Aquaculture

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1 . Aquulture Effets of methionine, ysteine nd medium hin triglyerides on nutrient digestiility, sorption of mino ids long the intestinl trt nd nutrient retention in Atlnti slmon ž Slmo slr L. / under pir-feeding regime,) Sigve Nordrum, Ashild Krogdhl, Cmill Røsjø, Jn J. Olli, Hlvor Holm d BioMr, 7484, Trondheim, Norwy Norwegin Shool of Veterinry Siene, Oslo, Norwy AKVAFORSK ( Institute of Aquulture Reserh ), As, Norwy d Institute for Nutrition Reserh, UniÕersity of Oslo, Oslo, Norwy Aepted 2 Deemer 1999 Astrt Atlnti slmon, kept in sewter 8 118C., initil weight 180 g, were pir-fed four high energy, low protein diets for 65 dys in triplite. The diets were supplemented isonitrogenously.. with mino ids: methionine 6.2 g kg, ysteine 5.0 g kg or lnine 3.7 g kg. ontrol.. In the fourth diet, lso supplemented with lnine, medium hin triglyerides MCT. 100 g kg. repled fish oil. Feed intke ws estimted nd feeding level djusted sed on the differene etween delivered nd wsted feed. Apprent digestiilities nd pnreti enzyme tivities long the intestinl trt were determined from nlyses of hyme from different regions nd of fees, using yttrium oxide s the mrker. Plsm mino id onentrtions were determined in lood from the udl vein. Whole fish tken t strt nd termintion of the experiment were used to estimte nitrogen nd energy retention. Feed intke in fish fed the MCT-diet determined the feeding level of fish on the other diets. MCT inresed digestiilities of protein nd strh nd inresed nitrogen retention from 52% to 66%, while energy retention ws signifintly redued. MCT used umultion of ysteine in the proximl regions of the post-gstri intestinl trt nd inresed pnreti proteolyti tivity of the hyme. Methionine ) Corresponding uthor. Tel.: q ; fx: q E-mil ddress: sigve.nordrum@iomr.no S. Nordrum r00r$ - see front mtter q 2000 Elsevier Siene B.V. All rights reserved.. PII: S

2 342 ( ) S. Nordrum et l.raquulture supplementtion inresed ft digestiility. Cysteine inresed digestiilities of protein, ft, strh, s well s nitrogen retention. Hene, sulphur mino ids seemed to e the first limiting mino ids nd methionine less effetive thn ysteine in relieving the limittion. Plsm levels of turine did not, however, indite ny limittion in the pility to onvert methionine into ysteine. q 2000 Elsevier Siene B.V. All rights reserved. Keywords: Methionine; Cysteine; Medium hin ftty id; Digestion; Nitrogen retention; Pir-feeding 1. Introdution The quulture feed industry is onstntly striving to improve ost effiieny through inresed utilistion of protein, the most expensive mronutrient of fish diets. Higher protein utilistion my e otined y inresing protein digestiility, improving mino id lne, nd reduing the mount of protein used for energeti purposes. Cysteine nd turine, whih is derived from ysteine, re very importnt digestive omponents. Digestive enzymes ontin high levels of ysteine Blow, 1974; Ikenk et l., 1974., digestive seret re rih in ysteine-ontining glutthione Gnpthy et l., 1991; Dhm nd Jones, nd ile slts re onjugted with turine Wlton et l., 1982; Stryer, 1988; Yokoym nd Nkzoe, The demnd for sulphur mino ids in digestive proesses represents mjor prt of the totl turnover of these sulphur-ontining mino ids. Cowey et l found enhned trnsulphurtion tivity with inresed methionine ontent in diets of rinow trout nd oserved higher level of hepti glutthione with dietry supplementtion of ysteine, ut not with methionine. There re, however, inditions tht onversion of methionine to ysteine my not lwys provide suffiient supply nd tht turine my e limiting mino id in fish nutrition under ertin onditions Yokoym nd Nkzoe, 1992; Yokoym et l., Skguhi et l found inresed growth in hum slmon 7 12 g. when diet sed on sein ws supplemented with turine. Moreover, in study with Atlnti slmon fed diets with inresing levels of stndrd soyen mel 0% to 40% soyen protein of totl protein. supplemented with methionine, plsm methionine onentrtion inresed while plsm turine onentrtions deresed Olli et l., Soyen inlusion my lso inrese exretion of proteolyti enzymes in fees Drowski et l., Yokoym nd Nkzoe oserved hepti nd musle methionine nd ystthionine onentrtions to inrese in rinow trout 20 g. when fed sein-sed diets supplemented with either 0.5%, 1.0% or 1.5% L-methionine, while glutthione nd turine levels were unffeted. Medium hin triglyerides MCT. hve reeived muh ttention in reent yers in niml nd humn nutrition euse of their rpid sorption nd oxidtion without deposition in the ody Mustf et l., 1991; Ppmndjris et l., 1998., nd for their potentil nitrogen-spring effet Tntihedhyngkul nd Hshim, MCT lso pper to e potentil stimultors of nutrient trnsport into enteroytes Tjim et l., 1993; Wng et l., It ppers tht MCT my hve the potentil to enhne nutrient sorption, derese mino id oxidtion, redue ody ft deposition nd therey llowing inresed protein utilistion. No informtion is ville inditing whether this potentil n e utilised in slmonids.

