Methionine requirement of juvenile Japanese flounder(AN,2001)

AquacultureNutrition20017;201^209

MethioninerequirementofjuvenileJapanese¯ounderParalichthysolivaceusestimatedbytheoxidationofradioactivemethionine

M.S.ALAM,S.TESHIMA,M.ISHIKAWA,S.KOSHIO&D.YANIHARTOLaboratoryofAquatic

AnimalNutrition,FacultyofFisheries,KagoshimaUniversity,Shimoarata,Kagoshima,Japan

Abstract

GrowthandaminoacidoxidationstudieswereconductedtoestimatemethioninerequirementofjuvenileJapanese¯ounder,Paralichthysolivaceus,byusingthepuri®eddietscontaining500gkg±1crudeproteinfromcasein,gelatineandcrystallineaminoacids(CAA).Dietswithsixgradedlevelsofmethionine(5.3,8.3,11.3,14.3,17.3and20.3gkg±1diet)werefedtotriplicategroupsofthejuvenile(initialweight2.8󰂋0.05g)twiceadayfor40days.Topreventleachinglosses,CAAwereprecoatedusingcarboxymethylcellulose(CMC),andfurtherdietswereboundbyCMCandj-carrageenan.Basedonbroken-lineanalysisofpercentageweightgainandfeedconversione󰂁ciency,themethioninerequirementsofJap-anese¯ounderinthepresenceof0.6gkg±1ofcystinewere14.9and14.4gkg±1drydiet,respectively.Afterthegrowthstudywas®nished,adirectestimateofmethioninerequire-mentwasmadebyexaminingthein¯uenceofdietarymethioninelevelon14C-methionineoxidationbydetermin-ingradioactivecarbondioxide,proteinandnonproteinfractionsofthewholebody.Thedose±responsecurvebetweenexpiredradioactiveCO2anddietarymethioninelevelsshowedthattheoptimummethioninelevelforthe¯ounderwasestimatedtobewithintherangeof14.3±17.3gkg±1ofdietinhighagreementwithvaluesobtainedfromthegrowthstudy.

KEYWORDS:

aminoacidoxidation,14CO2,Japanese¯ounder,

methionine,Paralichthysolivaceus,requirement

Received11September2000,accepted9February2001

Correspondence:Shin-ichiTeshima,LaboratoryofAquaticAnimalNutri-tion,FacultyofFisheries,KagoshimaUniversity,Shimoarata4-50-20,Kagoshima0-0056,Japan.E-mail:teshima@®sh.kagoshima-u.ac.jp

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Introduction

Somenutritionalstrategyfor®shfedwitharti®cialdietsistoprovideindispensableaminoacidsatalevelsu󰂁cienttomeetthedemandsformaximumproteinretentionwithoutavoid-inganexcessivesupply.Methionineisthesulphur-containingaminoacid,whichisrequiredfornormalgrowthofmany®shes(Wilson19).Methioninewasthemostlimitingaminoacidindietsforsomewarmwater®sh(Lovell19)andinsomesemipuri®eddietforthereddrum(Moon&Gatlin19).Methionineisconvertedtocystineinanimals,therefore,thepresenceofcystinesparestherequirementofmethionineformaximumgrowth.Thecystinereplacementvalueformethioninehasbeendeterminedformany®shes,suchaschannelcat®shIctaluruspunctatus(Hardingetal.1977),reddrumSciaenopsocellatus(Moon&Gatlin1991)andrainbowtroutOncorhynchusmykiss(Kimetal.1992a).MethionineisalsoaprecursorofcholineandKasperetal.(2000)reportedthatwhenmethionineisnotinexcessinthedietofNiletilapia,Oreochromisniloticus,cholineisrequiredforgrowth.Cataractswereobservedintherainbowtroutfedwithmethionine-de®cientdiets(Waltonetal.1982;Rumseyetal.1983).

