Effects of protein-, peptide- and free amino acid-based diets in fish nutrition

REVIEW ARTICLE

Effects of protein-, peptide- and free amino acid-based

diets in fish nutrition

Konrad Dabrowski1,YongFang Zhang1, Karolina Kwasek1,2, Piotr Hliwa2 & Teresa Ostaszewska3

1School of Environment and Natural Resources, Ohio State University, Columbus, OH, USA2University of Warmia and Mazury, Olsztyn-Kortowo, Poland3University of Life Sciences,Warsaw, Poland

Correspondence: K Dabrowski, School of Environment and Natural Resources, Ohio State University, Columbus, OH 43210, USA. E-mail:

[email protected]

Abstract

In the present review, we summarize data related tothe utilization of puri¢ed diets formulated with thepurpose of determining the amino acid requirementsin ¢sh independent of the ontogenetic stage and themorphological characteristics of the digestive tract.Expanding present knowledge on the formulation ofprotein, free amino acid (FAA) and synthetic dipep-tide-based diets can provide possible insights thatmight lead to a better understanding of the mechan-ismof amino acid utilization in the growth of ¢sh. Dif-ferences exist in the utilization of protein, dipeptidesor free amino acids for growth between stomach-pos-sessing and stomachless ¢sh with respect to their re-sponse to manipulating the proportion of protein anddipeptides in the formulas. Free amino acid-baseddiets are uniformly inferior. The e¡ects of diet manip-ulation on indispensable FAA concentrations in thebody (muscle) are not simply the result of deamina-tion or the protein synthesis/degradation ratio. Thehydroxyproline/proline ratio was con¢rmed to be ofvalue in quantifying muscle collagen degradation/synthesis and can perhaps be used to quantify theamino acid requirement necessary to maximize theutilization (deposition) of dietary amino acids. Insummary, indispensable amino acid requirements formaximumgrowth in ¢sh can be addressed using dietsformulated from protein/peptide/FAA sources.

Introduction

Many species of ¢sh develop a functional digestivetract but actually remain stomachless throughout

life (cyprinids, gobids); others metamorphose and afunctional stomachappears in juveniles (coregonids).Most cichlids and salmonids have a functional sto-mach at the time of their ¢rst exogenous feeding. In-formation on nutrient acquisition during transitionfrom endogenous (yolk reserves) nutrition to exogen-ous feeding was presented incorrectly in the pastwith respect to salmonids (Dabrowski 1984). This in-formation was based on the description provided byBurnstock (1959), who suggested the ‘yolk sac open-ing into the gut [is located] just behind the pyloricsphincter’and further that ‘the intestine and rectumare [. . .] full of yolk’. On the contrary, there is notransfer of yolk substances to the digestive tract in tel-eosts (Fig.1) including the most ancient order, Osteo-glossomorpha (Jaroszewska & Dabrowski 2009a), incomparison with non-teleost groups such as Acipen-seriformes (Ostaszewska & Dabrowski 2009) and Le-pisosteidae (Jaroszewska & Dabrowski 2009b). Insalmonids (Fig.1) and other teleosts (Wallace & Pack2003), yolk utilization and digestive tract formationare spatially and functionally (yolk cell cytoplasmicenzymes) separate throughout the morphogenesisof the digestive tract. This distinction is critical tounderstanding the di¡erence between yolk nutrientutilization via a process that may be considered to bethe equivalent of parenteral nutrition, whereinmacromolecules (lipoprotein) are subjected to theaction of cytoplasmic enzymes (Fig. 1a). Nutrientsare then released by the action of enzymes secretedinto the gastrointestinal lumen and pass via absorp-tive surfaces into circulation (enteroparenteral nutri-tion). In salmonids, the functional stomach appearsbefore the ¢rst feeding, although this feature does

Aquaculture Research, 2010, 41, 668^683 doi:10.1111/j.1365-2109.2010.02490.x

r 2010 TheAuthors668 Journal Compilationr 2010 Blackwell Publishing Ltd

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not ensure utilization of free amino acid (FAA) dietsand results in signs of undernourishment in the in-testine epithelial structures (Fig.1b) (Dabrowski, Lee& Rinchard 2003).This pathology is remarkably simi-lar in appearance to the atrophic intestinal epithe-lium in mammals subjected to parenteral nutrition(Ihara, Tsujikawa, Fujiyama & Bamba 2000). In ci-chlid ¢sh, the utilization of the yolk sac through theprocess of yolk hydrolysis and macromolecular parti-cle exocytosis to the vascular system (Fishelson1995)coexists with the process of active feeding and thisperhaps contributes to the ability of cichlids to utilizedietary free amino acids (Santiago & Lovell1988; Dab-rowski, Arslan, Terjesen & Zhang 2007a). However,with respect to midas (Amphilophus citrinellum), thefree amino acid-based diet weight gain was observedto be highly inferior (26%) in comparison with pro-tein-based diet (68%) weight gains (Dabrowski et al.2007a).We might conclude that to have a functionalstomach at the time of the ¢rst exogenous feeding isnot a prerequisite of FAA diet utilization for bodygrowth. Fyhn (1989) coined the hypothesis that ‘arti-¢cial ¢rst feed of ¢sh larva (will bene¢t from) a pool offree amino acids matching that consumed by the ¢shembryo during endogenous feeding’. R�nnestad,Thorsen and Finn (1999) followed this suggestionand argued that ‘amino acids, preferably in their freeform, should probably comprise a major componentof the diets of early larvae’. Further, based on a com-parison of absorption of free and protein-bond amino