3 ( ) S. Nordrum et l.raquulture The im of the present study ws to investigte the effets of dietry supplementtion with either methionine, ysteine or MCT on growth, digestiility, nitrogen nd energy retention, levels of plsm mino ids nd tivity of pnreti enzymes in Atlnti slmon under pir-feeding nd onditions of limited protein supply. 2. Mterils nd methods 2.1. Fish nd filities The tril ws onduted t the reserh sttion of AKVAFORSK, Sunndlsør, Norwy. Atlnti slmon Slmo slr L.. of the Sunndlsør reed, weighing on verge 180 g, were distriuted in 12 fireglss tnks ontining sewter. Eh tnk ontined n initil iomss of pproximtely 12 kg fish. Wter surfe in the tnks ws 1m 2, wter depth 58 m, nd the wter supply ws 19 lrmin. Oxygen level ws 80% of sturtion. At tril termintion, wter temperture hd risen to 118C from n initil 88C. Totl sum of degrees ws 632 nd the durtion of the tril ws 65 dys Diets nd feeding regimes The experiment involved four diets, eh fed to fish in three tnks under pir-feeding regime. Tles 1 nd 2 show feed omposition nd results of hemil nlysis. All diets were sed on the sme sl msh ontining low temperture dried LT. fish mel s the min protein soure. Yttrium oxide Y O. 2 3, 100 mgrkg, ws used s n externl inditor for digestiility determintions see elow.. To optimise onditions for detetion of improvements in protein utilistion, the protein level ws low g protein per MJ digestile energy. nd the diets were formulted to e isonitrogenous. Whet rn ws used s filler to e le to reh the intended protein-to-energy rtio without risking lekge of ft from the diets. The mino ids were dded on n equl nitrogen sis to the diet msh efore extrusion. The diet supplemented with lnine ALA-diet. served s the referene diet. Feed ws provided 24 h dy using utomti feeders delivering feed every 15 min. Feed delivery nd wste were reorded eh dy. The tnk of fish with the lowest feed intke determined the feeding level in the remining tnks. Feed ws delivered in exess during the first week of feeding to find the limiting group. Therefter, this group determined the mount of feed distriuted to the other tnks the next dy. Fish were fed the sme mount of feed ording to iomss in eh tnk. Feed intke ws lulted s the differene etween distriuted feed nd wste feed olleted Reords nd smpling All fish in eh tnk were ulk-weighed nd ounted t the strt nd end of the experiment. At the strt of the tril, three groups of 10 fish from the sme popultion s the experimentl fish were rndomly smpled, nd t the end, groups of 10 fish were smpled from eh tnk for reording of ody length nd of weight, nd for nlysis of

4 344 ( ) S. Nordrum et l.raquulture Tle 1 Formultions nd hemil omposition of the test diets ALA-diet MET-diet CYS-diet MCT-diet ( y 1) Ingredients g kg Whet Whet rn Fish mel Fish oil Sndeel MCT oil 100 Yttrium oxide Vitmin nd minerl premixes Pigment d L-Alnine d L-Methionine 6.2 e L-Cysteine 5.0 Chemil omposition s nlysed Wter g kg Protein g kg. nitrogen= Strh g kg Ash g kg Ft g kg Totl ny3 ftty ids of ft g kg Totl ny6 ftty ids of ft g kg Totl monounsturted ftty ids of ft g kg Totl sturted ftty ids of ft g kg Gross energy MJ kg f DPrDE g MJ Norse LT 94, Nordsildmel, Norwy. Prioritry to BioMr, Brnde, Denmrk. Crophyll Pink 8% stxnthin formultion Hofmnn-LRohe, Bsel, Switzerlnd.. d Sigm, St. Louis, MO, USA. e Fluk Chemie, Buhs, Switzerlnd. f DPrDEsgrm digestile protein MJ of digestile energy. Gross energy for the nutrients nd results of digestiility nlysis in the present experiments were used in the lultion. whole ody omposition. At the end, n dditionl 15 fish were smpled from eh tnk. Blood smples were drwn from five fish nd entrifuged. Plsm smples were frozen in liquid nitrogen nd stored elow y208c. Therefter, the fish were killed y low to the hed, nd the omplete gstrointestinl GI. trts of the 15 fish were disseted, wrpped in luminium foil, frozen in liquid nitrogen nd stored elow y208c. Body mesurements were tken. Livers from 10 of the 15 fish were weighed nd smpled. In hlf-thwed stte, the GI trts were divided into the following four regions: stomh ST, J-shped region with musulr losures in oth ends, the rdi nd the pylori sphinters., pylori region PR, the proximl region of the intestine, strting t the distl side of the pylori sphinter nd ending t the lotion of the distl-most e., mid-intestine MI, the region distl to the pylori region nd proximl to the inrese in intestinl dimeter. nd distl intestine DI, from the distl end of the mid-intestine to the nus.. When divided into two equl-sized piees, the mid- nd distl