Aminoacidrequirementsof®shesareusuallydeterminedandbasedonthegrowthratesof®shesfedwithgradedlevelsoftheparticularaminoacid(Wilsonetal.1978;Nose1979).Asgrowthisa󰂀ectedbymorecomplexfactorsthantheadequacyofthedietaryaminoacidcontentandbalance,manyphysiologicalresponsestochangesinaminoacidlevelsinthedietshavebeenusedtode®neaminoacidrequirement.Directoxidationstudiesonaminoacidshavebeencarriedoutattheendofdose-responsefeedingtrialsforcon®rma-tionoftheresultsofthegrowthcurve(Cowey1995).Comparedwithgrowthmeasurement,aminoacidoxidationtechniquehasgivenreliableresultsforseveral®sh(Waltonetal.1984a;Kaushiketal.1988;Andersonetal.1991;Lall

201

M.S.Alametal.

etal.1994).Aminoacidoxidationtechniqueisbasedonthemeasurementofexpired14CO2whenanintraperitoneallyinjectedpulseororaladministrationof14C-labelledaminoacidundertestisgivenatthedi󰂀erentlevelsoftestaminoacidintake.Whenanaminoacidislimitingorde®cientinadiet,themajorproportionwillbeutilizedforproteinsynthesis,andlittlewillaccumulateintheplasmaorbeoxidizedtoCO2,whereaswhenthequantityofanaminoacidissuppliedinexcess,andisthusnotalimitingfactorforproteinsynthesis,plasmalevelswillincreaseandmorewillbeavailableforoxidation(Brookesetal.1972;Aguilaretal.1974).

Therequirementsofalltheessentialaminoacidsareknownonlyforalimitednumberofjuvenilespeciessuchas,rainbowtroutOncorhynchusmykiss(Ogino1980),catlaCatlacatla(Ravi&Devaraj1991),chinooksalmonOncorhynchustshawytscha,channelcat®sh,commoncarpCyprinuscarpio,JapaneseeelAnguillajaponica,Niletilapia(NRC1993)andwhitesturgeonAcipensertransmotanus(Ng&Hung1995).Ontheotherhand,researchontherequirementsofmarine®shforessentialaminoacidsisscarceexceptforthestudiesontherequirementsofacoupleofaminoacidsincommonlyculturedspeciessuchasyellowtail(Ruchimatetal.1997).Japanese¯ounder,apopularfood®shinJapan,butfewstudieshavebeenconductedusingdi󰂀erentalternativeproteinsourceswhichmainlyfocusonthespeci®cnutritionalrequirements(Kikuchietal.1994a,b;Kikuchi1999).Exceptforlysine(Forster&Ogata1998),therehavebeennostudieswithregardtotheessentialaminoacidrequirementsofthejuvenileJapanese¯ounder.ThepurposeofthepresentexperimentwastoevaluateoptimumdietarymethioninelevelfortheJapanese¯ounder,Paralich-thysolivaceusbygrowthresponseandtheoxidationofatracerdoseof14C-methionine.

Materialsandmethods

GrowthstudyThedetailsofthepreparationofdietsandrearingconditionsofthegrowthstudieshavebeendescribedinourrecentpublication(Alametal.2000).Thejuveniles(2.8󰂋0.05g)werefedthesixisonitrogenoustestdiets(Table1)containing500gkg)1crudeproteinintriplicateatarationsize5%oftheirbodyweight(BW)twiceadayfor40days.Casein,gelatinandcrystallineaminoacids(CAA)wereaddedtothetestdietstoprovideanaminoacidpatternsimilartothatofthejuvenileJapanese¯ounderwholebodyproteinexceptformethionine.Diet1(basaldiet)containedminimumlevelof

methionine,5.3gkg)1ofdietor10.6gkg)1ofprotein,fromintactproteinandgelatin.Theotherdietsweresupplementedwithincrementallevelsof3gkg)1ofcrystallinemethioninetothediet1,resultinginmethionineconcentrationsrangingfrom8.3to20.3gkg)1ofdietor16.6to40.6gkg)1ofprotein.Thesedietarymethioninelevelswerebelowandabovethemethioninelevelofthejuvenile¯ounderwholebodyprotein(Table2).DietswerepreparedaccordingtoMillamenaetal.(1996)withslightmodi®cation.TopreventleachinglossofCAA,theywereprecoatedwithcarboxy-methylcellulose(CMC)andj-carrageenan.

