Aquaculture J. Alejandro Buentello, Delbert M. Gatlin III )

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1 Ž. Aquculture The dietry rginine requirement of chnnel ctfish ž Ictlurus puncttus/ is influenced by endogenous synthesis of rginine from glutmic cid J. Alejndro Buentello, Delbert M. Gtlin III ) Deprtment of Wildlife nd Fisheries Sciences nd Fculty of Nutrition, Texs A&M UniÕersity System, College Sttion, TX , USA Received 11 November 1999; received in revised form 24 Jnury 2000; ccepted 24 Jnury 2000 Abstrct Previous studies with young mmmls hve estblished tht rginine synthesis from glutmte-derived citrulline cn be mjor endogenous source of rginine. Therefore, n experiment ws conducted to re-ssess the dietry rginine requirement of juvenile chnnel ctfish nd to determine the metbolic effects of including glutmte or glycine to mintin isonitrogenous levels mong diets. Two sets of diets were formulted to contin 24 g crude proteinr100 g dry weight from cseinrgeltin nd crystlline mino cids with rginine supplementtion in 0.5 increments from 0.5 to 2.0 gr100 g diet. Amino cid nitrogen ws mintined equl, within sets, by replcing rginine with sprtte nd either glutmte or glycine. Ech diet ws fed to pprent stition to triplicte groups of 12 fish initilly verging 11.4 grfish for 8 weeks. Weight gin Ž WG., feed efficiency Ž FE., protein efficiency rtio Ž PER., protein retention Ž PR. nd survivl were significntly Ž P ffected by rginine. At the suboptiml level of dietry rginine, glutmte ppered to contribute rginine through internlly derived citrulline bsed on incresed plsm citrulline nd rginine concentrtions. WG nd plsm mino cid concentrtions of fish fed diets with glycine suggested tht it does not serve s precursor for citrulline. Bsed on WG nd FE, juvenile chnnel ctfish were found to require rginine t 3.3% to 3.8% of dietry protein, when glutmte ws included in the diet. The requirement estimte ws 33% higher when glycine replced glutmte in the diet nd ws similr to the previously determined rginine requirement of chnnel ctfish t 4.3 gr100 g of dietry protein. These results strongly suggest tht dietry ) Corresponding uthor. Tel.: q ; fx: q Ž. E-mil ddress: d-gtlin@tmu.edu D.M. Gtlin III r00r$ - see front mtter q 2000 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 312 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture glutmte is used for endogenous synthesis of rginine in chnnel ctfish, especilly when rginine is deficient in the diet. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Arginine requirement; Amino cid metbolism; Chnnel ctfish; Citrulline 1. Introduction The mino cid rginine hs importnt nutritionl nd physiologicl roles nd is estblished s indispensble in the diet of mny fish species Ž NRC, Experiments with young pigs, rts nd dogs hve indicted tht renl rginine synthesis is mjor endogenous source of rginine for these nimls Ž Wu nd Morris, The kidney redily synthesizes rginine from citrulline, which cn rise from the intestinl metbolism of glutmte, the so-clled intestinl renl xis tht converts glutmte to rginine. Endogenous synthesis vi the intestinl renl xis provides resonble explntion for rginine being dispensble for most mmmlin species s rginine biosynthesis is sufficient to meet the needs of dult nimls Ž Bker, However, young nimls my require dietry rginine becuse the rte of endogenous synthesis is low compred with the metbolic requirement for this mino cid Ž Fuller, Studies with young pigs Ž Wu nd Knbe, 1995; Wu et l., indicte tht the endogenous synthesis of rginine cn provide 50 69% of the totl dily rginine requirement. The quntittive rginine requirement of chnnel ctfish hs been determined to be 4.3% of dietry protein Ž Robinson et l., Diets in tht study were formulted to contin 24% crude protein nd were mintined isonitrogenous by djusting the levels of sprtic cid nd glutmte. Becuse glutmte hs proven to be n importnt precursor for endogenous rginine synthesis in mny species, this pproch could ffect the estimtion of dietry rginine requirements. Therefore, the present study ws conducted to re-evlute the dietry requirement of chnnel ctfish for rginine nd to ssess the metbolic effects of including glutmte or glycine in the experimentl diets. 2. Mterils nd methods 2.1. Feeding tril An 8-week feeding tril ws conducted in which juvenile chnnel ctfish, Ictlurus puncttus, were fed vrious levels of dietry rginine. Experimentl diets were similr to those used by Robinson et l. Ž The bsl diet Ž Tble 1. ws formulted to contin 24% crude protein from csein, geltin nd crystlline L-mino cids to meet the mino cid requirements of chnnel ctfish, except for rginine Ž NRC, The crude protein content of the experimentl diets ws confirmed by nlysis ccording to estblished procedures Ž AOAC, Two different sets of diets were prepred by dding grded mounts of rgininephcl to provide rginine t 0.5%, 1.0%, 1.5% or 2.0% of diet.