acids intubated to juvenile teleost ¢sh, the authors ar-gued that ‘free amino acids seem to be superior toprotein as a dietary source of amino acids . . .’. How-ever, R�nnestad, Conceic� a� o, Araga� o and Dinis(2000) and Rojas-Garc|¤ a and R�nnestad (2003) usedmethylated-bovine serum albumin, a protein used asa marker for radioimmunoassays, with dimethyl-ly-sine ester speci¢cally resistant to trypsin enzymaticdigestion. The authors overlooked the fact that amethylated protein transforms into a non-digestiblemolecule, and makes the comparison of protein andFAAabsorption useless.Evidence based oncomparisonof FAAdiets simulat-

ing amino acid pro¢les of proteins such as casein oregg albumin led to an unequivocal acceptance of thesuperiority of diets containing free amino acids overproteins when evaluated based onweight gains of ratsfed such diets (Table 1) (Forsum & Hambraeus 1978),although the di¡erences in feed intakes, favouringamino acid mixture diets, were clearly overlooked.However, nitrogen retention in rats fed proteins orthe amino acid mixtures simulating these proteins(casein, egg albumin) was not signi¢cantly di¡erent(Itoh, Kishi & Chibata 1973; Forsum & Hambraeus1978). Therefore, it has to be concluded that in thecase of ¢sh, and larval/juveniles stages in particular,a metabolic inadequacy occurs that prevents thedevelopment of puri¢ed diets that would ensureweight gains approaching optimal levels. Althoughan increased frequency of feeding FAAmixture diets

Figure 1 Schematic representation of endogenous nutrition in salmonid (modi¢ed fromWalzer & Sch˛nenberger 1979)and exogenous feeding in the early life stage of salmonid (Dabrowski et al.2003). (a) The yolk syncytial layer and cytoplas-mic enzymes are involved in the hydrolysis, transport and transfer of yolk nutrients into the blood vessel system ofthe yolk. (b) The intestine of the salmonid juvenile shows a well-folded mid-intestine section of ¢sh fed a protein-baseddiet, whereas the lower panel shows the individual with delayed di¡erentiation of the intestinal folding, and degenerativechanges in the undernourished enterocytes; this refers to the ¢sh maintained on the free amino acid-based diet.

Aquaculture Research, 2010, 41, 668^683 E¡ects of protein, peptide and FAA-based diets in ¢sh nutrition KDabrowski et al.

r 2010 TheAuthorsJournal Compilationr 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 668^683 669

led to signi¢cantly improved growth (Yamada,Tana-ka & Katayama1981), adjustment of the diet pH andthe concentration of electrolytes (Murai, Hirasawa,Akiyama & Nose 1983) would lead to further im-provements, but such tactics do not resolve this me-tabolic shortcoming. As shown for the ¢rst time byMurai, Ogata, Takeuchi,Watanabe and Nose (1984),losses of dietary FAA are taking place through gilland urinary excretion. Murai et al. (1984) reportedthat 36% of the total nitrogen excretion was com-posed of free amino acids when carp juveniles werefed with a 38.4% FAA diet. Although a sixfold in-crease in ammonia excretion was observed in juve-nile carp following FAA mixture ingestion (Kaushik& Dabrowski1983), metabolic losses due to deamina-tion accounted for o6% of ingested nitrogen. Re-cently, Bodin, Mambrini,Wauters, Abboudi, Ooghe,Le Boulenge, Larondelle and Rollin (2008) evaluatedsemi-puri¢ed diets based on16% protein and 27% offree amino acids in rainbow trout and Atlantic sal-mon in relatively low water temperatures, perhapsfavouring the latter species. The authors arrived atextensive conclusions related to the threonine re-quirements of the species even though no protein-based diet was included in the study, and dailyweight gains amounted to only1% and 2.6%, in sal-mon and trout respectively. It has been concludedthat small amounts of a protein supplement (5^16%) considerably improve amino acid mixtures(20^30%)-based diets in cat¢sh (Andrews, Page &Murray1977), tilapia (Santiago & Lovell1988), yellowperch (Twibell & Brown1997) or rainbow trout (Kim,Kayes & Amundson 1991). Kaczanowski and Beam-ish (1996) concluded that the infusion of free aminoacids into the dorsal aorta of rainbow trout was as-sociated with increased oxygen consumption andargued that mixtures de¢cient in essential aminoacids or providing excess lysine resulted in higher

oxygen consumption. However, the authors did notexamine an alternative hypothesis that the infusionof balanced amino acids (similar to trout body pro-teins) might have led to amino acid excretionwithoutdeamination and an enhanced oxygen uptake. Brownand Cameron (1991) provided data that suggest thatthe infusion of free amino acids to cat¢sh caudal aortamay be associated with increased oxygen consump-tion due to protein synthesis. In both studies, the siteof ‘dietary’ amino acid infusion was highly unusual,and not physiologically rational, and neither proteinsynthesis nor direct amino acid excretion was ac-counted for. Inotherwords, the conclusions arehighlyspeculative. The most recent results of Saavedra, Con-ceic� a� o, Helland, Pousa� o-Ferreira and Dinis (2008) canbe interpreted as providing evidence that ‘acute’ sup-plementation of FAA recognized as de¢cient, basedon its content in the compound (formulated) diet, doesnot ensure a signi¢cant impact on protein synthesis.The synthesis rate is due to‘recirculation’of the signi¢-cant pool of amino acids through protein degradation,both in the metabolic, regular proteolysis-renewalprocesses, and most importantly, through proteoso-mally degraded ‘defective’ proteins that are almost in-stantly hydrolysed after ‘faulty’ synthesis (Vabulas &Hartl 2005).It has been reported that species such as common

carp are unable to utilize exclusively FAA or peptide-based diets (Zhang, Dabrowski, Hliwa & Gomuzka2006) in comparison with stomach-possessing ¢shsuch as rainbow trout. In the latter species, syntheticdipeptide-based diets have sustained high growthrates (Terjesen, Lee, Zhang, Faila & Dabrowski 2006).Therefore, it is worth determining whether experi-mental, protein-, peptide- and free amino acid-baseddiets are adequate for larval stomachless ¢sh,whether commercial and puri¢ed diets are compar-able as the ¢rst/exclusive feed for growth and survi-val and to examine whether FAA concentrations inthe ¢sh body are potential indicators of the availabil-ity of amino acid sources.The objectives for the present revieware (1) to sum-

marize present knowledge on the formulation of pro-tein-, amino acid- and synthetic dipeptide-baseddiets and provide possible scenarios that lead to dif-ferences in utilization for growth, (2) to compare sto-mach-possessing and stomachless ¢sh with respectto their response to the manipulation of the propor-tion of protein and dipeptides in dietary formulasand (3) to examine the e¡ects of diet manipulationson FAA concentrations in the body (muscle) follow-ing ingestion of experimental diets.