AminoacidoxidationstudyExperimental®shAttheendofthegrowthexperiment,six®shfromeachdietarygroupwererandomlycollected,transferredtotheradioisotopelaboratoryandthe®sheswereplacedinthree10Lglassaquarium,calledfeedingchambers(eachchambercontainedtwo®shes).Triplicategroupsofthe®shforeachdietarytreatmentwerefedwiththerespectivetestdietsfor7daystoacclimatizetotheenviron-ment.Fiftypercentofwaterinthefeedingchamberswasexchangeddailyandaerationwassupplied.

Radioactivelylabelleddiets[1±14C]L-Methionine[speci®cactivity,55mCimmol±1(2.03GBqmmol±1)]waspurchasedfromAmericanRadiolabeledChemicalsInc.(StLouis,MO63146,USA)inethanol:water(7:3).Thecompositionsoftheradioactivedietswerethesameasforgrowthtrials,exceptfortheinclusionof14C-methionine.Toprepareradiolabelleddiets,1goftherespectivecolddietsweregroundinasmallmortaraddingwater(1:1w/v),14C-Methioninewasaddedandmixedwelltoensurethatallparticlesofdietswerecompletelymixedwith14C-methionine.Thedoughwasextrudedthrougha10-mLplasticdisposablesyringewitha3.0-mmdiameteroutlet.Thespaghetti-likestrandsweredriedandthenbrokenupintopellets.Thespeci®cactivityofthepelletswasapproximately3lCig±1(111KBqg±1).Administrationof14C-methioninedietsand14CO2collectionTodetermineoxidationof14C-methionine,expired14CO2werecollectedaccordingtoQuerijeroetal.(1997)withthefollowingmodi®cation.Thejuvenileswerestarvedfor24hbeforefeedingtheradiolabelleddiets.The®sheswerefedwiththerespectivelabelleddietatthedoseof20000dpmg)1BW.Thefeedswereconsumedbythejuvenileswithinfewminutesexceptthosefedwithdiets1and6,whichleftverysmallamountsofuneatenfeeds.After5minoffeeding,thejuvenileswereremovedfromthechamber,washedwithsea

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202MethioninerequirementofJapaneseflounder

Table1Compositionofthetestdiets

Ingredient

CaseinGelatine

Aminoacidmixture1Squidliveroil2Soyabeanlechithin3Vitaminmixture4Mineralmixture5a-Starch

Carboxymethylcellulose(CMC)j-Carrageenana-CelluloseAttractants6L-methionineTotalmethioninegkg)1ofdietgkg)1ofprotein

Crudeprotein(gkg)1,drybasis)CrudelipidCrudeash

1203

Testdiets(gkg)1drydiet)117080253505050501204425881005.310.6502.297.2.3

217080250505050501204425881038.316.6503.397.355.0

3170802475050505012044258810611.322.95.798.555.2

4170802445050505012044258810914.328.6504.294.756.2

51708024150505050120442588101217.334.6507.396.053.3

61708023850505050120442588101520.340.6500.6.355.1

SeeTable2..2FeedoilW,RikenV|tamin,Tokyo,Japan.3KantoChemicalCo.,Inc.,Tokyo,Japan.4(gkg)1diet)q-Aminobenzoicacid,1.60;biotin,0.02;inositol,16.02;nicotinicacid,3.20;Ca-pantothenate,1.12;pyridoxine-HCl,0.19;ribo£avin,0.80;thiamine-HCl,0.24;menadione,0.19;vitaminA-palmitate,0.77;a-tocopherol,1.60;cyanocobalamine,1.10;calciferol,0.04;ascorbyl-2-phosphate-Mg,0.28;folicacid,0.06andcholinchloride,32.75.5(gkg)1diet)NaCl,1.838;MgSO4á7H2O,6.850;NaH2PO4á2H2O,4.360;KH2PO4,11.990;Ca(H2PO4)2á2H2O,6.790;Fe-citrate,1.485;Ca-lactate,16.350;AlCl3á6H2O,0.009;ZnSO4á7H2O,0.179;CuCl2,0.005;MnSO4á4H2O,0.040;KI,0.008andCoCl2,0.050.6(gkg)1diet)Taurine,5;betaine,4;andinosine-5-monophosphate,1.