5 ( ) S. Nordrum et l.raquulture Tle 2 Amino id omposition of diets g kgy 1. Amino id ALA-diet MET-diet CYS-diet MCT-diet Alnine Arginine Asprti id Glutmi id Glyine Cysteinerystine Histidine Isoleuine Leuine Lysine Methionine Phenyllnine Proline Serine Threonine Tyrosine Vline Sum intestines were termed MI1 proximl hlf. nd MI2 distl hlf., DI1 nd DI2, respetively. The intestines were opened nd the luminl ontents seprted from the intestinl wll nd ph mesured diretly in the hyme efore it ws olleted in test tues. After smpling of fish for registrtion nd nlysis, the remining fish were nesthetised nd fees stripped ording to Austreng for determintion of pprent digestiility of dietry protein, ft, strh, nd energy nd of pprent mino id sorility Anlyses Feed, hyme, fees nd fish Nitrogen, ft grvimetrilly. nd sh nlysis were rried out s desried y the Assoition of Offiil Chemists Ft in feed, hyme nd fees ws extrted with dihloromethne nd homogentes of fish with ethylette. Protein ontent ws estimted s nitrogen N. = Feed dry mtter ws determined y oven drying t 1058C until the weight ws stle. Energy ontent in feed nd fees ws nlysed y om lorimetry Assoition of Offiil Chemists, Fees nd hyme were pooled within eh tnk nd intestinl setion nd sujeted to freeze-drying efore nlysis. Dry mtter ws otined from weight redution during freeze-drying. Strh ws determined s gluose fter hydrolysis with het stle gluosidse under oiling onditions employing hexokinse, lierting redued niotinmide dinuleotide NADH., deteted spetrophotometrilly Boehringer Mnnheim, kit. no Determintion of mino ids ws rried out ording to the methods desried y Spkmn et

6 346 ( ) S. Nordrum et l.raquulture l nd Moore Smples of pproximtely 50 mg were hydrolysed with 5 ml 6 M HCl ontining 1 mgrml phenol nd 5000 nmol norleuine s internl stndrd for 24 h t 110 8C in thoroughly evuted, seled glss tues. Following hydrolysis, 0.5 ml of eh hydrolyste ws evported with rottory evportor in in vuo nd the residues were tken up in the 2 ml ph 2.2 uffer. Aliquots of 50 ml were nlysed with BIOTRONIK LC 5001 mino id nlyser using the extended physiologil system with lithium itrte uffers nd ninhydrin detetion. Dt olletion ws done with two-hnnel Shimdzu C-R2AX integrtor. Methionine nd hlf-ysteine were determined s methionine sulfone nd ystei id, respetively, in seprte smples hydrolysed following oxidtion with performi id. The ysteine results therefore represent the sum of ysteine nd ystine in the smple. The oxidised smples were nlysed using LKAB Alph-Plus mino id nlyser using the stndrd protein hydrolyste system with sodium itrte uffers. The results were normlised on the sis of the smple weight nd the reovery of the dded internl stndrd norleuine. The mino id nlysis ws rried out t the Swedish Agriulturl University, Uppsl, Sweden. Ftty id omposition of the feeds ws determined y use of gs hromtogrphy s previously desried y Røsjø et l Yttrium ws deteted with n ICP spetrometer model 1100, Termo JrrellrAsh, Frnklin, MA, USA. Refstie et l., Enzyme tivities were determined in 10% wrv. wter extrts of the freezedried smples. Totl proteolyti tivity ws ssyed ording to Holm et l with sein s sustrte. Csein hydrolysis ws used to express the totl tivity of the intrluminl proteinses nd peptidses long the intestinl trt. Lipolyti tivity ws ssyed ording to Gjellesvik et l using 4-nitrophenyl myristte s sustrte. All enzyme tivities re expressed per milligrm of yttrium. As the diets ontined the sme onentrtion of yttrium, the given enzyme tivities indited tivities per unit of food eten Blood Free mino ids nd turine were determined in deproteinised trihloroeti id. smples of plsm fter prederivtistion with phenyl isothioynte PITC. using Wters HPLC mino id nlyser system Pio Tg w. Cohen et l., t the Institute of Nutrition, Diretorte of Fisheries, Bergen, Norwy Clultions The following lultions were performed: Speifi growth rte SGR. s lnw y lnw. rd = where W sweight t end, W sweight t strt, dsnumer of dys in the period. 2 1 Apprent digestiility ( or sorility) of ompounds1 yompound in feesryttrium in fees. r ompound in dietryttrium in diet... = 100. In the present study nitrogen sorility ws tken s n estimte of protein digestiility.

