Asian-Aust. J. Anim. Sci. Vol. 24, No. 7 : July doi: /ajas

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1 974 Asin-Aust. J. Anim. Sci. Vol. 24, No. 7 : July doi: /js Re-evlution of Dietry Methionine Requirement y Plsm Methionine nd Ammoni Concentrtions in Surgiclly Modified Rinow Trout, Oncorhynchus mykiss Jun-Young Be, Im-Ho Ok, Seunghyung Lee, Sils S. O. Hung 1, Te Sun Min 2 nd Sungchul C. Bi* Deprtment of Mrine Bio-mterils nd Aquculture / Feeds nd Foods Nutrition Reserch Center, Pukyong Ntionl University, Busn , Kore ABSTRACT : This study ws designed to re-evlute the dietry methionine requirement y mens of the plsm methionine nd mmoni concentrtions in surgiclly modified rinow trout, Oncorhynchus mykiss. A totl of 35 rinow trout verging 505±6.5 g (initil ody weight, men±sd) were rndomly distriuted into seven groups with five fish in ech group. After 48 h of feed deprivtion, ech group of fish ws fed one of seven L-mino cid sed diets contining 0.5% cystine nd grded levels of methionine (0.25, 0.40, 0.50, 0.60, 0.70, 0.80 or 0.95% of diet, dry mtter ses) y intution t 1% ody weight on dry mtter sis. Blood smples were tken t 0, 5 nd 24 h fter intution. Post-prndil plsm free methionine concentrtions (PPmet, 5 h fter intution) nd postsorptive plsm free methionine concentrtions (PAmet, 24 h fter intution) of fish fed diets contining 0.60% or higher methionine were significntly (p<0.05) higher thn those of fish fed diets contining 0.50% or lower methionine. PPmet nd PAmet in fish fed diets contining 0.60% or higher methionine were not significntly different except PPmet of fish fed diet contining 0.95% methionine. Postprndil plsm mmoni concentrtions (PPA, 5 h fter intution) of fish fed diets contining 0.70% or higher methionine were significntly higher thn those of fish fed diets contining 0.60% or lower methionine, nd PPA of fish fed diets contining 0.25 nd up to 0.60% methionine were not significntly different from ech other. Broken-line model nlyses on PPmet, PAmet, nd PPA indicted tht the dietry methionine requirement of rinow trout ws etween 0.59 (1.69) nd 0.67 (1.91) % of diets (% dietry protein ses) when the diets contined 0.5% cystine. (Key Words : Methionine, Rinow trout, Requirement, Intution, Plsm, Ammoni) INTRODUCTION Methionine is sulfur contining nd n essentil mino cid (EAA) required y fish s well s terrestril vertertes for norml growth nd metolic functions. This sulfur contining EAA, in the form of S-denosyl-methionine serves s principl donor of methyl group, mjor contriutor to the whole ody pool of one cron units tht re required for trns-methyltions, the iosynthesis of choline, thymidine, polymines nd cretine (Alm et l., 2000; Mi et l., 2006). Methionine deficiency resulted in reduced growth nd feed efficiency, s well s ctrct in * Corresponding Author : Sungchul C. Bi. Tel: , Fx: , E-mil: sci@pknu.c.kr 1 Deprtment of Animl Science, University of Cliforni, One Shields Avenue, Dvis, CA , USA. 2 Ntionl Reserch Foundtion of Kore (NRF), Dejeon Kore. Received My 17, 2010; Accepted April 6, 2011 rinow trout (Poston et l., 1977; Wlton et l., 1982) nd hyrid striped ss (Keemiyehetty nd Gtlin, 1993). Therefore it is very importnt to determine the methionine requirement of cultured fish for norml growth nd feed utiliztion. Dietry methionine requirements hve een estimted rnging from 1.8 to 4.0% of dietry protein for commonly cultured species of fish (Wilson, 2002). Severl reserchers hve reported the quntittive methionine requirements of rinow trout rnging etween 0.5 (1.0) % nd 0.8 (2.35) % of diets (% of dietry protein ses) (Wlton et l., 1982; Rumsey et l., 1983; Cowey et l., 1992; Kim et l., 1992; Rodehutscord et l., 1995). The ove requirements were determined y feeding trils. Feeding trils re expensive nd lst for severl weeks or months efore pprecile responses re oserved. Determintion of mino cid requirements y mesuring plsm mino cid nd mmoni concentrtions on the other hnd lsts for just week, with insignificnt lor nd mteril costs. This method hs een employed in severl