3 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture Tble 1 Composition of the bsl diet Ingredients Amount Ž %. Csein 10.0 Geltin 2.5 b Amino cid premix 6.7 Dextrin 24.3 Celufil 30.3 Corn oil 4.0 Cod liver oil 6.1 c Vitmin premix 3.0 c Minerl premix 4.0 CŽ PO Crboxymethylcellulose 2.0 L-Asprtte 3.0 Glycine or L-glutmte 3.0 L-ArgininePHCl United Sttes Biochemicl, Clevelnd, OH. b Consisted of L-mino cids from Nutri-Quest, Chesterfield, MO nd provided Ž s % of diet.: histidine, 0.14; isoleucine, 0.19; leucine, 0.06; lysine, 0.64; methionine, 0.32; phenyllnine, 0.42; serine, 1.57; threonine, 0.13; tryptophn, 0.02; vline, 0.11; proline, 1.57; lnine, Amino cids lso were provided by 10% csein nd 2.5% geltin which in combintion with the crystlline mino cids met chnnel ctfish requirements Ž NRC, c Sme s Buentello et l. Ž Dietry rginine levels were determined by reversed phse HPLC; the bsl diet contined 0.48% rginine nd supplementl levels were confirmed by HPLC nlysis. Amino cid nitrogen ws mintined equl mong diets, within ech set, by djusting the levels of rginine nd either glutmte or glycine in combintion with sprtte, resulting in totl of eight experimentl diets. Although diets within ech set were isonitrogenous, those contining glutmte hd 6.9% less totl nitrogen thn those contining glycine, due to the lower nitrogen content of glutmte reltive to glycine. Diets were supplemented with complete minerl nd vitmin premixes nd djusted to ph 7.0 with 6 N NOH. Procedures for diet preprtion nd storge were s previously described Ž Buentello et l., All diets were fed twice dy to pprent stition to triplicte groups of chnnel ctfish ech consisting of 12 fish initilly weighing 137.5" 2.2 grgroup. These juvenile chnnel ctfish were obtined from ponds t the Aquculturl Reserch nd Teching Fcility of the Texs A& M University System Ž Burleson, Texs. nd cclimted to stndrdized conditions in quri for 2 weeks prior to the feeding tril, during which they were fed the bsl diet contining glycine. The feeding tril ws conducted in 24, 38-l quri operted s flow-through system with well wter flowing t 1 lrmin. Wter temperture ws mintined t 27" 28C. Supplementl ertion ws provided to ech qurium to mintin dissolved oxygen levels ner ir sturtion. Fluorescent illumintion controlled by timer provided 12:12 h light:drk cycle. Wter qulity ws monitored weekly for mmoni, nitrte, nitrite, hrdness nd ph nd remined within

4 314 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture recommended levels for chnnel ctfish Ž Lee, throughout the feeding tril. Procedures used in this study were pproved by the Texs A&M University System Animl Cre nd Use Committee. Estblished formuls Ž Hlver, were used to compute weight gin Ž WG., feed efficiency Ž FE. nd protein efficiency rtio Ž PER. for fish t the end of the tril Smple collection nd nlyses A smple of three fish t the strt of the feeding tril nd three fish per qurium t termintion were homogenized in blender nd stored frozen Ž y208c. in polyethylene bgs before determining proximte composition of crcss ccording to estblished procedures Ž AOAC, Bsed on crcss protein content, protein retention Ž PR. ws computed s PR s 100 = ŽŽ fish finl protein concentrtion= finl biomss. yžfish initil protein concentrtion= initil biomss.. rprotein intke. At the end of week 8, blood smples from three fish per qurium were obtined from the cudl vsculture by heprinized needles, 14 h fter the lst feeding. Blood plsm ws seprted by centrifugtion Ž 2000 = g, 10 min. nd stored t y808c. Anlysis of plsm-free mino cids ws performed using HPLC s described by Wu nd Knbe Ž but with modified grdient progrm nd plsm filtrtion to llow for better seprtion of threonine, citrulline nd rginine. Briefly, plsm smples were deproteinized by ddition of 1.5 molrl HClO Ž 1 ml HClO rml plsm. 4 4 followed by neutrliztion