7 ( ) S. Nordrum et l.raquulture Feed onõersion rtio FCR. s feed eten. rfinl iomssqiomss of ded fishy initil iomss. Condition ftor CF. sfish weight g. rfish length m.. 3. = 100 Heptosomti index HSI. s liver weightrody weight. = 100 Retention of nitrogens nitrogen inrese in fishrnitrogen intke. = 100 Retention of energy s energy inrese in fishrenergy intke. = Sttistis Sttistil evlution of differenes etween diets ws onduted using nlysis of vrine generl liner model. SAS Institute, Multiple omprisons of growth, speifi growth rte, feed onversion, plsm mino ids, ody omposition, nutrient retention nd digestiility of protein, ft, strh nd energy, of mino ids sorility, nd of enzyme tivities etween diets were onduted using the Dunn multiple rnge test, with tnk men s oservtion. For lipolyti nd proteolyti tivity, omprisons were lso rried out within eh intestinl region seprtely. A signifine level of PF0.05 ws hosen for ll tests. As the vrine of the mino id sorilities within eh intestinl segment were firly similr, omprison of the sorilities ws rried out on the sis of 95% onfidene rnge lest signifint differene., estimted s 4=SEM. 3. Results 3.1. Feed intke, growth nd FCR Feed intke inresed during the first prt of the experiment, inditing tht the fish needed ertin period of limtistion efore intke stilised t 1.3% of BW. A pre-experimentl period of limtistion ws not inluded in the experiment, s it would hve ffeted the strt weights of the fish. At the end, fish fed the MCT-diet turned out to hve eten slightly less 6%. thn fish fed the other diets. No signifint growth differenes were oserved mong fish fed the four diets Tle 3., nd ll fish showed similr ondition ftor fter the tril, pproximtely 1.3. Fish fed the MCT-diet hd signifintly lower gutted weight perentges nd higher HSI thn ny of the other groups. Fish fed the MCT-diet showed signifintly lower FCR thn fish fed the ALA-diet nd MET-diet Tle 3.. FCRs for fish fed the CYS-diet were of intermedite mgnitude, signifintly different only from the ALA-diet group Tle Digestiility nd sorility Dietry inlusion of MCT improved energy digestiility y improving protein digestiility 5% units, ft digestiility y 3% units nd strh digestiility y 13% units, ompred to the ALA-diet Tle 3.. Both methionine MET-diet. nd ysteine CYS- diet. improved ft digestiility signifintly. Cysteine lso improved strh, protein nd energy digestiility signifintly.

8 348 ( ) S. Nordrum et l.raquulture Tle 3 Growth, FCR, pprent nutrient digestiility, ody omposition, liver index, gutted weight nd nutrient retention for Atlnti slmon fed diets mixed with lnine, methionine, ysteine or MCT for 65 dys ) ALA-diet MET-diet CYS-diet MCT-diet P vlue SEM ns12. Growth. g FCR 1.07, 1.04, Digestiilityrsorility (%) )) Strh )) Nitrogen 80, Sum of mino ids 76, 79, )) Ft )) Energy 72, 74, Body omposition Ft g kg Protein g kg , Condition ftor Ash g kg HSI Gutted weight % Nutrient retention (%) Nitrogen retention Energy retention ) Numers re tretment mens. Results from nlysis of vrine, where SEM is the stndrd error of the men, nd the P-vlue is the signifine level for the effet of dietry tretment. Men vlues with different supersript letters re signifintly different. )) Fees were olleted y stripping Austreng, Determined s sum of mino id residues in hyme olleted y dissetion from the distl most prts of the intestine. Fig. 1 illustrtes the umultive pprent totl mino id sorility long the GI trt. The results indite tht ysteine nd MCT supplementtion inresed sorility of mino ids in the distl intestinl segments. Amino id sorption ws signifintly improved for the MCT-diet ompred to the ALA-diet P s Tle 4 presents sorility of ll mino ids long the GI trt in the ALA-diet, wheres Fig. 2 depits umultive sorility long the GI trt for four seleted mino ids: ysteine, glyine, lnine nd methionine in this diet. Totl mino id sorility, estimted s umulted sorility in the DI2 segment, ws highest for glutmi id nd lowest for ysteine. Cysteine sorility ws low throughout the intestinl trt, prtiulrly in the pylori region Fig. 2.. Turine onentrtions "1 SEM. in hyme dry mtter from the ALA-diet group were 1"0.6, 20"0.8, 18"1.1, 16"0.5, 11"0.1 nd 7"0.6 mg g in ST, PR, MI1, MI2, DI1 nd DI2, respetively. The CYS-diet showed higher sorility of ysteine in ll segments ompred to the ALA- nd MET-diets Fig. 3.. Also MCT ft inresed totl pprent sorility of ysteine signifintly P s , lthough pprent sorility of ysteine ws

9 ( ) S. Nordrum et l.raquulture Fig. 1. Cumultive pprent sorility of totl mino ids in the GI trt of Atlnti slmon fed diets mixed with lnine ALA-diet., methionine MET-diet., ysteine CYS-diet. or MCT MCT-diet.. Dt points represent the men of three tnks. Brs indite "SEM ns3.. Tle 4 Cumultive sorility of individul mino ids in intestinl trt of Atlnti slmon fed the ALA-diet PR MI1 MI2 DI1 DI2 Alnine 59.0" " " " "3.1 Arginine 56.0" " " " " 4.6 Asprti id 30.7" " " " "4.1 Cysteine 4.7" " " " "3.2 Glutmi id 57.3" " " " "3.9 Glyine 41.7" " " " "3.8 Histidine 46.3" " " " "4.2 Isoleuine 50.0" " " " "4.4 Leuine 54.3" " " " "4.3 Lysine 56.7" " " " "4.2 Methionine 55.7" " " " "3.8 Phenyllnine 50.7" " " " "3.9 Proline 53.0" " " " "3.7 Serine 41.0" " " " "4.2 Threonine 40.7" " " " "4.2 Tyrosine 33.3" " " " "4.4 Vline 49.0" " " " "4.3 Tryptophn ws not nlysed. PRspylori region, the proximl region of the intestine, strting t the distl side of the pylori sphinter nd ending t the lotion of the distl-most e; MIsmid-intestine, the region distl to the pylori region nd proximl to the inrese in intestinl dimeter; DIsdistl intestine, from the distl end of the mid-intestine to the nus. Results re mens for tnks"sem of men ns3..