2 Be et l. (2011) Asin-Aust. J. Anim. Sci. 24(7): species nd results re similr to those otined y feeding trils (Cowey, 1995; Ok et l., 2002). Hence, it is dvisle tht this procedure e confirmed in importnt species so tht they could e employed in determintion of yet undetermined mino cid or other nutrient requirements in those species. Plsm free mino cids (PFAAs) concentrtions were used to investigte mino cids metolism nd to evlute the qulity of dietry protein in rinow trout (Nose, 1972; Wlton nd Wilson, 1986; Schuhmcher et l., 1997), common crp, Cyprinus crpio (Plks et l., 1980; Drowski, 1982) nd Atlntic slmon, slr (Espe et l., 1993; Sunde et l., 2003). Evlution of PFAAs concentrtions hs led to the discovery of genetic defects of mino cid metolism s result of primry renl or liver disese nd the effects of mino cid deficiencies, imlnces nd toxicities on mino cid metolism (Zicker nd Rogers, 1990). The reltionships etween the concentrtion of PFAAs nd intke of dietry mino cids hve een investigted y severl reserchers (Plks et l., 1980; Muri et l., 1987; Bi et l., 2003). Although the effects of dietry protein or mino cid mixtures on PFAAs concentrtions in rinow trout (Muri et l., 1987; Schuhmcher et l., 1997; Vermeirssen et l., 1997) nd se ss, Dicentrrchus lrx (Theult, 1985) hve een reported, the complete dose response reltionships for mino cids hve not een investigted. In our previous studies, dietry essentil mino cids such s rginine, lysine, vline, nd threonine requirements for rinow trout were otined y using the surgiclly modified methods in our lortory (Ok et l., 2001; Ok, 2002; Bi et l., 2003; Prk et l., 2005). As prt of our studies on essentil mino cid requirements of rinow trout, the purpose of the present study ws to estimte the dietry methionine requirement sed on the plsm free methionine nd mmoni concentrtions in dorsl ort cnnulted rinow trout. MATERIALS AND METHODS Experimentl fish Rinow trout, O. mykiss verging 505±6.5 g (initil, men±sd) were otined from Ewhjung trout frm in Sngju, Kore. In the present experiment, seven net cges ( m) were plced in flow-through concrete rcewy with wter flow of 60 L min -1. Fish were kept in the net cges nd fed commercil rinow trout diet (Woo-sung Feed Co. Ltd., Dejon, Kore) for 72 h until the fish recovered from the opertion of dorsl ort cnnultion. Supplementl ertion ws lso provided to mintin the dissolved oxygen t 6.7±0.5 mg L -1, nd wter temperture ws mintined t 15±0.3 C. Experimentl diets A sl diet ws formulted y modifiction of Kim (1997) nd it contined 36.6% crude protein (29.6% crystlline mino cids mixture, 5% csein nd 2% geltin). Seven experimentl diets were formulted sed on the sl diet nd they contined grded levels of methionine (0.25, 0.40, 0.50, 0.60, 0.70, 0.80 or 0.95% of diet, on dry mtter se) nd ll diets contined 0.50% cystine. Equl mount of sprtic cid nd glutmic cid y weight were sustituted for the grded mounts of methionine in the sl diet to mintin the isonitrogenous of the seven experimentl diets. Formultion nd mino cid composition of the sl diet re shown in Tle 1 nd 2, respectively. The ingredient mixtures without oil were stored t -80 C until used nd the diets were prepred y dding fish oil (10% of diets) nd wter (4 prts of distilled wter: 10 prts of diet, w/w) efore intution. Dorsl ort cnnultion nd intution (force-feeding) Rinow trout were nesthetized with 200 mg L -1 3-minoenzoic cid ethyl ester methnsulfonte (MS-222, Sigm Chemicl Compny, St. Louis, MO) for 3 to 5 min, plced on V-shpe tle, nd gills were irrigted continuously with 16 C wter contining 100 mg L -1 of MS-222 during the opertion. A 50 cm-long cnnul (Cly Tle 1. Composition of the sl diet 1 Ingredients % of dry mtter EAA NEAA Csein Geltine Dextrin Dextrose α-cellulose Fish oil Other ingredients L-methionine Asprtic cid Glutmic cid Diets were neutrlized with NOH to give finl ph of EAA, essentil mino cids (g/100 g diet): rginine, 1.924; histidine, 0.725; isoleucine, 1.674; leucine, 2.702; lysine, 1.904; cystine, 0.481; phenyllnine, 1.742; tyrosine, 1.335; threonine, 1.601; tryptophn,0.462; vline, (Ajinomoto, Tokyo, Jpn). 