waterandtransferredtothemetabolicchamber,a4-Lglasscontainercontaining2.5Lseawater.Theopeningofeachmetabolicchamberwascoveredwiththeplasticcoverwithaninletandoutlettube.Airenteringtheinlettubeintothewaterofmetabolicchamberpassedthrough250mLof1.0MNaOHsolutiontoremoveCO2fromair.Theairleavingfromthemetabolicchamberpassedthroughfour-testtubetrapsinseriestocollectalltheexpired14CO2,eachcontaining2mLof0.36MNaOHsolutiontoabsorbmetabolic14CO2leavingthemetabolicchamber.Theairtubeleavingthemetabolicchamberwas®ttedwithathree-wayjunctioncorktoenablealternatecollectionofmetabolic14CO2atvariouscollectionperiods,withoutpermittingtheescapeofmetabolic14CO2attheendofeachperiod.The®sheswerekeptinthemetabolicchambersfor48handexpired14CO2wassampledat24and48h.After48h,the®sheswereremovedfromthemetabolicchambers,freezedriedandstoredforradioactivitymeasurements.Afterthe®sheswereremovedfromthemetabolicchambers,30mLof0.5MHClwasaddedinchamberstodecreasethepHofthe

waterto5.Furthercollectionof14CO2thatmighthavebeentrappedinthewaterwascarriedoutforanother12h.Ingestedradioactivitywasobtainedbydeterminingthesumofthetotalradioactivityofexpired14CO2,whole®shbody,andwaterfromthemetabolicchambers.Theradioactivityofthefeedingchamberwasnotanalysed.

SeparationofproteinandfreeaminoacidfractionfromthewholebodyFishsampleswereseparatedintoprotein,non-protein(mainlyfreeaminoacids),andremainderfractionsbytreatmentwithtrichloroaceticacid(TCA)asfollows:thewholebodysampleswerefreezedried(LABCONCO,AshahilifeScienceFreezeDrySystem,Japan),cutintosmallpiecesandhomogenizedwith10volumesofdistilledwaterusingaPolytronhomogenizer(KINEMATICA,GmbhLITTAU,Switzerland).ThehomogenatewasseparatedintotheTCA-precipitable(protein)andTCA-soluble(mostlyfreeaminoacids)fractionafteradditionof10volumesof10%TCAbycentrifugation(3000´g,10min).TheradioactivityofTCA-solublefractionwasmeasuredafterremovingTCA

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204M.S.Alametal.

Table2Aminoacidcompositionofingredientsusedinthetestdietsandthewholebodyprotein(gkg)1dryweight)oftheJapanese¯ounder

Suppliedby

AminoacidsEAA2ArginineHistidineIsoleucineLeucineLysine3MethioninePhenylalanineThreonineTryptophanValineNEAA6AsparticacidGlutamicacidSerineProlineGlycineAlanineTyrosineCystine

Hydroxyproline

12Casein

(170gkg)1)6.04.98.115.016.04.88.46.5ND9.910.638.47.117.62.44.310.10.57^

gelatine(80gkg)1)6.50.81.22.42.40.51.61.7ND2.04.88.72.010.017.68.30.20.179.2

CAA118.86.912.821.432.9variable411.013.13.613.732.234.710.2011.420.49.7^^

Total31.312.622.138.851.3

variable421.021.33.625.7.681.819.327.631.433.020.0^9.2

50%flounderwholebodyprotein31.312.622.138.851.314.221.021.33.6525.7.681.819.323.431.433.020.0ND^

ThemixtureofcrystallineL-aminoacids(AjinomotoCo.,Inc.,Japan).Essentialaminoacids.3SuppliedasL-LysineáHCl.4SeeTable1.5Kanazawaetal.(19).6Nonessentialaminoacids.7NRC(1993),ND=Notdetected.

withdiethylethertreatment.TheTCA-precipitablefractionwaswashedwith5volumesofcoldethylalcohol,followedbycoldethylalcoholandchloroform(3:1),andcolddi-ethylether.Theproteinfractionssoobtainedwerefreezedried.Thewashingsobtainedbythesetreatmentswereconsideredastheremainderfraction.