with 2 molrl K CO Ž 0.5 ml K CO rml plsm Deproteinized nd neutrlized smples were filtered through 0.22-mm polycrbonte syringe filters followed by precolumn derivtiztion with o-phthldildehyde Ž Sigm, St. Louis, MO.. Identifiction nd quntifiction of mino cids were ccomplished using externl stndrds Ž Sigm.. All sttisticl nlyses were conducted using the Sttisticl Anlysis System ŽSAS Institute, A 4=2 fctoril nlysis of vrince ws conducted to determine the effects of dietry rginine level nd source of blncing nitrogen Ž glutmte or glycine. on WG, FE, PER, PR, plsm-free mino cids nd survivl of chnnel ctfish. The minimum dietry rginine requirement of chnnel ctfish ws determined by subjecting the response vribles to lest-squres regression nlysis using the broken-line method Ž Robbins, Contrst comprisons lso were mde between the diets contining 0.5% rginine nd either glutmte or glycine. A P vlue of less thn 0.05 ws tken to indicte sttisticl significnce. 3. Results Survivl ws high for fish fed ll diets Ž 97% on verge. except for those fed the diet contining 0.5% rginine with glycine s nitrogen source Ž 0.5 Arg Gly. t only 73% Ž Tble 2.. Grded concentrtions of dietry rginine significntly Ž P ffected WG, FE, PER nd PR of juvenile chnnel ctfish, with fish fed both diets contining

5 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture Tble 2 WG, FE, PER, PR nd survivl of chnnel ctfish fed diets contining four rginine levels nd either Gly or Glu to mintin them isonitrogenous Dietry Nitrogen WG Ž% initil FE Žg ginrg feed PER Žg ginrg PR Ž %. Survivl Ž %. rginine source weight. offered. protein fed. Ž % of diet. 0.5 Gly Gly Gly Gly Glu 88 b,y Glu Glu Glu b,x Fctoril ANOVA, Pr ) F c Arg level Nitrogen source Arg=Nitrogen Pooled SEM Mens of three replicte groups. b Contrst comprisons between 0.5 glycine nd 0.5 glutmte yielded significnt Ž P differences for feed efficiency nd survivl s noted by the different superscripts while P vlues for WG, PER nd PR were 0.09, 0.34 nd 0.09, respectively. c Probbility ssocited with the F sttistic. b,x b,y 0.5% rginine hving the lowest responses Ž Tble 2.. WG of fish fed the diet with 0.5 Arg Gly ws bout 66% of tht chieved by fish fed the diet contining 0.5 Arg-Glu. Thus, glutmte in the diet limited the severity of rginine deficiency. FE, PER nd PR of fish fed ech diet contining 0.5% rginine followed pttern similr to WG with fish fed 0.5 Arg Glu hving greter responses compred to those fed 0.5 Arg Gly Ž Tble 2.. Broken-line nlysis of WG nd FE dt from fish fed diets contining glycine s nitrogen source provided rginine requirement estimtes Ž "SE. of 1.0% Ž "0.07. nd 1.2% Ž "0.05. of diet, respectively. These vlues correspond to 4.2% nd 5% of dietry protein. However, using the sme responses from fish fed diets contining glutmte, the broken-line nlysis, fitted by the lest-squres method, yielded rginine requirement estimtes of 0.8 Ž "0.06. nd 0.9 Ž "0.05., which correspond to 3.3% nd 3.8% of dietry protein. Plsm rginine concentrtion in fish ws significntly Ž P incresed 97% nd 55% with n elevtion of dietry rginine from 0.5% to 1.5% of diet in the presence of glycine nd glutmte, respectively Ž Tble 3.. The highest concentrtion of plsm rginine ws found in fish fed the 1.5 Arg Glu diet. The use of glycine or glutmte s the nitrogen source lso yielded significnt Ž P differences in plsm concentrtions of glutmte, glutmine, citrulline, rginine nd ornithine Ž Tble 3.. Plsm glutmte levels did not vry significntly Ž P in fish fed diets contining glycine; however, ner significnt Ž P step-wise reduction in plsm glutmte