3 NEEA, Non-essentil mino cids (g/100 g diet): lnine, 1.741; glycine, 0.758; proline, 0.568; serine, (Ajinomoto, Tokyo, Jpn). 4 United Sttes Biochemicl (USB), Clevelnd, Ohio, USA. 5 Ewh Oil Compny, Busn, Kore. 6 Other ingredients include: croxymethyl cellulose, 1.00% (United Sttes Biochemicl, Clevelnd, Ohio, USA); C(H 2 PO 4 ) 2 H 2 O, 3.00%; choline itrtrte, 1.20% (United Sttes Biochemicl, Clevelnd, Ohio, USA), vitmin mixture, 3.00% nd minerl mixture, 4.00% (Prk et l., 2005). 7 Ajinomoto, Tokyo, Jpn.

3 976 Be et l. (2011) Asin-Aust. J. Anim. Sci. 24(7): Tle 2. Amino cid composition of the sl diet (% of dry mtter se) Amino cids Essentil mino cids From csein nd geltin From crystlline mino cids Totl 1 Arginine Histidine Isoleucine Leucine Lysine Methionine Cystine Phenyllnine Tyrosine Threonine Tryptophn Vline Non-essentil mino cids Alnine Asprtic cid Glycine Glutmic cid Proline Serine The mino cid profile ws simulted with tht of 35% whole chicken egg protein (Roinson et l., 1981). 2 Seven experimentl diets were formulted to hve grded levels of methionine (0.25, 0.40, 0.50, 0.60, 0.70, 0.80 or 0.95%); equl mount of sprtic cid nd glutmic cid y weight were sustituted for the proper mounts of methionine in the sl diet. Adms PE 50 tuing, Prsippny, NJ, USA) with ule of out 5-6 cm from one end ws wshed with the heprinized Cortlnd sline solution (Houston, 1990), nd 13-guge needle ws used to pierce hole on the right nostrum (ventrl side up) for the cnnul to exit. A 19-guge needle ws used to ore smll hole in the roof of the mouth t the mid-line ehind the third gill rc t 30 ngle, nd pino wire ws inserted into the PE 50 tuing s guide. The proper insertion ws verified y the oservtion of slow lood flow fter the wire ws withdrwn from the cnnul. A 3 ml syringe with 23- guge needle ws used to remove ir nd lood clot nd the cnnul ws flushed with the heprinized sline solution. The cnnul ws sutured ehind the ule on the roof of the mouth, led out from the right nostrum, plugged with color hed pin, nd sutured t the dorsl fin (Ok et l., 2001; Bi et l., 2003; Prk et l., 2005). Thirty five dorsl ort cnnulted rinow trout were rndomly distriuted into seven groups with five fish per group nd they were fed one of the seven experimentl diets per group y intution t 1% ody weight on dry mtter sis. After 24 h feed deprivtion, these fish were nesthetized with 200 mg L -1 of MS-222 nd fed the experimentl diet y the stomch intution method (diet plus 0.4 prts of distilled wter per diet) using 3 ml syringe. Smple collection nd nlysis Five fish per group were nesthetized with 200 mg L -1 MS-222 nd lood ws smpled using 3 ml syringe from ech fish t 0, 5 nd 24 h fter intution of the experimentl diets. Plsm smples were prepred y centrifugtion t 3,000 g for 10 min t room temperture. For deproteiniztion, plsm smples were mixed with 10% 5-sulphoslicylic cid solution in the rtio of 4:1 (v/v), cooled on ice for 30 min nd re-centrifuged. The proteinfree superntnt ws dissolved in ph 2.2 lithium citrte smple dilution uffer in the rtio of 1:1 (v/v), nd the smples were stored t -80 C until nlysis. Plsm free mino cids were quntified using S433 mino cid nlyzer (Sykm, Gilching, Germny) using the ninhydrin method. Plsm mmoni concentrtions were nlyzed using the Berthelot rection (Sigm). Sttisticl nlysis Dt were sujected to one wy nlysis of vrince test using Sttistix 3.1 (Anlyticl Softwre, St Pul, MN, USA). When significnt tretment effect ws oserved, Lest Significnt Difference test ws used to compre mens. Tretment effects were considered significnt t p<0.05. The rekpoints for PPmet, PAmet, nd PPA were estimted y using the roken line model of Roins et l. (1979). RESULTS Plsm free methionine concentrtions Results of post-prndil plsm free methionine concentrtions (PPmet, 5 h fter intution) nd postsorptive plsm free methionine concentrtions (PAmet, 24 h fter intution) in