Table 1 Weight gains and utilization of proteins and theircorresponding amino acid mixtures in rats (Forsum &Ham-braeus1978)

DietWeight gain(g in 3 weeks)

Nitrogenretention (%)

Casein 41.4 70.8

AA simulating casein 63.7 67.6

Soybean flour 40.7 14.7

AA simulating soybean 84.2 53.4

Egg albumin 86.2 80.0

AA simulating egg albumin 115.5 76.8

AA, amino acid.

E¡ects of protein, peptide and FAA-based diets in ¢sh nutrition K Dabrowski et al. Aquaculture Research, 2010, 41, 668–683

r 2010 TheAuthors670 Journal Compilationr 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 668^683

Stomach-possessing fish response todietary nitrogen sources

To our knowledge, no synthetic dipeptide diets wereever tested in ¢sh in comparison with free aminoacid-based diets until the work of Dabrowski et al.(2003). Already the next year following this ¢nding,Araga� o, Conceic� a� o, Martins, R�nnestad, Gomes andDinis (2004) showed that juvenile Senegale sole feda diet enriched with dipeptides containing indispen-sable amino acids (IDAA) (Leu-Gly and Phe-Ala) ex-hibited improved general amino acid deposition inthe body and decreased catabolism (excreted CO2)when protein-hydrolysate was used as the majorsource of radiolabelled amino acids.Terjesen et al. (2006) used six semi-puri¢ed diets

that were formulated based on casein^gelatin as thesole protein source (control), two proportions of syn-thetic dipeptides to protein, 50:50 and 75:25 respec-tively (50,75P). One diet was based only on syntheticdipeptides (100P), and one diet was without argi-nine-containing dipeptides (100P w/o Arg). A dietcomposed of FAA mixture as the main amino acidsource was also included. Diets were formulated tobe isonitrogenous and isolipidic. After yolk-sac ab-sorption, the alevins of rainbow trout were randomlydistributed into glass aquaria at the ¢rst-feedingstage. The mean weight of alevins at the start of thefeeding trial was124 � 8mg.The relative growth of rainbow trout, when using

synthetic dipeptide (100P)-based diets, was only34% of the growth observed when alevins were fedthe casein^gelatin control diet. The FAA-based dietresulted in no growth in the alevins. In contrast, a1:1 ratio of synthetic dipeptides to casein^gelatin,the 50P diet, resulted in growth similar to that ofthe control diet-fed ¢sh. This is the ¢rst time thatpuri¢ed, semi-synthetic diets generated growthrates equal to protein-based diets of ¢rst-feeding sal-monids. This formulation can be used to validateamino acid requirements at maximum growth ratesin salmonids and perhaps other stomach-possessing¢sh species where a series of diets can be preparedwithout a speci¢c dipeptide (containing IDAA) orsupplemented with graded levels of dipeptide(IDAA).The concentrations of indispensable FAA (IDAA)

in muscle were, with a few exceptions, similar in thecontrol and the 50% peptide group, while the100% dipeptide- or FAA-based diet fed ¢sh showedlower levels of free IDAA in muscle. Alevins fed di-peptide-based diets without arginine (100P w/oArg)

exhibited high mortality rates within 2 weeks andnegative or minimal growth. This dietary group wasrestarted at 2 and 4 weeks, and similar results wereobtained. As both glutamate and arginine were es-tablished as metabolic precursors of proline in ani-mals (Fig. 2), it was interesting to point out that adiet devoid of arginine did not result in a decrease infree proline in ¢sh muscle. Furthermore, althoughproline is one of the most abundant amino acids incasein, a primary response to dietary proline hasbeen demonstrated (Fig. 3) when it was provided inthe free form.The results of the present analysis showthat highgrowth rates are correlatedwith high ratiosof hydroxyproline over the proline (5^10 fold-) con-centration in muscle. If the amount of degraded ma-ture collagen in muscle can be estimated based onthe free hydroxyproline in the free pool (Laurent,McAnulty & Gibson1985), and during muscle-accel-erated hypertrophy collagen degradation also in-creases (although at a slower rate than synthesis),then perhaps these results warrant further studieswhere amino acid utilization in the ‘muscle compart-ment’can be estimated based on the postprandial hy-droxproline/proline ratio.In conclusion,Terjesen et al. (2006) indicated that a

50% replacement of the protein portion with syn-thetic dipeptides is an acceptable diet formulationfor stomach-possessing rainbow trout in terms ofthe growth, survival and muscle levels of indispensa-ble FAA. The e¡ects of arginine-containing dipeptidewithdrawal that resulted in growth depression andhigh mortality indicate that arginine is an IDAA in¢rst-feeding alevins. Therefore, the expression of theornithineûrea cycle and related enzymes that mayfunction in the net de novo synthesis of arginine(Wright, Felskie & Anderson 1995) is marginal andnot nutritionally relevant. The synthetic dipeptidediets used byTerjesen et al. (2006) were able to identi-fy limiting IDAA and may be used to re-evaluateIDAA requirements in the early life stages of salmo-nids and other stomach-possessing ¢sh species (atlarval or juvenile stages) characterized by highgrowth.