6 316 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture Tble 3 Plsm free rginine, citrulline, ornithine, glutmine nd glutmte in juvenile chnnel ctfish fed diets contining grded levels of rginine with either Gly or Glu to mintin them isonitrogenous Dietry rginine Nitrogen source Amino cid Ž mmolrl. Ž % of diet. Glutmte Glutmine Citrulline Arginine Ornithine 0.5 Gly Gly Gly Gly Glu Glu Glu Glu Fctoril ANOVA, Pr ) F b Arg level Nitrogen source Arg=Nitrogen Pooled SEM Vlues re mens of three individul fish from ech of three replicte groups. b Probbility ssocited with the F sttistic. ws observed when glutmte ws decresed to mintin the diets isonitrogenous s rginine ws incresed. 4. Discussion 4.1. Arginine requirement nd glutmte spring WG chieved by chnnel ctfish in this experiment ws similr to tht obtined by Robinson et l. Ž Reltively low-protein diets comprised primrily of crystlline mino cids typiclly support less rpid growth of fish thn diets composed of intct protein do Ž Wilson nd Hlver, Requirement estimtes of 4.2% nd 5.0% of dietry protein for glycine diets in the present study gree with the previously estimted rginine requirement of chnnel ctfish t 4.3% of dietry protein ŽRobinson et l., However, glutmte ppered to spre up to 33% of the rginine requirement in chnnel ctfish nd hd beneficil effects on fish fed the rginine-deficient diet compred to those fed the rginine-deficient diet with glycine. Presumbly, fish fed the rginine-deficient diets hd greter need for rginine, nd glutmte provided n dequte substrte for de novo synthesis of rginine. Retrded growth, high mortlity nd fin erosion hve been reported s signs of rginine deficiency in rinbow trout Ž Ketol, nd chnnel ctfish ŽRobinson et l., Similr conditions were observed in the present experiment only in fish fed the 0.5 Arg Gly diet. The bsence of these nd other clinicl deficiency signs in fish fed the 0.5 Arg Glu diet my hve been due to the use of glutmte for synthesis of ornithine

7 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture nd citrulline before incorportion in rginine, s observed in one other fish species Ž Chiu et l., Although the glycine series diets contined bout 6.9% more totl nitrogen thn the glutmte series, the glutmte-contining diets significntly Ž P out-performed the glycine-contining diets in terms of FE nd survivl s ssessed by the contrst comprison Ž Tble Plsm mino cids Plsm mino cid concentrtions of chnnel ctfish in the present study were comprble to those noted in other experiments with juvenile chnnel ctfish ŽRobinson et l., 1981; Wilson et l., Post-prndil chnges in plsm-free mino cid concentrtions hve been used to investigte mino cid vilbility nd utiliztion in fish species becuse these ptterns reflect the net results of digestion, bsorption nd subsequent utiliztion Ž Wilson nd Hlver, However, the use of plsm-free mino cid responses to confirm dietry requirements ssessed by growth trils hve filed Ž Robinson et l., 1981; Wlton et l., 1986; Cho et l., or succeeded Ž Kushik, 1979; Lll et l., 1994; Berge et l., In the present study, blood collection took plce 14 h fter feeding nd plsm rginine exhibited dose-dependent response. This post-prndil time point ws selected bsed on the time course of plsm mino cid concentrtions of fish fed csein cseinte mixes ŽSchuhmcher et l., Robinson et l. Ž collected blood h fter feeding nd found reltively constnt levels of serum rginine. It lso ws suggested by Robinson et l. Ž tht serum levels of free rginine my hve been dependent not only on dietry rginine concentrtion, but lso on interreltions with other dietry mino cids. Indeed, circulting free rginine levels nd overll rginine homeostsis in nimls is influenced by endogenous synthesis nd degrdtion, intrcellulr protein turnover nd physiologicl stte Ž Wu et l., Plsm-free rginine ws responsive to dietry rginine in the present study by incresing with grded levels of rginine up to 1.5% of diet, then slightly declining t the highest rginine level. Griffin et l. Ž noted similr response while ssessing the dietry rginine requirement of hybrid striped bss. Berge et l. Ž lso noted tht rginine bove 4.0% of dietry protein resulted in reduced Ž from pek vlues. circulting levels of rginine. This trend suggests the presence of homeosttic mechnism by which fish regulte free plsm rginine. The liver would be likely site for this regultory ction due to the presence of reltively high rginse ctivity Ž Anderson, 1995; Berge et l., exerting control on circulting levels of rginine. This heptic regultory effect hs been proposed s mens by which extrheptic tissues of fish cn optimize the utiliztion of dietry mino cids for protein synthesis Ž Wilson et l., Endogenous synthesis of rginine Results from the present study suggest tht diets contining glutmte contribute rginine through de novo synthesis, especilly if dietry rginine is limited. This ws reflected in generl improvement in WG, FE, PER nd PR tht occurred t suboptiml levels of rginine Ž 0.5% of diet. if glutmte ws included in the diet. It is now

8 318 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture generlly ccepted tht endogenous synthesis of rginine in humns, pigs, dogs, sheep nd rts involves the intestinl renl xis in which citrulline relesed by the smll intestine is converted to rginine in the kidney vi rgininosuccinte synthse nd rgininosuccinte lyse Ž Dhnkoti et l., 1990; Wu nd Morris, Moreover, jejunl cells of newborn pigs synthesize rginine from glutmine to compenste for limited rginine present in the sow s milk Ž Blchier et l., 1993; Wu nd Knbe, The enteric section of the intestinl renl xis for de novo rginine synthesis involves the conversion of dietry or circulting glutminerglutmte long the following pthwy: glutmine glutmte glutmyl-g-phosphte glutmyl-g- semildehyde pyrroline-5-crboxylte Ž P5C. ornithine citrulline, ctlyzed by phosphte-dependent glutminse, D-P5C synthse, D-P5C synthse Žbifunctionl enzyme., spontneous rection, ornithine minotrnsferse Ž OAT. nd ornithine crbmoyltrnsferse Ž OCT., respectively Ž Wu et l., In mmmls, these enzymes re found in liver s prt of the ure cycle, but heptic rginse ctivity is so high tht rginine is split into ornithine nd ure, preventing ny net relese of rginine from liver into circultion. Huggins et l. Ž found pprecible levels of ll enzymes of the ure cycle in severl species of teleost fish. Wilson Ž reported perceptible quntities of OCT nd crbmoylphosphte synthse in chnnel ctfish tissues. The fct tht most fish species excrete mmoni s the end product of nitrogen metbolism seems to hve n energetic bsis, rther thn the bsence of required enzymes. Chiu et l Ž reported incorportion of L-w1- Cx ornithine nd L-wcrbmoyl- Cx citrulline into tissue rginine when rinbow trout were injected intrperitonelly. These uthors, however, concluded tht rginine biosynthesis occurred in the trout s liver s prt of the ure cycle. The high rginse ctivity in the trout liver found by Chiu et l. Ž nd by Berge et l. Ž mkes the conclusion of Chiu et l. Ž inconsistent with the current understnding of interorgn Ž intestine kidney. de novo synthesis of rginine. It is well estblished tht the kidney possesses low rginse-to-rginine synthse rtio Ž Fetherston et l., nd rginine synthse Žrgininosuccinte synthse plus rgininosuccinte lyse. is found predominntly in the renl cortex, mking the kidney mjor biosynthetic source of circulting rginine in rts nd other mmmls ŽDhnkoti et l., The full complement of enzymes of the ure cycle hve been shown to exist in number of typicl teleost species ŽHuggins et l., 1969; Red, 1971; Wilson, 1973; Chiu et l., 1986; Mommsen nd Wlsh, 1989, 1991; Anderson, Tken together, these reports suggest the presence of ll enzymes required for synthesizing rginine from glutmte in fish tissues Ž Wkbyshi et l., However, ssessment of the enzymtic ctivities of the intestinl renl xis in fish wrrnts further reserch. Interestingly, proline lso my be quntittively importnt source of citrulline nd rginine on the bsis of recent findings tht the -mino group nd the crbon skeletons of ornithine, citrulline nd rginine cn come from proline rther thn from glutmine, glutmte or sprtte Ž Wu, In the present study, plsm-free glutmte, glutmine, citrulline nd ornithine in fish fed the Arg Gly diets did not show significnt chnges s rginine ws incresed in diet Ž nd glycine ws decresed.. This suggests tht glycine does not hve n importnt physiologicl role in the metbolism of these mino cids. In contrst, glutmine

9 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture concentrtion in plsm ws significntly Ž P higher in fish fed diets contining glutmte compred to glycine. This elevtion Ž 71.2% on verge. of plsm glutmine could be explined by n up-regulted conversion of glutmte into glutmine by cytosolic glutmine synthse s observed in perivenous heptocytes ŽHussinger, or by reduced muscle-relesed glutmine utiliztion s substrte for citrulline, ornithine nd rginine synthesis Ž Wu nd Knbe, Dt on circulting plsm glutmte, glutmine, citrulline nd ornithine indicte trends ŽP- 0.06, 0.09, 0.08 nd 0.08, respectively. of n incresed citrulline production in fish fed diets with glutmte, suggesting up-regultion of the rections glutmte P5C OAT ornithine citrulline, in chnnel ctfish fed diets with glutmte. The elevted circulting citrulline could be used for endogenous synthesis of rginine, possibly t the kidney, s in dult rts Ž Dhnkoti et l., The extent to which enteric citrulline is funneled into renl tissue Ž or elsewhere. or used for enterocyte conversion into rginine, for locl use, remins to be elucidted. Becuse most of the structurl fetures nd physiologicl functions of the mmmlin kidney re llocted to both the trunk kidney nd hed kidney in fish Ž Bond, 1979., the site of rginine biosynthesis needs to be further investigted in vrious fish species. Acknowledgements This study ws supported in prt by the Texs Agriculturl Experiment Sttion. The uthors wish to thnk Nutri-Quest Ž Chesterfield, MO. for providing mino cids for the diets. Funding for Mr. Buentello ws provided in prt by the Consejo Ncionl pr l Cienci y l Tecnologı Ž CONACYT-Mexico.. References Anderson, P.M., Ure cycle in fish: moleculr nd mitochondril studies. In: Cellulr nd Moleculr Approches to Fish Ionic Regultion. Acdemic Press, New York, pp Assocition of Officil Anlyticl Chemists Ž AOAC., Officil Methods of Anlysis. 16th edn. AOAC, Arlington, VA. Bker, D., Utiliztion of precursors for L-mino cids. In: D Mello, J.P.F. Ž Ed.., Amino Acids in Frm Animl Nutrition. CAB Interntionl, Wllingford, UK, pp Berge, G.E., Lied, E., Sveier, H., Nutrition of Atlntic slmon Ž Slmo slr.. The requirement nd metbolism of rginine. Comp. Biochem. Physiol., Prt A 117, Blchier, F., M Rbet-Touil, H., Posho, L., Drcy-Vrillon, B., Duee, P.H., Intestinl rginine metbolism during development. Evidence for the de novo synthesis of L-rginine in newborn pig enterocytes. Eur. J. Biochem. 216, Bond, C.E., In: Biology of Fishes. Sunders College Publishing, USA, pp Buentello, J.A., Gtlin, D.M. III, Dle, B.E., Evlution of costl Bermud grss protein isolte s substitute for fishmel in prcticl diets for chnnel ctfish Ictlurus puncttus. J. World Aqucult. Soc. 28, Chiu, Y.N., Austic, R.E., Rumsey, G.L., Ure cycle ctivity nd rginine formtion in rinbow trout Ž Slmo girdneri.. J. Nutr. 116,