dorsl ort cnnulted rinow trout force-fed with diets contining seven grded levels of methionine re shown in Figure 1. PPmet of fish fed diets contining 0.60% or higher methionine were significntly (p<0.05) higher thn those of fish fed diets contining 0.50% or lower methionine. PPmet of fish fed diet contining 0.95% methionine ws significntly higher thn those of fish fed the other diets (p<0.05). PPmet of fish fed diets contining 0.60, 0.70 nd 0.80 (%) methionine were not significntly (p>0.05) different from ech other. Broken-line model nlyses on the sis of PPmet indicted tht the dietry methionine requirements of rinow trout were 0.60 (1.71) % of diets (% of dietry protein on dry mtter se) (Figure 1). PAmet of fish fed diets contining 0.60% or higher methionine were significntly (p<0.05) higher thn those of fish fed diets contining 0.50%

4 Be et l. (2011) Asin-Aust. J. Anim. Sci. 24(7): Post prndil plsm free methionine concentrtions (nmol ml -1 ) e d c y = x y = r 2 = % Dietry methionine level (%) Figure 1. Broken line nlysis of post-prndil plsm methionine concentrtions (nmol ml -1 ) in rinow trout fed grded levels of dietry methionine. Vlues of the X-xis re the methionine levels in the experimentl diets. Vlues of the Y-xis re post-prndil plsm free methionine concentrtions. Vlues re mens±sd of 5 replictes. methionine. PAmet of fish fed diets contining 0.60% or higher methionine in the diets were not significntly (p>0.05) different from ech other. Broken-line model nlyses on the sis of PAmet indicted tht the dietry methionine requirements of trout could e 0.67 (1.91) % of diets (% of dietry protein on dry mtter se) (Figure 2). Plsm mmoni concentrtions Post-prndil plsm mmoni concentrtions (PPA, 5 h fter intution) nd post-sorptive plsm mmoni concentrtions (PAA, 24 h fter intution) in rinow trout force-fed grded levels of dietry methionine re shown in Figure 3. Post-prndil plsm mmoni concentrtion from fish fed diets contining methionine from 0.25% (168±5 nmol ml -1 ) to 0.60% (156±12 nmol ml -1 ) were not significntly different from ech other. However, PPA were significntly incresed with dietry methionine levels from 0.70 (195±8 nmol ml -1 ) to 0.95% (251±11 nmol ml -1 ). PAA in the present study were not significntly (p>0.05) different mong the groups. Broken-line model nlyses on the sis of PPA indicted tht the dietry methionine requirement of rinow trout could e 0.59 (1.69) % of diets (% of dietry protein on dry mtter se) (Figure 3). DISCUSSION In the present study, the dietry methionine requirements for the rinow trout sed on the plsm free methionine concentrtions (PPmet nd PAmet) could e etween 0.60 (1.71) nd 0.67 (1.91) % of diets (% of dietry protein ses) if the diet contined 0.5% cystine. In the previous studies, severl reserchers hve reported the quntittive methionine requirements of rinow trout with rnge etween 0.50 (1.00) % nd 0.80 (2.35) % of diets (% of dietry protein ses) (Wlton et l., 1982; Rumsey et l., 1983; Cowey et l., 1992; Kim et l., 1992; Rodehutscord et l., 1995). These results re similr to those in other fresh wter fish species, such s chnnel ctfish, Ictlurus puncttus (0.56%, Hrding et l., 1977), Nile tilpi, Oreochromis niloticus (0.75%, Sntigo nd Lovell, 1988), nd common crp, C. crpio (0.86% of diet, Schwrz et l., 1998). Furthermore, the methionine requirements of rinow trout ws lower thn those of mjor mrine finfish species, such s hyrid striped ss, Morone chrysops M. sxtilis (1.00%, Keemiyehetty nd Gtlin, 1993), red drum, Scienops ocelltus (1.06%, Moon nd Gtlin, 1991), yellowtil, Seriol quinquerdit (1.11%, Ruchimt et l., 1997), grouper, Epinephelus coioides (1.31%, Luo et l., 2005), coi, Rchycentron cndum (1.19%, Zhou et l., 2006), nd yellow croker, Pseudoscien croce (1.44%, Mi et l., 2006). The vritions of methionine requirement my hve resulted from fish size nd ge, feed ingredients, pltility of diets, feeding regime, nd culture conditions mong the different experiments (Tcon nd Cowey, 1985; Rodehutscord et l., 1997).