10 350 ( ) S. Nordrum et l.raquulture Fig. 2. Cumultive pprent sorility of methionine, lnine glyine nd ysteine in Atlnti slmon fed. the ALA-diet. Dt points represent the men of three tnks. Brs indite "SEM ns3. low in the pylori e Fig. 3.. Fig. 4 depits pprent sorility of methionine for the four diets. Totl pprent methionine sorility inresed 15% in the MCT-diet, ompred to the ALA-diet nd the MET-diet Ps Fig. 4.. Fig. 3. Cumultive pprent sorility of ysteine in the GI trt of Atlnti slmon fed diets mixed with lnine ALA-diet., methionine MET-diet., ysteine CYS-diet. or MCT MCT-diet.. Dt points represent the men of three tnks. Brs indite "SEM ns3..

11 ( ) S. Nordrum et l.raquulture Fig. 4. Cumultive pprent sorility of methionine in the GI trt of Atlnti slmon fed diets mixed with lnine ALA-diet., methionine MET-diet., ysteine CYS-diet. or MCT MCT-diet.. Dt points represent the men of three tnks. Brs indite "SEM ns3.. Of ll the mino ids, sprti id nd glutmi id were present in highest onentrtions in ll prts of the intestinl trts. Conentrtion of totl mino ids in the hyme deresed from 256 mg g in the stomh to 114 mg g in the DI2 segment Body omposition Body ft level ws found to e signifintly lower in the MCT-fed fish ompred to the other groups Tle 3., while there were no signifint differenes mong the ALA-diet, MET-diet nd CYS-diet groups. The ody nitrogen level ws signifintly higher for the MCT group ompred to ALA nd MET groups. The CYS-diet group ws intermedite of these nd not signifintly different from either group Retention of nitrogen nd energy Nitrogen retention ws signifintly higher for the MCT-diet thn for ll other diets Tle 3.. Fish fed the CYS-diet hd signifintly higher nitrogen retention thn those fed the ALA- nd MET-diets. The ltter groups did not differ signifintly from eh other. On the other hnd, retention of energy ws onsiderly nd signifintly lower in the MCT-fed groups thn in ll the other groups Enzymes nd luminl ph. No differene in GI ph profile ws found. Averge ph "1 SEM vlues were 5.1"0.53, 8.3"0.40, 8.5"0.31 nd 8.6"0.27 in the stomh, the pylori, the mid-

12 352 ( ) S. Nordrum et l.raquulture nd distl intestine, respetively. Proteolyti tivity of hyme in the pylori region ws 75% higher in fish fed the MCT-diet ompred to fish fed ny of the other diets P s A differene in proteolyti tivity ws mintined throughout the entire length of the intestine. The only exeption ws the proximl mid-intestinl segment Fig. 5.. Totl proteolyti tivity mmolrminrmg yttrium. in five postgstri regions of Atlnti slmon fed diets mixed with lnine ALA-diet., methionine MET-diet., ysteine CYS-diet. or MCT MCT-diet.. Totl proteolyti tivity ws mesured with sein s sustrte nd tivity is expressed s DOD nm. 280 r10 min in the TCA superntnt. Brs represent the men of three tnks, nd error rs indite "SEM ns3.. Mens with the sme letter re not signifintly different.. Lipolyti tivity mmolrminrmg yttrium. in the postgstri regions of Atlnti slmon fed diets mixed with lnine ALA-diet., methionine MET-diet., ysteine CYS-diet. or MCT MCT-diet.. Ativity is expressed s mmol 4-nitrophenyl myristte hydrolysedrmin. Brs represent the men of three tnks, nd error rs indite "SEM ns3..

13 ( ) S. Nordrum et l.raquulture MI1. where the totl proteolyti tivity for the MET-diet pprohed tht of the MCT-diet Fig. 5.. A similr pttern of inresed enzyme tivity ws seen for lipolyti tivity, lthough not signifint Fig. 5.. There were, however, signifint orreltions etween oth lipolyti tivity mesured in the pylori region nd ft digestiility r 2 s 0.73, P s nd etween proteolyti tivity in the pylori region nd protein digestiility r 2 s 0.81, P s Moreover, fish fed MCT showed signifintly lower Ps dry mtter ontent in hyme ompred to the three other diets in the MI1 region, 16.2% nd 20.6%, respetively Plsm mino ids nd turine Signifint differenes were oserved mong fish fed the different diets for plsm threonine, glutmine, hydroxyproline, glyine, methionine nd turine Tle 5.. Methionine supplementtion elevted plsm methionine signifintly. In ontrst, lnine supplementtion did not ffet plsm lnine signifintly. Cysteine supplementtion used mrked inrese in plsm glyine level. This diet lso inresed glutmte level ompred to the ALA-diet nd hydroxyproline level ompred to the ALA nd MCT-diets plsm ysteine itself ws not nlysed.. Both ysteine nd methionine supplementtion used elevtion of plsm turine. Fish fed MCT ft showed lower hydroxyproline levels thn fish fed the other diets. Tle 5 Plsm onentrtion of mino ids nd turine mmolr100 mol. in Atlnti slmon fed diet with lnine, ALA-diet MET-diet CYS-diet MCT-diet P vlue SEM ns60. methionine, ysteine or MCT supplementtion Alnine Arginine Asprgine Asprti id Glutmi id Glutmine Glyine Histidine Hydroxy proline Isoleuine Leuine Lysine Methionine Ornithine Phenyllnine Proline Serine Threonine Tryptophn Tyrosine Vline Turine Vlues with different supersript letters re signifintly different.