10 320 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture Cho, C.Y., Kushik, S., Woodwrd, B., Dietry rginine requirement of young rinbow trout Ž Oncorhynchus mykiss.. Comp. Biochem. Physiol., Prt A 102, Dhnkoti, S.N., Brosnn, J.T., Herzberg, G.R., Brosnn, M.E., Renl rginine synthesis: studies in vitro nd in vivo. Am. J. Physiol. 259, E437 E442, Endocrinl. Metb. 22. Fetherston, W.R., Rogers, Q.R., Freelnd, R.A., Reltive importnce of kidney nd liver in synthesis of rginine by the rt. Am. J. Physiol. 224, Fuller, M.F., Amino cid requirements for mintennce, body protein ccretion nd reproduction in pigs. In: D Mello, J.P.F. Ž Ed.., Amino Acids in Frm Animl Nutrition. CAB Interntionl, Wllingford, UK, pp Griffin, M.E., Wilson, K.A., Brown, P.B., Dietry rginine requirement of juvenile hybrid striped bss. J. Nutr. 124, Hlver, J.E., In: Fish Nutrition. 2nd edn. Acdemic Press, New York, p Hussinger, D., Nitrogen metbolism in liver: structurl nd functionl orgniztion nd physiologicl relevnce. Biochem. J. 267, Huggins, A.K., Skutch, G., Bldwin, E., Ornithine-ure cycle enzymes in teleosten fish. Comp. Biochem. Physiol. 28, Kushik, S., Appliction of biochemicl method for the estimtion of mino cid needs in fish: quntittive rginine requirements of rinbow trout in different slinities. In: Hlver, J.E., Tiews, K. Ž Eds.., Finfish Nutrition nd Fishfeed Technology vol. 1 Heenemnn Verlgsgesellschft, Berlin, pp Ketol, H.G., Requirement for dietry lysine nd rginine by fry of rinbow trout. J. Anim. Sci. 56, Lll, S.P., Kushik, S.J., Le Bil, P.Y., Keith, R., Anderson, J.S., Plisetsky, E., Quntittive rginine requirement of Atlntic slmon Ž Slmo slr. rered in se wter. Aquculture 124, Lee, J.S., Commercil ctfish frming. In: 3rd edn. Interstte Publishers, IL, USA, p Mommsen, T.P., Wlsh, P.J., Evolution of ure synthesis in vertebrtes: the piscine connection. Science 243, Mommsen, T.P., Wlsh, P.J, Ure synthesis in fish: evolutionry nd biochemicl perspectives. In: Hochchk, P.W., Mommsen, T.P. Ž Eds.., Biochemistry nd Moleculr Biology of Fishes vol. 1 Elsevier, Amsterdm, pp Ntionl Reserch Council Ž NRC., Nutrient Requirements of Fish. Ntionl Acdemy Press, Wshington, DC. Red, L.J., The presence of high ornithine-ure cycle enzyme ctivity in the teleost Opsnus tu. Comp. Biochem. Physiol., Prt B 39, Robbins, K.R., A method, SAS progrm nd exmple for fitting the broken-line to growth dt. In: University of Tennessee Agriculture Experiment Sttion Reserch Report. University of Tennessee, Knoxville, TN, pp Robinson, H.E., Wilson, R.P., Poe, W.E., Arginine requirement nd pprent bsence of lysine rginine ntgonist in fingerling ctfish. J. Nutr. 111, SAS Institute, SAS User s Guide: Sttistics. 5th edn. SAS Institute, Cry, NC. Schuhmcher, A., Schon, J., Goldberg, M., Gropp, J.M., Plsm mino cid levels in rinbow trout Ž Oncorhynchus mykiss.. J. Appl. Ichthyol. 11, Wkbyshi, Y., Ymd, E., Hsegw, T., Ymd, R., Enzymologicl evidence for the indispensbility of smll intestine in the synthesis of rginine from glutmte: I. Pyrroline-5-crboxylte synthse. Arch. Biochem. Biophys. 291, 1 8. Wlton, M.J., Adron, J.W., Coloso, R.M., Cowey, C.B., Dietry requirements of rinbow trout for tryptophn, lysine nd rginine s determined by growth nd biochemicl mesurements. Fish Physiol. Biochem. 2, Wilson, R.P., Nitrogen metbolism in chnnel ctfish Ictlurus puncttus: II. Evidence for n pprent incomplete ornithine-ure cycle. Comp. Biochem. Physiol., Prt B 46, Wilson, R.P., Gtlin, D.M. III, Poe, W.E., Postprndil chnges in serum mino cids of chnnel ctfish fed diets contining different levels of protein nd energy. Aquculture 49, Wilson, R.P., Hlver, J.E., Protein nd mino cid requirements of fishes. Annu. Rev. Nutr. 6, Wu, G., Synthesis of citrulline nd rginine from proline in enterocytes of postntl pigs. Am. J. Physiol. 272, G1382 G1390, Gstrointest. Liver Physiol. 35.

11 ( ) J.A. Buentello, D.M. Gtlin IIIrAquculture Wu, G., Dvis, P.K., Flynn, N.E., Knbe, D.A., Dvidson, J.T., Endogenous synthesis of rginine plys n importnt role in mintining rginine homeostsis in postwening growing pigs. J. Nutr. 127, Wu, G., Knbe, D.A., Arginine synthesis in enterocytes of neontl pigs. Am. J. Physiol. 269, R621 R629. Wu, G., Morris, S.M., Arginine metbolism: nitric oxide nd beyond. Biochem. J. 336, 1 17.