5 978 Be et l. (2011) Asin-Aust. J. Anim. Sci. 24(7): Post sorptive plsm free methionine concentrtions (nmol ml -1 ) c y = x y = r 2 = % Dietry methionine level (%) Figure 2. Broken line nlysis of post-sorptive plsm methionine concentrtions (nmol ml -1 ) in rinow trout fed grded levels of dietry methionine. Vlues of the X-xis re the methionine levels in the experimentl diets. Vlues of the Y-xis re post-sorptive plsm free methionine concentrtions. Vlues re mens±sd of 5 replictes. 350 Post sorptive plsm free methionine concentrtions (nmol ml -1 ) c y = x y = r 2 = % Dietry methionine level (%) Figure 3. Broken line nlysis of post-prndil plsm mmoni concentrtions (nmol ml -1 ) in rinow trout fed grded levels of dietry methionine. Vlues of the X-xis re the methionine levels in the experimentl diets. Vlues of the Y-xis re post-prndil plsm mmoni concentrtions. Vlues re mens±sd of 5 replictes.

6 Be et l. (2011) Asin-Aust. J. Anim. Sci. 24(7): The effects of dietry methionine intke on PPmet nd PAmet were dependent upon the reltive dequcy of the dietry methionine supply. Similr results hve shown tht dietry methionine levels ffected plsm free mino cids in severl fish species such s se ss, Dicentrrchus lrx (Hidlgo et l., 1987), common crp, C. crpio (Schwrz et l., 1998), nd yellow perch, Perc flvescens (Twiell et l., 2000). Muri et l. (1987) nd Schuhmcher et l. (1997) reported tht the plsm concentrtions of rginine, leucine, isoleucine, vline, phenyllnine nd threonine in rinow trout force-fed crystlline mino cids t 1% ody weight (dry mtter) peked t 6-9 h nd returned to the sl level t h post feeding. Ok et l. (2001) demonstrted tht most of the plsm free mino cids peked pproximtely 4 h fter feeding, nd returned to ner sl level in 24 h in rinow trout tht force-fed the experimentl diets fter. In chicks, Zimmermn nd Scott (1967) found tht lysine, vline, nd rginine concentrtions in the plsm incresed in dose response mnner when the intke of the dietry mino cid just exceeds the level required for mximum growth. In the present study, PPmet nd PAmet incresed with dietry methionine incresed from 0.25% to 0.60% of diets (dry mtter se) nd they only incresed slightly therefter. In nother words, PPmet nd PAmet incresed with incresing dietry methionine when their level is elow requirement. Their concentrtions remined constnt when dietry methionine is higher thn requirement vlues. Post-prndil plsm mmoni concentrtions were ffected y dietry methionine levels wheres postsorptive plsm mmoni concentrtions were not. Similr oservtions were reported tht PPA ws significntly ffected y dietry rginine, vline, lysine, nd threonine level in rinow trout (Ok, 2002). This hs lso een reported in studies of methionine requirements in rinow trout (Kim et l., 1992) nd yellowtil (Ruchimt et l., 1997). Much of the excess mino cids eyond the requirement were tken up y the liver nd degrded. The liver of fish is the min site of mmoni production nd where most enzymes of mino cid ctolism re concentrted (Wlton nd Wilson, 1986). Judging from lrge increse of mmoni excretion in rinow trout fter eing fed high protein diet, Brett nd Zl (1975) suggested tht the excess dietry mino cid ws rpidly ctolized y the liver. The ptterns of most plsm free mino cid concentrtions from force-fed rinow trout with nd without dorsl ort cnnultion were similr, nd the dorsl ort cnnultion did not ffect the pttern of plsm free mino cid concentrtions in rinow trout (Ok et l., 2001). These results indicte