14 354 ( ) S. Nordrum et l.raquulture Disussion 4.1. Generl spets Awre of the influene of growth rte on nutrient requirement nd retention, we designed our experiment with pir-feeding regime to otin similr growth rtes, restriting feed intke to the intke of the tretment groups of fish eting the lest. The 6% differene oserved in feed intke ws within the vriility inherited in the experimentl proedure nd onsidered unimportnt for the differenes oserved etween diets in the present experiment. In the present study, protein digestiility ws 80 85%, whih is firly low ompred to the digestiilities oserved in previous studies with fish mel-sed diets for Atlnti slmon Opstvedt et l., 1984; Anderson et l., 1992, 1993, The most likely explntion for the low protein digestiilities is the firly high ontent of whet rn Hilton nd Slinger, 1986; Hilton et l., Whet rn, used s filler, ws inluded to otin low protein to energy rtio, mrginlly elow requirement, in order to optimise the possiilities of reveling differenes in nitrogen deposition etween diets. The results of the present study onfirm previous investigtions reveling tht the pylori e ply mjor role in terms of nutrient uptke Drowski nd Drowsk, 1981; Lied nd Nj, 1982; Buddington nd Dimond, 1987; Smith, 1989; Ahern et l., 1992; Krogdhl et l., However, our study shows tht lso the more distl intestinl regions, from MI2 to DI2, my ply importnt roles in nutrient sorption. This seemed to e true in prtiulr for resorption of turine. The importne of the distl intestine in nutrient sorption ws lso pprent in studies of Drowski et l oserving ler dereses in ysteine onentrtion in hyme of the distl-most intestinl regions. Any impirment in funtion of the distl intestines, s n e seen for exmple with feeds ontining ertin soy produts Ingh et l., 1991, 1996; Bæverfjord nd Krogdhl, 1996., my use lrge drins in turine s well s ysteine, resulting in n inresed requirement of sulphur-ontining mino ids. The 20-fold higher turine onentrtion in the hyme of the pylori region thn of the stomh minly reflets turine seretion from the liver, inorported in ile s turoholtes. The present oservtions re in ordne with results found in studies with od y Lied nd Nj 1982., who found 17-fold inrese in turine onentrtion in the pylori hyme ompred to tht of feed nd stomh ontents. Ringø determined pprent turine sorility in Arti hr nd found vlues etween 65% nd 80%. The present study, reveling signifint, positive orreltion etween proteolyti tivity nd protein digestiility nd etween lipolyti tivity nd lipid digestiility, indites tht onditions triggering enzyme seretion or delying enzyme intivtion, lso inrese protein nd lipid digestiilities. Similr results hve een found in juvenile. Atlnti od nd rinow trout Kwi nd Iked, 1973; Lemieux et l., Replement of fish oil y MCT The positive effets of MCT on oth hyme enzyme tivities nd digestiility of protein, lipid nd strh indite tht MCT influene the digestive proesses onsider-

15 ( ) S. Nordrum et l.raquulture ly. The effet my involve oth inresed enzyme seretion nd inresed stility. The high ysteine ontent found in the pylori region of fish fed MCT lso indites tht the inresed enzyme tivity ws, t lest prtly, used y inresed seretion, s ystine is present in high proportions in pnreti proteolyti enzymes ompred to fish mel Blow, 1974; Ikenk et l., 1974; Asgeirsson et l., 1989; Pike et l., The higher wter ontent in the hyme in PR of MCT fed fish lso supports the hypothesis tht inresed pnreti seretion my explin the inresed enzyme tivities. The mehnism of the MCT effets in slmon my involve stimultion of holeystokinin CCK.. Experimentl inditions tht suh mehnisms re involved exist for mmmls. Tkd et l oserved tht dietry supplementtion of MCT to pigs inresed seretion of CCK, result in greement with erlier oservtions y Stus nd Stile Among diets ontining orn oil, eef tllow, fish oil or MCT oil the diet with MCT oil ws shown to e the most powerful stimultor of CCK seretion in rts Dougls et l., Also, in humns, MCT seem to inrese CCK seretion Iss et l., The higher nitrogen retention oserved for the MCT-diet ompred to the ALA-diet seemed minly to e the result of higher rss inorportion stimulted y n inrese in mino id sorption. The negtive effet of MCT on ft deposition nd energy utilistion ws most likely refletion of the positive effet of MCT on nitrogen retention, whih mens protein synthesis, highly energy requiring proess. The results onfirm tht dietry protein level ws limiting protein deposition, s intended. The diets of the present study ontined 14 g DPrMJ DE, whih is lower thn ssumed to e optiml Wilson, 1989; Johnsen nd Wndsvik, 1991; Johnsen et l., 1993; Einen nd Roem, Experiments with yu onfirm our oservtion of opposite effets of MCT on protein nd energy retention Mustf et l., Also, in humns, MCT hs een found to hve nitrogen-spring effets Tntihedhyngkul nd Hshim, Moreover, the energy ontined in MCT seems to e utilised well in mmmls suh s rt nd mn Gelieter et l., 1983; Teo et l., The improvements in nutrient digestiility nd nitrogen retention y MCT inlusion oserved in the present study re of interest for the slmon-frming industry nd points to potentils for improvement in oth protein utilistion nd rss omposition. On the other hnd, the inresed liver index nd deresed gutted weight perentge indite tht MCT hllenges the liver nd hnges energy prtitioning. Our results lso indited tht MCT, under n d liitum feeding regime, might ffet feed intke negtively. Redued feed intke, due to dietry inlusion of MCT, hs een oserved previously in red drum y Crig nd Gtlin However, there ppers to e speies differenes, s yu hve shown higher feed intke when fed MCT-ontining diets Mustf et l., Supplementtion with ysteine nd methionine The improvement in pprent ysteine sorility oserved with the CYS-diet ws most likely due to high sorility of the supplemented, free ysteine. Asorility of the supplemented, free ysteine ws estimted to e 94%, ssuming similr sor-