Stomachless fish response to dietarynitrogen sources

The design of the experiment with common carp ju-veniles was dictated by earlier experiences with sto-mach-possessing rainbow trout alevins, where dietsbased on 50% protein replacement with peptides



proved to be acceptable and resulted in signi¢cantweight gains (Terjesen et al. 2006).The earlier studiesalso indicated that complete, synthetic dipeptidediets were not nutritionally adequate for commoncarp larvae or juveniles (Zhang et al. 2006).In the follow-up experiment, common carp larva

were hatched, and at the ¢rst-feeding stage(1.4 � 0.16mg wet weight), were o¡ered live brineshrimp, Artemia salina nauplii.Water was maintainedat 24 1C and oxygen was maintained at 7.3 � 0.3mg L�1.The commoncarp juveniles fed on live naupliifor 2 weeks were then (individual weight 20.5 �4.7mg) gradually adjusted to a compound diet (Prot-40; Table 2) for 4 days. The experiment was carriedout using 6 formulated diets (Table 2), with live brineshrimp and one commercial diet (AgloNorse, AN,Stavanger, Norway) used as controls. All formulated

diets were isonitrogenous and isolipidic (Dabrowskiet al.2003) andmet the requirements for IDAA in com-mon carp (NRC1993). Diets were in the 0.7^1mm par-ticle size category and were fed at 3-h intervals totriplicate groups.The formulated diets included positive (Prot-40)

and negative (Prot-20) control diets based solely onprotein nitrogen, and diets supplemented withsynthetic peptides that replaced 50% protein. In atwo-factorial design, we speci¢cally addressed thequestion of proline requirement and the interconver-sion of arginine (Arg) via ornithine to proline. Prolinehas been suggested to serve as a conditionally IDAAin juvenile salmonids (Dabrowski, Terjesen, Zhang,Phang & Lee 2005). In other words, we examinedthe dependence of proline requirement in the ab-sence of supplemented arginine. Because we used

Figure 2 Proline metabolism and theoretical conditionally indispensable amino acid status based on ontogenetical de-¢ciency in P5C reductase activity (modi¢ed from Phang1985). Interconversion of glutamate/proline into arginine is alsodemonstrated. Conversion of proline to arginine and arginine to proline has nearly 50% e⁄ciency when amino acids aredelivered in the diet (intestine), whereas an intravenous infusion of the same amino acids results in no conversion inpiglets (Bertolo, Brunton, Pencharz & Ball 2003).



Arg-Phe peptides, in order to balance for supplemen-ted indispensable Phe, Gly-Phe was added instead inthe control (Table 2).

Juvenile carp were divided into 24 tanks, 4 L vo-lume each, 50 ¢sh per tank (individual weight53.5 � 12.8mg) divided into triplicate ¢sh groups

Figure 3 E¡ect of dietary levels of proline and hydroxyproline (Hpro) on rainbow trout muscle levels of free amino acids(modi¢ed fromTerjesen et al. 2006). The response of free proline in the muscle to a dietary source of free proline is notice-able. Their appears to be a linear response of free Hpro in muscle to the dietary source of Hpro (casein). Di¡erent lettersindicate signi¢cant di¡erences among groups.

Table 2 Composition of the experimental diets�

Ingredients

Diets

Prot140 Prot120 1Arg 1Pro 1Arg �Pro �Arg 1Pro �Arg �Pro

Casein 40.01 18.51 18.51 18.51 18.51 18.51

Gelatin 6.11 2.62 2.62 2.62 2.62 2.62

Dipeptide mixw 0.0 0.0 22.41 22.41 22.41 22.41

Dextrin 20.43 45.42 21.43 21.43 21.43 21.43

CPSPz 5.27 5.27 5.26 5.26 5.26 5.26

Vitamin mix 4.08 4.08 4.08 4.08 4.08 4.08

Mineral mix 3.06 3.06 3.06 3.06 3.06 3.06

Vitamin C 0.05 0.05 0.05 0.05 0.05 0.05

Carboxymethylcellulose 2.04 2.04 2.04 2.04 2.04 2.04

L-lysine 0.80 0.80 1.30 1.30 1.30 1.30

L-methionine 0.40 0.40 0.40 0.40 0.40 0.40

Taurine 0.50 0.50 0.50 0.50 0.50 0.50

Lecitin 6.0 6.0 6.0 6.0 6.0 6.0

Fish oil 6.0 6.0 6.0 6.0 6.0 6.0

Choline chloride 1.0 1.0 1.0 1.0 1.0 1.0

Cellulose 4.25 4.25 5.34 5.34 5.34 5.34

�Dipeptide diets were formulated to meet all the amino acid requirements in juvenile common carp based on NRC (1993).wCompositions (g/22.41g) of experimental diets (% dry matter). Diet (1Arg1Pro) Arg-Phe, 1.72; Pro-Gly, 4.27; Gly-Phe, 0; Ala-Gly, 0; diet(1Arg�Pro) Arg-Phe, 1.72; Pro-Gly, 0; Gly-Phe, 0; Ala-Gly, 4.27; Gly-Val, 1.91; b-Ala-His, 0.82; Diet (�Arg1Pro) Arg-Phe, 0; Pro-Gly, 4.27;Gly-Phe, 1.72; Ala-Gly, 0; diet (�Arg�Pro) Arg-Phe, 0; Pro-Gly, 0; Gly-Phe, 1.72; Ala-Gly, 4.27; For all four experimental diets: Gly-Val,1.91; b- Ala-His, 0.82; Gly-Leu, 2.73; Gly-Ile, 1.97; Met-Ala, 0.74; Lys-Tyr, 1.05; Gly-Phe, 0.28; Lys-Gly, 1.75; Lys-Asp, 0.75; Gly-Trp, 0.28; Gly-Thr, 0.78; Ala-Gln, 3.36.zSoluble ¢sh protein concentrate (attractant) (CPSP 90, Sopropeche SA, Boulogne-sur-Mer, France).



per dietary treatment. Fishwere fed10% biomass thatwas readjusted for weight gain based on weeklyweighing; studies were terminated after 4 weeks.Whole ¢sh were sampled and wet weight was mea-sured within 0.1mg accuracy, frozen on dry ice ra-pidly and stored at �83 1C. Methods of FAA analysisin the dorsal muscle of ¢shwere described in detail inprevious work (Zhang et al. 2006; Kwasek, Zhang &Dabrowski 2010). Arginase activity was analysed asdescribed earlier (Tesser, Terjesen, Zhang, Portella &Dabrowski 2005).The ¢nal weight of carp fed either live food (Art

group) or the commercial starter diet (AgloNorse,AN) was signi¢cantly greater than that of ¢sh o¡eredthe semipuri¢ed casein^gelatin-based diet (40% pro-tein) (Fig. 4a). Fish in all the remaining treatmentsshowed an increase in body weight during the ex-periment (the initial weight is shown by a dashedline); however, a 20% peptide supplement did not im-prove ¢sh performance in comparison with a nega-tive control (20% protein).The total FAA concentrations in dorsal muscle of

carp indicated higher levels of these amino acids in¢sh fed semipuri¢ed diets than those fed a commer-cial feed, particularly with respect to IDAA (Fig. 4b).Numerically, the highest concentrations of total andIDAAwere found in ¢sh de¢cient in both Argand Pro.Lysine concentrations were higher in the Art and

AN groups, and in general, the lysine concentrationacross all the dietary treatments was inversely pro-portional to the arginine levels in muscle (Fig. 5).Lysine concentrations were 3^12-fold higher thanthose of arginine, and de¢ciency in arginine (�Argdiets) did not result in a decrease in muscle concen-trations.When analysed in individual ¢sh, no signi¢-cant correlation was found between lysine andarginine levels (r5 �0.346, P50.114, NS).Adecrease in the protein levels from40% to 20% of

the diet did not result in any signi¢cant decrease inthe free proline levels in carp muscle (Fig.5). Diets de-void of proline had numerically lower free proline le-vels than ¢sh fed diets with supplemented proline.The ratio of proline to hydroxyproline in fast-grow-ing ¢sh fed a commercial diet was reversed in com-parison with ¢sh fed semipuri¢ed (casein^gelatinbased) diets. It is puzzling why proline concentra-tions in fast-growing ¢shwould be10-fold lower thanthose in ¢sh fed semipuri¢ed diets. If hydroxyprolineis symptomatic of collagen degradation, why did pur-i¢ed diets (casein) appear to enhance the processes?In the general pool of free amino acids in the carp

body, taurine and histidine are the major compo-

nents (Fig. 5) besides glycine (1.3^3.2mmol kg�1).However, both Tau and His are present in carp juve-niles in signi¢cantly higher concentrations than injuvenile rainbow trout (Terjesen et al. 2006) whenfed similar diet formulations. In contrast, the glycineconcentrations in rainbow trout muscle are 10-foldhigher (10^14mmol kg�1) than that in carp muscle.Arginase activity in carp muscle was signi¢cantly

a¡ected by dietary treatments (Fig.6). However, diet-ary supplementation of Arg-containing peptide didnot result in any change in enzyme activity in com-parison with controls. These e¡ects of diet composi-tion are most certainly in£uenced by the ¢sh size(Fig. 6 inset). Muscle arginine concentration showeda signi¢cant correlationwith arginine-degrading en-zyme, suggesting that an excess of substrate mightlead to enzyme activity inhibition. The product of ar-ginine degradation, free ornithine, correlated posi-

Figure 4 (a), Mean body weight and (b), free amino acidlevels incommoncarpmuscle following the feedingexperimentwith semi-puri¢ed diets based on dipeptide and protein. Notethe increase in body weight from the initial level (dashedline). The lack of increase in the mean body weight in ¢shfed diets supplemented with an equivalent of 20% of pro-tein in the form of dipeptides is notable. Di¡erent lettersindicate signi¢cant di¡erences among groups.



Figure 5 Free amino acid levels in the dorsal muscle of common carp fed diets based on protein and dipeptides. (a) In-verse correlation between lysine and arginine in muscle. The10-fold higher level of lysine in comparisonwith arginine isnotable. (b) In fast-growing carp freeHpro is higher thanproline, whereas the opposite is true in ¢sh fed semipuri¢ed diets,resulting in a slower growth rate. (c) Proportion between taurine and histidine might describe di¡erences between fast-and slow-growing carp. The mmol levels of these two amino acids,5^10-fold higher than other amino acids, are notable.Di¡erent letters indicate signi¢cant di¡erences among groups.



tively with muscle arginase activity, although thebiological signi¢cance of this relationship seems tobe uncertain if one takes into account the enormousvariation observed among the individual ¢sh withinone treatment (dietary group).Based on earlier ¢ndings regarding the positive role

of taurine in the growth of larval stages of marine ¢sh(Takeuchi 2001), we supplemented diets for commoncarp (Table 2) with taurine.Therefore, it is appropriatetomake a reference that would relate the dietary levelof taurine to the level of this non-protein amino acidin ¢sh tissues. Indeed, taurine is almost uniformlythe most abundant FAA in ¢sh tissues (Takeuchi2001; Zhang et al. 2006). Silver bream, a cyprinid ¢shexamined in related studies by Kwasek, Zhang, Hli-wa, Gomuzka, Ostaszewska and Dabrowski (2009) inwhich peptide- and free amino acid-based diets wereused, demonstrated an increase in taurine levels inthe body that corresponded to observedweight gains.This was true in both postprandial ¢sh body and fol-lowing fasting (Fig.7). In light of the ¢ndings of Kim,Takeuchi, Akimoto, Furuita, Yamamoto, Yokoyamaand Murata (2005) that taurine supplemented up to1% of the diet increased body weight gain in marine¢sh and the corresponding taurine levels in severaltissues (muscle, liver), we suggest that some positivee¡ects of dietary taurine might also be achieved infreshwater ¢sh. Interestingly, the taurine level inbrine shrimp (Artemia) nauplii is one of the highest

among free amino acids, whereas the level of taurinein rotifers is very low and alanine is the most abun-dant FAA (Araga� o et al. 2004). This does not seem toimpact the high nutritional value of both feeds forlarval ¢sh.In summary, positive results with some stomach-

less larvae/juvenile of one cyprinid ¢sh species (Kwa-sek et al. 2009) and negative results with commoncarp (present results) indicate that synthetic dipep-tides in diet formulations need further investigation.It needs to be emphasized that the comparison of sto-machless and stomach-possessing ¢sh must expli-citly recognize the fact that most species of ¢sh atthe larval stage are stomachless (non-functional sto-mach). The lack of a stomach does negatively a¡ectprotein digestion in the case of certain type of pro-teins; however, it remains unclear how this might af-fect the utilization of dipeptides. The e¡ect ofdipeptide-based diets on the absorption/metabolismof amino acids cannot be equated with the responsefollowing feeding of protein hydrolysate, frequentlycalled peptide-containing diets. It has been docu-mented that parenteral administration of dipeptidesresults in an increase in IDAA, such as arginine,whereas enteral delivery of Ala-Gln cannot be usedas a precursor for arginine synthesis at the intestinallevel (Melis, Boelens, van der Sijp, Popovici, De Bandt,Cynober & van Leeuwen 2005). If parental nutritioncan be considered to simulate the absence of a sto-

Figure 6 Arginase-speci¢c activity in dorsal muscle of commoncarp. Arginase activity is increased in ¢sh fed diets withdipeptide supplementation but no signi¢cant e¡ect of dietary Arg-containing dipeptide on enzyme activity is found.Di¡erent letters indicate signi¢cant di¡erences among groups.



mach, then formulations enrichedwith peptides mayrepresent an interesting avenue to pursue.

Optimum protein/peptide/FAAin the diets

Kwasek et al. (2010) demonstrated that most of theIDAA concentrations in the body of larval/juvenilecarp, independent of size (ranging from 2.9 to23.7mg) and nutritional history (puri¢ed or live fooddiets), decreased signi¢cantly after ingestion of FAA-or peptide-based diets. This is a counterintuitive re-sult in many respects because intracellular depletionof free amino acids is regarded as a sign of de¢ciency.The results of many studies on the utilization of FAA-based diets that have been carried out with commoncarp have indicated inferior utilization of aminoacids in comparison with equivalent protein-baseddiets with respect to ¢sh growth. This dilemma wasin part resolved when it was determined that in sto-machless ¢sh as well as stomach-possessing ¢sh,such as sturgeon, the majority of dietary free aminoacids are excreted directly through the gills and/orwith the urine. Dabrowski et al. (2003) observed thatthe FAA-based diet resulted in no bodyweight gain in¢rst-feeding rainbow trout alevins, whereas juvenile

trout showed some growth when the same diet waso¡ered. In Fig. 8, we demonstrate theoretical plots oftissue amino acid concentration following the deliv-ery of protein (A) or free amino acid-based (B) meals.Fast absorption, and admittedly that includes manydipeptides, results in the accelerated formationof me-tabolic over£ow or hyperaminoacidaemia. A ¢shdeals with this condition by direct amino acid excre-tion; the metabolic costs, however, are unclear. It hasalso been observed in mammals that an amino acid-based diet results in adaptation of the intestinaltransporters. Furthermore, absorption has been seento decrease two to threefold in animals exposed to anamino acid diet for several weeks (Nolles, Peeters,Bremer, Moorman, Koopmanschap, Verstegen &Schreurs 2007). In the work of Kwasek et al. (2010),however, the consumption of an FAA-diet resultedin severe hypoaminoacidaemia as demonstrated incarp. It is also important to note that in common carplarvae, a peptide-based diet has been shown to fail tosupport ¢sh growth (Zhang et al. 2006) and signs of adecrease in the body amino acid pool were similar tothose exhibited by ¢sh fed an FAA-based diet.Dietary dipeptides become gradually accepted as

supplements or signi¢cant portion of semi-puri¢eddiets that result in ¢sh growth and would allow forvalidation of IDAA requirement in ¢sh (Terjesen

Figure 7 Taurine concentrations in the whole bodyof silver bream fed commercial and semi-puri¢ed diets (Kwasek et al.2009). The inverse proportion between taurine concentrations and weight gains of juvenile silver bream is notable. Taur-ine concentration is higher in fasted ¢sh in comparison with ¢sh following feeding (postprandial). This is contrary to ageneral rule with respect to free amino acid levels in tissues.



et al. 2006). Ostaszewska, Kamaszewski, Grochowski,Dabrowski, Verri, Aksakal, Szatkowska, Nowak andDobosz (2010) showed that in juvenile rainbow trout,the speci¢c intestinal transporter PEPT1was signi¢-cantly more expressed in ¢sh fed a diet supplementedwith Lys-Gly in comparison with control diets with-out a dipeptide.It is critical to consider in diet formulation for the

early life stages of ¢sh that the feed is supplementedwith free or peptide-based amino acids, and all IDAAare provided. The requirement has to satisfy theneeds for growth (synthesis) and apparent catabolicneeds. For instance, Fauconneau, Aguirre and Pergot(1986) demonstrated that arginine catabolism, esti-mated as the rate of CO2 excretion, decreased severalfolds, in parallel to the10^20-fold decrease in the pro-tein synthesis rate between yolk-sac larvae stage (en-dogenous nutrition) and feeding larvae or juveniles.We have indicated that larval/juvenile ¢sh might notdi¡er in the conditional proline requirement (Dab-rowski et al. 2005) as concluded earlier in piglets(Ball, Atkinson & Bayley 1986). The enzymes leadingfrom proline to ornithine, pyrroline-5-carboxylatereductase (P5CR) and ornithine aminotransferase(OAT), are expressed in trout alevins (P5CR, Dab-rowski et al. 2005) and in adult trout (OAT,Wekell &Brown1973). This pathway, as long as arginase is ex-pressed in di¡erent tissues, is theoretically able toproduce net arginine de novo, as inmammals (Flynn,Meininger, Haynes & Wu 2002). However, the studypresented here (Fig.5) indicates that any such synth-esis, if at all present in alevins, does not have the ca-pacity to provide all the arginine needed for growthand homeostasis. This ¢nding lends support to thehypothesis (Wright et al.1995) that the OUC enzymesare expressed during the early life stages of teleosts inorder to control endogenously produced ammonia.Although Buentello and Gatlin III (2000) suggestedthat in the case of low-arginine diets, some arginine

may be synthesized via glutamate, P5C and the or-nithine pathway (Fig. 2) in cat¢sh intestine, the evi-dence is rather weak as it was based on someinsigni¢cant increase in blood plasma ornithine, ci-truline and arginine. Glutamate and glycine increasein the diets from 0.5% to 1.0% had a similar additivee¡ect on ¢sh growth though.Dabrowski et al. (2005) suggested that proline may

be a conditionally indispensible amino acid in theearly life stages of ¢sh as it has been documented tobe in pigs (Ball et al. 1986).Our conclusion was basedon a negligible concentration of free proline in the¢sh body when the ¢shwere fed diets actually devoidof proline (Dabrowski et al. 2005). Hpro levels in thetissues of rainbow trout were also insigni¢cant de-spite the high content of Pro in amino acid-baseddiets that did not promote ¢sh growth. Perhaps, theuse of Hpro in the diet formulation needs to be ex-panded and placed in the context of the possibility ofproline being essential during the early ontogeneticstages in ¢sh, and, the larger context of aquatic foodchain transfer of Hpro. Hpro is found in multicellularalgae such as Volvox, and Hpro-rich proteins maycontain up to 26 Hpro molecules in a single stretch(Sumper & Hallmann 1998). Hpro is also present insome aquatic invertebrates in ¢bril-forming col-lagens (Mann, Gaill & Timpl 1992). Therefore,Hpro may be transferred in the aquatic foodchain, although there is no evidence of Hpro in crus-taceans (Helland,Terjesen & Berg 2003). However, itis inexplicable how the claim can be made that freehydroxyproline (Hpro) can be utilized for ¢sh growth(Aksnes, Mundheim,Toppe & Albrektsen 2008). Thelatter authors suggest an improvement in rainbowtrout growth as a result of a diet that was supplemen-ted with free hydroxyproline (Hpro), although it wasclear whether greater consumptionof feed with Hprowas responsible for only a slightly higher weightgain. Perhaps Hpro acts as an attractant (Yamashita,

Figure 8 Schematic representation of ‘slow’and ‘fast’nutrient (amino acids) absorption (arrows) processes in salmonids(stomach-possessing ¢sh). (a) Slower evacuationof protein-containing diets and hydrolysis of protein result inan extendedtime of amino acid and di- tri-peptide release and absorption. (b) Fast absorption generates a‘metabolic over£ow’or hyper-aminoacidaemia and excretion of amino acids via the gills and urinary tract.



Yamada & Hara 2006). However, the authors impliedthat ‘the whole vertebral column showed increasedHpro content,’ which may be related to ‘increaseddietary Hpro inclusion’. It has been known for nearly50 years (Adams & Goldstone 1960; Valle, Goodman,Harris & Phang 1979) that although the ¢rst stepsof proline and Hpro degradation are di¡erent, thesecond step is catalysed by a common enzyme. Thesecond reaction in proline and hydroxyproline degra-dation is the oxidation of pyrroline-5-carboxylic acid(P5C) and 3-OH-P5C to glutamate and g-hydroxyglu-tamate (Fig. 2) respectively (Adams & Goldstone1960). In fact, Aksnes et al. (2008) are also wrong inconcluding that Hpro ‘can be synthetized in vivo’.Hpro is not synthesized as a free imino acid. Hpro isproduced by hydroxylation of the tripeptide of theprocollagen polypeptide chain. Free hydroxyprolinein ¢sh tissues is derived largely from endogenouscollagen degradation and from hydrolysis of dietaryproteins. There is no return of Hpro into proteinsynthesis. Therefore, speculations by Aksnes et al.(2008) that ‘dietary Hpro inclusion may [. . .] reducewounds in [the] skin’of salmon or that ‘Hpro may beincorporated directly into the (¢sh) protein’ do nothave any corroborating evidence.Aksnes et al. (2008) criticized the classical work of

Halver (1957) for not using Hpro in their diets whendetermining amino acid requirements in salmon.Clearly, the authors of the work completed over 52years ago were familiar with research performedwith other animals, the lack of nutritional value ofHpro in the diets and also understood the process ofpeptide-bound prolinehydroxylation. AddingHpro tothe ¢sh diets as the nutrient was not worth it then,and it certainly is not worth it today.These erroneous conclusions published byAksnes

et al. (2008) are further propagated by Li, Mai,Trush-enski andWu (2009) with the formation of comple-tely unfounded concepts regarding the possiblepositive e¡ects of Hpro supplementation in plant pro-tein-based diets on the growth and survival of ¢sh. Inthis latest review, the lack of a critical understandingof Hpro degradation and biochemical pathwaysadded to misconceptions of Hpro function, synthesisand possible role in ¢sh nutrition.

Imbalance/complementary IDAA diets/feeding strategy

Under practical farmingconditions, feed formulationsthat include plant proteins, pellet processing and

storage usually result in dietary IDAA imbalancesand de¢ciency. Previous studies have shown thatIDAA-de¢cient diets decrease feed intake, changefeeding behaviour and thus lower the growth andsurvival of animals. However, no information isavailable on how IDAA-de¢cient/imbalanced dietsa¡ect the feed intake, feed utilization and di¡erencesin feed acceptance in the short term (within hours)or over longer periods (several weeks) in ¢sh. Wehypothesize that the use of sequential disproportion-ate amino acid rations will reduce or eliminate thenegative e¡ects of IDAA de¢ciency on ¢sh growth.We have investigated how IDAA-de¢cient dietsa¡ect the FAA pro¢le in the ¢sh body in cichlid ¢sh,midas (A. citrinellum) (Zhang 2007). This work, forthe ¢rst time, addressed amino acid imbalances in¢sh and aimed to establish the physiological regula-tory mechanism that governs diet acceptance or re-jection in ¢sh. This study (see the experimentaldesign in Dabrowski et al. 2007a) aimed to developfeeding strategies to reduce the adverse e¡ects ofIDAA-de¢cient diets on ¢sh growth in the aquacul-ture industry.The concentrations of nine (except Arg) of ten

IDAA decreased in the whole ¢sh body from 30minto 6 h after the meal with FAA. This in turn resultedin a trend towards decreased total IDAA.The levels oftotal FAAand total DAA showed an increasing trendduring this period. This suggests selective utilizationof IDAA for protein synthesis. In the (� )Arg group,the concentrations of eight (except Arg and Lys) outof ten IDAAs decreased numerically between 30minand 6 hafter feeding.The total IDAAat 6-h postpran-dial was lower than that at 30min postprandial,whereas total FAAand total DAA showed an increas-ing trend during this period. The level of total DAAnumerically increased in the (� )Lys group. Six outof ten IDAAs showed an increasing trend from30min to 6 h in the protein-fed group, suggestinggreater proteolysis than utilization for synthesis. Thetotal FAA, total DAAand total IDAA showed increas-ing trends during this period.This section aims to emphasize that besides

the diet composition and proportion of amino acidsdelivered in di¡erent forms, the strategy used thusfar, a diet composed of protein with an ideal aminoacid composition (meeting requirements), and deliv-ered with predetermined frequency, is inadequate. Itis quite removed from the conditions the ¢sh speciesin question experiences in the wild and that havebeen evolutionarily imprinted in a diurnal, seasonalor age-related life span. The physiological mechan-



ism(s) involved in an imbalanced amino acid feedingstrategy requires further investigation. It cannot bedenied that this strategy might have practical impli-cations with respect to nutrient delivery in general(for instance, vitamins; Blom & Dabrowski1998), butin particular with respect to another facet of ‘com-pensatory growth’ (Dabrowski, Zhang, Arslan & Ter-jesen 2007b).

Further research

Our preliminary data and critical review of the re-sults in stomachless and stomach-possessing ¢sh in-dicate that both approaches, use of the mixtures ofprotein, dipeptides and free amino acids, in combina-tion with imbalanced IDAA diet formulations, needfurther exploration. Both diet formulations and alter-native feeding strategies have the potential to becometools that will lead to a better understanding of thephysiological and regulatory processes of nutrientacquisition in the digestive tract of ¢sh.With respectto stomach-possessing ¢sh, a combination of the two,dipeptide supplements and imbalanced/complemen-tary nutrient diet formulations, alreadyappears to bethe most attractive proposition.We hypothesize thatthe use of sequentially disproportionate amino acid(dipeptide) rations will reduce or eliminate the nega-tive e¡ects of IDAA de¢ciency on ¢sh growth and ine¡ect up-regulate transporter systems at the intest-inal level and decrease the catabolism of IDAAs. Thisis based on evidence that (1) a dietary supplement ofdipeptides up-regulates expression of the peptidetransporter (PEPT1) and (2) IDAA utilization (viachanges in enzymeVmax or Km) e⁄ciency increasesunder a dietary amino acid imbalance despite a de-crease in the substrate (AA concentrations in blood).These in turn resulted in increased muscle and liverprotein synthesis. This approach, for the ¢rst time,addresses amino acid imbalances in lower verte-brates that appear to di¡er strikingly from mammals(Hao, Sharp, Ross-Inta, McDaniel, Anthony,Wek, Ca-vener, McGrath, Rudell, Koehnle & Gietzen 2005),which respond with anorexia to diets with missingsingle IDAA. The physiological regulatory mechan-ism that governs diet acceptance or rejection in ¢shstill remains to be established. Based on preliminaryexperiments, this direction of research aims to devel-op alternative feeding strategies that might reducethe adverse e¡ects of IDAA-de¢cient diets on ¢shgrowth andmight provide new solutions to the aqua-culture industry.

Acknowledgments

The data presented here were the result of several re-search projects. These projects were supported by thePolish Science Foundation, Scienti¢c Research Com-mittee grant KBN 2P06Z 05126 (to K. D.), and grantN31103032/2256 (to T. O.). The US AID AquacultureCooperative Research Support Program, OregonState University, grant LAG-G-00-96-9015-00, pro-vided a fellowship toYongfang Zhang.We thank Mar-ta Jaroszewska and Kornelia Dabrowska in preparingthe ¢rst draft of this paper.

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Effects of protein-, peptide- and free amino acid-based diets in fish nutrition

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