tht force-fed rinow trout might recover from the stress of dorsl ort cnnultion within 48 h of the opertion, nd the dorsl ort cnnultion would llow repeted smpling on the sme individul fish to study nutrient metolism in the lood circultion. Plsm mino cid concentrtions my e useful in determining the optimum lood smpling time nd evluting protein qulity nd essentil mino cid requirements. Furthermore, this technique cn e used in phrmceuticl nd toxicologicl, s well s in nutritionl reserch in rinow trout. Since results in this nd other studies involving mesurement of plsm free mino cids nd mmoni re similr to vlues otined y feeding tril, it could e suggested tht this technique could e employed in estimting other mino cid requirements in rinow trout. In conclusion, quntittive estimtion of the methionine requirement using the response of roken line model with plsm free methionine or mmoni concentrtions should e n cceptle method for determining essentil mino cid requirements of other fish species. Broken-line model nlyses on the sis of PPmet, PAmet, nd PPA concentrtions indicted tht the dietry methionine requirements of rinow trout could e etween 0.59 (1.69) % nd 0.67 (1.91) % of diets (dietry protein) when the diet contined 0.5% cystine. ACKNOWLEDGEMENTS This reserch ws supported in prt y the funds of the Feeds nd Foods Nutrition Reserch Center, Kore Se Grnt Progrm nd Brin Kore 21 Progrm t Pukyoung Ntionl University, Busn, Kore. REFERENCES Alm, M. D., S. I. Teshim, M. Ishikw nd S. Koshio Methionine requirement of juvenile Jpnese flounder, Prlichthys olivceus. J. World Aquc. Soc. 31: Bi, S. C., I. H. Ok, G. J. Prk, K. W. Kim nd S. M. Choi Development of modeling system for ssessing essentil mino cid requirements using surgiclly modified rinow trout. J. Aqucult. 16(1):1-7. Brett, J. R. nd C. A. Zl Dily pttern of excretion nd consumption of sockeye slmon under controlled conditions. J. Fish. Res. Bord Cn. 33: Cowey, C. B Protein nd mino cid requirements: critique of methods. J. 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Tn nd D. H. Liu Dietry l-methionine requirement of juvenile grouper, Epinephelus coioides t constnt dietry cystine level. Aquculture 249: Mi, K., J. Wn, Q. Ai, W. Xu, Z. Liufu, L. Zhng, C. Zhng nd H. Li Dietry methionine requirement of lrge yellow croker, Pseudoscien croce. Aquculture 253: Moon, H. Y. nd D. M. Gtlin III Totl sulfur mino cid requirement of juvenile red drum, Scienops ocelltus. Aquculture 95: Muri, T., H. Ogt, Y. Hirsw, T. Akiym nd T. Nose Portl sorption nd heptic uptke of mino cids in rinow trout force fed complete diets contining csein or crystlline mino cids. Nippon Suisn Gkkishi 53(10): Nose, T Chnges in ptterns of free plsm mino cids in rinow trout fter feeding. Bull. Fresh. Fish. Res. L. Tokyo 22: Ok, I. H., S. C. Bi, G. J. Prk, S.M. Choi nd K. W. Kim The ptterns of plsm free mino cids fter force-feeding in rinow trout, Oncorhynchus mykiss with nd without dorsl ort cnnultion. Aquc. Res. 32: Ok, I. H Determintion of essentil mino cid requirements y using plsm free mino cid concentrtions in rinow trout, Oncorhynchus mykiss. Ph.D. Thesis, Pukyong Ntionl University, Busn, Kore. Prk, G. J., S. C. Bi, I. H. Ok, K. M. Hn, S. O. Hung, Q. R. Rogers nd T. S. Min Post prndil plsm free rginine concentrtions increse in rinow trout fed rginine deficient diets. Asin-Aust. J. Anim. Sci. 18(3): Plks, S. M., T. Ktym, Y. Tnk nd O. Deshimru Chnges in the levels of circulting plsm free mino cids of