MeasurementofradioactivityRadioactivitiesweremeasuredasdescribedbyQuerijeroetal.(1997).Tomeasuretheradioactivityof14CO2,TCA-solublefractionandseawater,2mLofsamplewasaddedtoa10-mLofscintillationcocktailSCSII(Amersham,USA)inascintillationvial.Thediets,wholebody,proteinandremainderfractionweredigestedinSOLUENE-350(Packard,USA)for24hina55°Cwaterbathwithcontinuousstirring,thenwereadded8mLoftoluenebasedscintillationcocktailcontaining3.2gPPO(2,5diphenyloxazole),0.25gPOPOP{1.4-bis-2(5-phenyloxazolyl)benzene},inamixtureof500mLtoluene,200mLtritonX-100(polyoxyethyleneoctylphenylether)and100mLdistilledwater.Afteraddingthescintillationcocktail,allsamplesweresetasidefor6h,thenradioactivity

wasreadusingaliquidscintillationcounter(AlokaLSC3000,Aloka,Japan).

OtherschemicalandstatisticalanalysisAminoacidanalysiswasperformedbyHPLCasdescribedbyTeshimaetal.(1986).Approximately2mgdrysamplewasweighedandhydrolysedwith4N-methanesulphonicacidfor22hat110°C.ThepHofthehydrolysatewasadjustedto2.2andinjectedintoaHPLCunitwithanionexchangeresincolumn.Norleucinewasusedasaninternalstandard(0.06ng100lL±1).ThecrudeproteinofthedietswasdeterminedbyKjeldahlmethodwithaTecatorKjeltecSystem(1007Digestionsystem,1002Distillingunit,andTitrationunit)usingboricacidtotrapammonia,andthecrudelipidwasdeterminedusingBligh&Dyer(1959)method.AshandmoisturecontentswereanalysedasperAssociationofO󰂁cialAnalyticalChemists(AOAC1990)methods.Dataongrowthperformanceandtheradioactivemeasurementsweretestedusingone-wayanalysisofvariance(ANOVA).Signi®cantdi󰂀erencesbetweenmeanswereevalu-atedbyDuncanNewMultipleRangeTest(packageSuper

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ANOVA,

AbacusConcepts,Berkeley,CA,USA).Probabilities

ofP<0.05wereconsideredsigni®cant.Theoptimumdietarymethioninerequirementbasedonweightgaindatawasdeterminedusingthebroken-lineregressionmethod(Zeitounetal.1976;Robbinsetal.1979).RegressionanalysiswasperformedusingsoftwarepackageStatViewTM(AbacusConcepts).Therequirementwasestimatedtobethatleveleliciting95%ofthemaximumresponses.

Results

GrowthperformanceTheresultsofthegrowthperformanceoftheJapanese¯ounderwerealreadyreportedindetailselsewhere(Alametal.2000).Inbrief,asshowninTable3,thelowestweightgain,survivalandfeede󰂁ciency(FE),wereobtainedinthegroupfeddietwithoutsupplementalmethionineandthehighestvalueswererecordedwithdiet5containing17.3gkg)1ofmethionineindiet.Survivalratewasalsosigni®cantlya󰂀ectedbymethioninesupplementation.TheoptimumdietarylevelofmethionineforthejuvenileJapan-ese¯ounderbasedonbroken-linewasestimatedtobe14.9gkg)1ofdietor29.8gkg)1ofproteinfromtheweightgaindata.