16 356 ( ) S. Nordrum et l.raquulture ilities of the ysteine ound to protein in the two diets. Consequently, ysteine trnsport did not pper to e the limiting ftor for ysteine sorption in our study. Not only MCT, ut lso ysteine supplementtion improved strh nd ft digestiilities, wheres methionine improved ft digestiility. These inreses in lipid nd strh digestiilities my reflet inresed seretion of lipse nd mylse. The positive orreltion etween lipse tivity nd lipid digestiility suggests tht this mehnism my e relevnt for the effet of methionine nd ysteine supplementtion s well s for effets of MCT. Amylse tivity ws not mesured in the present study, s erlier efforts hve een unsuessful with hyme from Atlnti slmon Brudeseth, There is possiility tht ysteine supplementtion lso improved mylse stility or other onditions of importne for strh hydrolysis nd sorption. Suh effets my e relted to ysteine s role in glutthione nd its undne in the intestinl seretions nd in severl digestive enzymes. Our disussion regrding dietry effets on strh digestion is limited y the lk of informtion on fish mylses. Amylses from fish hve not gined muh ttention from the sientifi ommunity nd further studies re gretly needed reviewed y Krogdhl nd Sundy, The improved nitrogen retention stimulted y ysteine supplementtion my e due to similr improvements in nutrient digestiility s those disussed regrding effets of MCT. Our experiment indites tht methionine supplementtion my e less effiient in stisfying needs for sulphur mino ids thn ysteine, s only ysteine supplementtion improved strh digestiility nd nitrogen retention signifintly. A limited pity to onvert methionine into ysteine s desried y Yokoym nd Nkzoe nd Yokoym et l might explin this differene. However, these results, showing limited trnsulphurtion pities, hve ome under disussion Cowey et l., Plsm mino ids nd turine Results from previous study Krogdhl et l., indited tht surplus mino ids re tolised rpidly in Atlnti slmon. From the present results, it ppers tht rte of oxidtion my differ etween mino ids. Alnine supplementtion did not ffet plsm lnine onentrtion in ontrst to the elevtion oserved for methionine with the MET-diet. Plsm mino id levels re the result of lne etween influx from digested protein, endogenous tolism, mino id oxidtion nd protein synthesis. Our results showing inreses in lood turine level with oth methionine nd ysteine supplementtion suggest tht Atlnti slmon re le to onvert methionine to turine effetively. Hene, the differene in effets of ysteine nd methionine supplementtion ppers to e due to other spets of metolism of these mino ids suh s the differene in their role s methyl donors Stryer, Conluding remrks The present experiment indites tht nitrogen retention nd ody omposition n e improved eyond wht is generlly otined tody with high-qulity fish mel-sed diets. The potentilly positive effets of lning fish diets with sulphur-ontining mino ids should e exploited. The potentil for improvements y inluding MCT in