crp, Cyprinus crpio fter feeding protein nd n mino cid diet of similr composition. Aquculture 21: Poston, H. A., R. C. Riis, G. L. Rumsey nd H. G. Ketol The effect of supplementl dietry mino cids, minerls nd vitmins on slmonids fed ctrctogenic diets. Cornell Vet. 67: Roins, K. R., H. W. Norton nd D. H. Bker Estimtion of nutrient requirements from growth dt. J. Nutr. 109: Roinson, E. H., R. P. Wilson nd W. E. Poe Arginine requirement nd pprent sence of lysine rginine ntgonist in fingerling chnnel ctfish. J. Nutr. 111: Rodehutscord, M., J. Stephn, P. Michel nd P. Ernst Growing from 50 to 150 g to supplements of DL-methionine in semipurified diet contining low or high levels of cystine. J. Nutr. 125: Rodehutscord, M., A. Becker, M. Pck nd E. Pfeffer Response of rinow trout, Oncorhynchus mykiss to supplements of individul essentil mino cids in semipurified diet, including n estimte of the mintennce requirement of essentil mino cids. J. Nutr. 126: Ruchimt, T., T. Msumoto, H. Hosokw nd S. Shimeno Quntittive methionine requirement of yellowtil, Seriol quinquerdit. Aquculture 150: Rumsey, G. L., J. W. Pge nd M. L. Scott Methionine nd cystine requirements of rinow trout. Progressive Fish- Culturist 45: Sntigo, C. B. nd R. T. Lovell Amino cid requirements for growth of Nile tilpi. J. Nutr. 118: Schuhmcher, A., C. Wx nd J. M. Gropp Plsm mino cids in rinow trout, Oncorhynchus mykiss fed intct protein or crystlline mino cid. Aquculture 151: Schwrz, E. J., M. Kirchgessner nd U. Deuringer Studies on the methionine requirement of crp, Cyprinus crpio. Aquculture 161: Sunde, J., A. Kiessling, D. Higgs, J. Opstvedt, G. Venturini nd K. R. Torrissen Evlution of feed protein qulity y mesuring plsm free mino cids in Atlntic slmon, Slmo slr fter dorsl ort cnnultion. Aqucult. Nutr. 9: Tcon, A. G. nd C. B. Cowey Protein nd mino cid requirements. In: Fish Energetics nd New Perspectives (Ed. P. Tytler nd P. Clow). Croom-Helm, London. pp Theult, H Plsm essentil mino cid chnges in se ss, Dicentrrchus lrx fter feeding diets deficient nd supplemented in L-Met. Comp. Biochem. Physiol. 82A: Twiell, R. G., K. A. Wilson nd P. B. Brown Dietry sulfur mino cid requirement of juvenile yellow perch fed the mximum cystine replcement vlue for methionine. J. Nutr. 130: Vermeirssen, E. L. M., A. P. Scott nd N. R. Liley Femle rinow trout urine contins pheromone which cuses rpid rise in plsm 17α, 20β-dihydroxy-4-pregnen-3-one levels nd milt mounts in mles. J. Fish Biol. 50: Wlton, M. J., C. B. Cowey nd J. W. Adron Methionine metolism in rinow trout fed diets of differing methionine nd cystine content. J. Nutr. 112: Wlton, M. J. nd R. Wilson Postprndil chnges in plsm nd liver free mino cids of rinow trout fed complete diets contining csein. Aquculture 51: Wilson, R. P Amino cids nd proteins. In: Fish Nutrition, 3rd Ed. (Ed. J. E. Hlver nd R. W. Hrdy). Acdemic Press, Inc., New York, USA. pp Zhou, Q. C., Z. E. Wu, B. P. Tn, S. Y. Chi nd Q. H. Yng Optiml dietry methionine requirement for juvenile coi, Rchycentron cndum. Aquculture 258:

8 Be et l. (2011) Asin-Aust. J. Anim. Sci. 24(7): Zicker, S. C. nd Q. R. Rogers Use of plsm mino cid concentrtion in the dignosis of nutritionl nd metolic diseses in veterinry medicine. In: Proceeding of 6th Congress of the Interntionl Society for Animl Clinicl Biochemistry (Ed. J. J. Knedo). Dvis, Cliforni, USA. pp Zimmermn, R. A. nd H. M. Scott Effect of fsting nd of feeding nonprotein diet on plsm mino cid levels in the chick. J. Nutr. 91: