented in purified form. In suchcases, it is necessary to feed the fish an increas-ing number of daily meals, allowing more stableplasmatic concentration of amino acids, elic-iting better utilization, and reducing nitrogenwastes (Tibaldi et al. 1994; Schumacher et al.1997; Rodehutscord et al. 2000). Supplement-ing fish diets with coated l-lysine HCl improveutilization of this amino acid in comparison touncoated form (Zhou et al. 2007).

The close relationship between dietary aminoacids allowance and availability, and freeEAA in tissues and plasma (NRC 1993) leadto differences in absorption efficiency amongdietary amino acids. For instance, Gomez-Requeni et al. (2004) showed that when gilt-head sea bream, Sparus aurata, fed diets withdifferent protein sources and similar amountsof arginine and lysine, fish fed plant pro-tein sources retained lower levels of theseamino acids than fish fed fish meal-based diets.Feeding post-larval Senegal sole, Solea sene-galensis, diets containing radio-labeled EAA,Rønnestad et al. (2001) reported that higherproportions of EAAs lysine and arginine wereretained in the body (>81%) than catabolized(<16%). Finally, Atlantic cod, Gadus morhua,fed fish meal-based diets, retained 39.2% argi-nine, 40.5% histidine, 39.4% isoleucine, 35.8%leucine, 46.7% lysine, 39.9% methionine,40.8% phenylalanine, 47.6% threonine, 44.7%tryptophan, and 30.2% valine. These retentionvalues were higher than those observed whenthe Atlantic cod was fed diets containing eitherdehulled, extracted soybean meal or biopro-cessed extracted soybean meal as main proteinsource, but no difference was recorded for 1-or 2-yr-old age groups (Refstie et al. 2006).

Data on EAA requirements of curimbata andmatrinxa derived from dose–response experi-ments are not yet available. Therefore, infor-mation herein presented can be useful forresearchers and aquafeed industry alike. Addi-tionally, determination of digestibility valuesas well efficiency on amino acids utiliza-tion will further contribute to the precisionon formulation and processing of diets to

maximizeprotein synthesis and reduce nitrogenexcretion in aquatic environment.

Acknowledgments

A. J. A. Bicudo would like to thank Fundacaode Amparo a Pesquisa do Estado de SaoPaulo (Sao Paulo State Research Foundation -FAPESP) for granting his Doctoral scholarship(Proc. 05/51968-9).

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