Oxidationof14C-methionineDataonoxidationoflabelledmethioninearepresentedinTable4.Signi®cantlylower14CO2wasreleasedinthe®shwhichhadreceivedthetwolowestlevelsofmethionine,5.3and8.3gkg±1,thaninthose®shreceivinghigherlevels

Table3Weightgain,feede󰂁ciency(FE)andsurvivalofthejuvenileJapanese¯ounderfeddietsgradedlevelsofmethioninefor40days.Valuesaremeansofthreereplicategroups.Meanwithdi󰂀erentletterinthesamecolumndi󰂀ersigni®cantly(P<0.05)Methioninelevelgkg)1ofdietgkg)1Weightgain1ofprotein(%)FE(%)2Survival(%)53106.7a28a78a83166133.8b48b79a113226196.9c67c78a143286344.5d74cd88ab173346345.2d78cd98b203

406

338.1d82d88abPooledSEM

21.9

4.0

3.2

1Weightgain(%)=(Mean¢nalbodyweight)Meaninitialbodyweight)2/Meaninitialbodyweight´100.Feede¤ciency(FE)=weightgain(g)/totalfeedintakeindrybasis(g)´100.

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MethioninerequirementofJapaneseflounder

205

ofmethionine,14.3,17.3and20.3gkg±1.Anadditionofmethioninetothebasaldietuptothelevel11.3gkg±1ofdietenhancedslightlytheoxidationof14C-methioninebutnostatisticaldi󰂀erenceswereobservedamongthegroupsreceivingmethionineatthelevelsof5.3,8.3,11.3gkg±1ofdiet.Juvenilesfedwiththedietscontainingmorethan11.3gkg±1methionineshowedsigni®cantlyhigherexpired14CO2thanthosefedlowerlevelsofmethionine.Nostatisticaldi󰂀erenceswereobservedamongthegroupsreceivingmethionineatthelevelsof14.3and17.3gkg±1ofdiet.TheproductionofradioactiveCO2rapidlyincreasedabovealevelof17.3gkg±1ofdiet(Table4).Therefore,fromtheANOVAofexpired14CO2,therequirementofJapanese¯ounderformethioninewassuggestedtobewithintherangeof14.3±17.3gkg±1ofdiet(28.6±34.6gkg±1ofprotein).

Distributionofthewholebodyradioactivityafter48hTheradioactivityretainedinthewholebody,TCA-preci-pitable,TCA-solubleandremainderfractionsarepresentedinTable4.Theradioactivityofthewholebody(%ofingestion)decreasedwithincreasinglevelsofdietarymethi-onine.Thehighestradioactivityinthewholebodyproteinfraction(%ofthewholebody)wasobtainedinthejuvenilesfedwiththebasaldiet(diet1).Therewasatrendthattherecoveredradioactivityintheproteinfractiondecreasedwithincreasingdietarymethioninesupplement.Ontheotherhand,supplementationofmethioninetothebasaldietledtoanincreaseintheradioactivityoftheTCA-solublefraction.

Discussion

Theoptimumdietarymethioninelevelestimatedbytheoxidationmethodusingthe®shfedwithdi󰂀erentlevelsofmethioninewasfoundtobewithintherange14.3±17.3gkg±1ofdietor28.6±34.6gkg±1ofprotein.ThisrangewasclosetotherequirementvalueobtainedfromdataonweightgainandFE(Fig.1,Alametal.2000).TheoptimumdietarymethioninelevelfortheJapanese¯ounderwaslessthanthattherequirementvalueof40(gkg±1protein)reportedforthespeciessuchasgiltheadseabreamSparusaurata(Luquet&Sabaut1974)andchinooksalmon(Halveretal.1959),butwereclosetothoseformostofthecommerciallyimportant®n®shspecies,namely,rainbowtrout(30,Rumseyetal.1983),commoncarp(31,Nose1979),Japaneseeel(32,NRC1993),Niletilapia(32,Santiago&Lovell1988),milk-®shChanoschano(32,Borlongan&Coloso1993),cohosalmonOncorhynchuskiutch(27,Arai&Ogata1993),yellowtail

206M.S.Alametal.

sources,crystallineaminoacids,environmentalconditionsandexperimentaldesign(Tacon&Cowey1985).

TheresultsofthepresentstudyalsoshowedthattheJapanese¯ounderiscapableofutilizingcrystallinemethion-inesupplementsforgrowthasindicatedbyAlametal.(2000).Thehighe󰂁ciencyofcrystallinemethionineinimprovingthegrowthoftheJapanese¯ounderiscausedbytheimprovementofitswater-stabilitybycoatingwithCMCandj-carrageenan.Choetal.(1992),whenquantifyingtheargininerequirementofrainbowtrout,addedagar-coatedfreeaminoacidstothedietandgavehighratesofgrowthcomparablewiththecompletetestdiet.

Theaminoacidoxidationtechniquehasbeensuccessfullyappliedtodetermineaminoacidrequirementorverifyingdietaryaminoacidrequirementsestimatedbygrowthstudyforsheep(Brookesetal.1973),pigs(Chavez&Bayley1976;Kimetal.1983),rainbowtrout(Waltonetal.1984a,b;Kaushiketal.1988)andAtlanticsalmon(Lalletal.1994).Inthepresentexperiment2fortheestimationofmethioninerequirementbytheoxidationtechnique,increaseindietarymethioninelevelsfrom5.3to11.3gkg±1hadnomarkedincreasesintheproductionofCO2(Table4).Inotherwords,theJapanese¯ounderexpiredCO2slightlywhentheywerefedwiththedietsde®cientinmethionine.Thiscon®rmedthatthelevelsofessentialaminoacidsin¯uencethereleaseof14CO2from14C-labelledaminoacid(Kimetal.1983;Kaushiketal.1988).

Figure1ExpiredradioactivecarbondioxideandgrowthresponseofJapanese¯ounderfedgradedlevelsofdietarymethionine.Valuesshownaremeans󰂋SEoftriplicategroups.ThemethioninerequirementforJapanese¯ounderwasestimatedtobewithintherangeof14.3to17.3gkg)1ofdietfromtheexpiredradioactivecarbondioxide.

Seriolaquinqueradiata(25.6,Ruchimatetal.1997)andreddrum(26.9,Moon&Gatlin1991).Lowerdietaryrequire-mentsofmethioninehavebeenreportedforMossambictilapiaOreochromismossambicus(9.9,Jaunceyetal.1983)andseabassDicentrarchuslabrax(20,Thebaultetal.1985)ascomparedwiththatfor¯ounderinthispresentstudy.Therearemanyfactorswhichmaya󰂀ectaminoacidrequirements,includingspeciesandage,dietaryprotein

Testdiets1

Administrationof14C-methionineLevelofcoldmethionine(gkg^1)Radioactivityofprepareddiet(dpm´103mg^1)Givenradioactivity(dpm´103g^1BW)

Uneatendiets(%ofdietgiven)Ingestedradioactivity(%ofdietgiven)Radioactivityrecovered(%ofingestion)TotalexpiredCO2Wholefishbody

Waterinmetabolicchamber

5.33.11206.2659.24

28.33.2320061.07

311.33.5120058.48

414.33.1620057.08

517.33.3120057.76

620.33.52202.2056.19

SEM1Table4Oraladministrationofradioactivedietscontaining14C-methionineanddistributionofradioactivityintheJapanese¯ounder.Valuesaremeansofsix®shes.Meanwithdi󰂀erentlettersinthesamecolumndi󰂀ersigni®cantly(P<0.05)

3.09

6.19a88.25.56.29a83.910.18.52a,b9.95b86.381.75.19.0

31.353.815.0

9.97b77.712.322.457.819.8

14.40c79.16.520.660.219.2

0.72.82.72.74.33.4

Distributionofradioactivityinthewholebody(%ofwholebody)Proteinfraction51.743.638.6(TCA-precipitable)Nonproteinfraction22.937.046.0(TCA-soluble)Remainderfraction25.419.415.4

1SEM=Pooledstandarderrorofthemean.

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Theresultsofthedirectdeterminationofmethioninerequirementusing14CO2releasedfrom14C-methionineby®shreceivingdietswithgradedlevelsofmethioninedidnotallowtheconstructionofabroken-lineplottoshowrequirement.However,Table4indicatedthatproductionof14CO2fromorallyadministrated14C-methionineincreasedsharplywhenthemethioninelevelexceeded17.3gkg±1ofdiet.BytheANOVA,therequirementformethioninewasestimatedwithintherangeof14.3±17.3gkg±1ofmethionineinthediet.Therequirementvaluemostlyagreedwiththatestimatedbytheweightgain(14.9gkg±1)andFE(14.4gkg±1).Waltonetal.(1986)wasincapableofshowingthebreakpointfortherainbowtroutinthedose-responsefor14CO2from14C-lysineand14C-arginine,whereasinthecaseof14Ctryptophanitwassuccessful.Also,Kimetal.(1992b)didnot®ndanyclearbreakpointforrainbowtroutwhenanalysis14C-phenylal-anineoxidation.Waltonetal.(1986)suggestedthattheoxidationtechniquewouldnotbesuitableforuseintheabsenceofgrowthdatabecauseofitslackofprecisionindeterminingrequirementvaluesfromgraphicalplots.ThevariabilityinthereleaseofradioactivityasCO2from14C-methionineafteringestionoffeedmaybeexplainedbyvariationineithertherateofabsorptionofthemethionine;orintheratesofuptakebythetissueswhichwereobservedbyBali&Bayley(1984)inthecaseofpiglets.Theexperimentalprocedureinthepresentstudywasdesignedtominimizevariationinratesofingestionbetweenthe®shes;theyhadbeenfastedovernight,the®sheswereadjustedbyrespectivecolddietsfor7daysinthefeedingchamberandfeedswereconsumedinlessthan5min.Metabolicchamberswerealsoensuredtobewithoutanyleakageof14CO2.Inthepresentstudy,however,othergroupsof®sheswerefedwithrespectivegradeddietcontaining14C-methioninetocomparewiththeingestionrateofthetreated®shes.Theingestionratewasmoreorlesssameforbothstudies.

Therecoveredradioactivityoftheprotein(TCA-precipi-tate)fraction(%ofwholebodyradioactivity)intheJapanese¯ounderfedwithde®cientmethioninewashigherthanthatfedwithsupplementedmethionine.Thissuggeststhatthe®shreceivingmethionine-de®cientdietsutilizealargeproportionofdietarymethionineforproteinsynthesis,therebyreducingmethionineoxidation.Asthedietarysupplyofmethionineexceedsthe®shneedsforproteinsynthesis,partofdietarymethioninecouldbeoxidizedtoCO2forenergyproduction.However,Kimetal.(1983)reportedthatthepartitionofaminoacidsbetweenproteinsynthesisandaminoacidcatabolismmustdependonmanyfactors,particularly

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207

proteinturnover,thecombinede󰂀ectsofwhichmaybetoocomplextopermitthede®nitionofasinglerequirementforeachnutrient.

Insummary,theJapanese¯ounderjuvenilesareabletoutilizecrystallineaminoacidinthecoatedformandshowoverallgoodgrowthofthe¯ounder.Inthepresenceof0.6gkg±1ofdietarycystine,methioninerequirementforjuvenileJapanese¯ounderobtainedbyweightgain(14.9gkg±1ofdietor29.8gkg±1ofprotein)andfeede󰂁ciency(14.4gkg±1ofdietor28.8gkg±1ofprotein(Alametal.2000))weresupportedbytheclosevalues(14.3±17.3gkg±1ofdietor28.6±34.6gkg±1ofprotein)obtainedfromtheoxidationtechnique.

Acknowledgements

TheauthorswishtoacknowledgeAjinomotoCo.,Inc.,Japanfordonationofcrystallineaminoacids.The®nancialsupportreceivedbythe®rstauthorforthisresearchfromtheMinistryofEducation,CultureandSports(Monbusho)ofJapanisgratefullyacknowledged.

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