17 ( ) S. Nordrum et l.raquulture the feed formultions nnot e fully relised until we understnd the speifi effets on regultion of feed intke, digestive proesses nd energy metolism to greter extent. Aknowledgements Thnks re due to o-workers from the tehnil stff t AKVAFORSK, BioMr Teh Centre, Brnde, Norwegin Shool of Veterinry Siene nd University of Oslo for skilful nd effiient ssistne with the experimentl work. Thnks re due lso to Prof. Einr Lied nd o-workers t the Institute of Nutrition, Diretorte of Fisheries, Bergen, Norwy, for helping out in diffiult nlytil sitution with nlysis of mino ids in plsm. Our study ws finned in prt y BioMr, the Norwegin Shool of Veterinry Siene, the University of Oslo nd the Norwegin Reserh Counil NFR r122.. Referenes Ahern, G.A., Behnke, R.D., Zonno, V., Storelli, C., Kineti heterogeneity of N-D-gluose otrnsport in teleost gstrointestinl trt. Am. J. Physiol. 263, R1018 R1023. Anderson, J.S., Lll, S.P., Anderson, D.M., Chndrsom, J., Apprent nd true vilility of mino-ids from ommon feed ingredients for Atlnti slmon Slmo slr. rered in se-wter. Aquulture 108, Anderson, J.S., Lll, S.P., Anderson, D.M., MNiven, M.A., Evlution of protein-qulity in fish mels y hemil nd iologil ssys. Aquulture 115, Anderson, J.S., Lll, S.P., Anderson, D.M., MNiven, M.A., Avilility of mino ids from vrious fish mels fed to Atlnti slmon Slmo slr.. Aquulture 138, Asgeirsson, B., Fox, J.W., Thorgrd, G.H., Purifition nd hrteristion of trypsin from the poikilotherm Gdus morhu. Eur. J. Biohem. 180, Assoition of Offiil Chemists A.O.A.C.., Offiil Methods of Anlysis. Assoition of Offiil Chemists, Wshington, DC. Austreng, E., Digestiility determintions in fish using hromi oxide mrking nd nlysis of ontents from different segments of the gstrointestinl trt. Aquulture 13, Bæverfjord, G., Krogdhl, A., Development nd regression of soyen mel indued enteritis in Atlnti slmon, Slmo slr L., distl intestine: omprison with the intestines of fsted fish. J. Fish Dis. 19, Blow, D.M., Stereohemistry of sustrte inding nd hydrolysis in the trypsin fmily of enzymes. In: Fris, H., Tshetshe, H., Greene, L.J., Trusheit, E. Eds.., Pro. Int. Res. Conf. Proteinse Inhi., 2nd Springer, Berlin, pp Brudeseth, L., Hydrolysis of rohydrtes in intestinl muos of Atlnti slmon Slmo slr., hliut Hippoglossus hippoglossus. nd mink Mustel Õison.. Thesis, Mster of Siene, University of Oslo, pp Buddington, R.K., Dimond, J.M., Pylori e of fish: new sorptive orgn. Am. J. Physiol. 252, G65 G76. Cohen, A., Meys, M., Trvin, T.L., The Pio w Tg Method. A Mnul of Advned Tehniques for Amino Aid Anlysis. Wters Chromtogrphy Division, Milford, MA. Cowey, C.B., Cho, C.Y., Sivk, J.G., Weerheim, J.A., Sturt, D.D., Methionine intke in rinow-trout Onorhynhus mykiss., reltionship to trt formtion nd the metolism of methionine. J. Nutr. 122,

18 358 ( ) S. Nordrum et l.raquulture Crig, S.R., Gtlin, D.M., Coonut oil nd eef tllow, ut not triprylin, n reple menhden oil in diets of red drum Sienops oelltus. without dversely ffeting growth or ftty id omposition. J. Nutr. 125, Drowski, K., Drowsk, H., Digestion of protein y rinow trout Slmo girdneri. nd sorption of mino ids within the limentry trt. Comp. Biohem. Physiol. 69, Drowski, K., Lery, C., Nonnotte, G., Colin, D.A., Protein digestion nd ion onentrtions in rinow trout Slmo girdneri Rih.. digestive trt in se- nd fresh wter. Comp. Biohem. Physiol. 83A, Drowski, K., Pozyzynski, P., Kok, G., Berger, B., Effet of prtilly or totlly repling fish mel protein y soyen mel protein on growth, food utilistion nd proteolyti enzyme tivities in rinow trout Slmo girdneri.. New in vivo test for exorine pnreti seretion. Aquulture 77, Dhm, L.J., Jones, D.P., Seretion of ysteine nd glutthione from muos to lumen in rt smll intestine. Am. J. Physiol. 267, G292 G300. Dougls, B.R., Jnsen, J.B., de Jong, A.J., Lmers, C.B., Effets of vrious triglyerides on plsm holeystokinin levels in rts. J. Nutr. 120, Einen, O., Roem, A.J., Dietry proteinrenergy rtios for Atlnti slmon in reltion to fish size: growth, feed utilistion nd slughter qulity. Aquult. Nutr. 3, Gnpthy, V., Brndsh, M., Leih, J.H., Intestinl trnsport of mino ids nd peptides. In: Johnsen, L.R. Ed.., Physiology of the Digestive Trt. 3rd edn. Rven Press, New York, pp Gelieter, A., Tory, N., Bro, E.F., Hshim, S.A., Vn Itllie, T.B., Overfeeding with medium-hin triglyeride diet results in diminished deposition of ft. Am. J. Nutr. 37, 1 4. Gjellesvik, D.R., Lomrdo, D., Wlther, B.T., Pnreti ile slt dependent lipse from od Gdus morhu. purifition nd properties. Biohem. Biophys. At 1124, Hilton, J.W., Atkinson, J.L., Slinger, S.J., Effet of inresed dietry fier on the growth of rinow trout Slmo girdneri.. Cn. J. Fish. Aqut. Si. 40, Hilton, J.W., Slinger, S.J., Digestiility nd utilistion of nol mel in prtil-type diets for rinow trout Slmo girdneri.. Cn. J. Fish. Aqut. Si. 43, Holm, H., Hnssen, L.E., Krogdhl, A., Florholmen, J., High nd low inhiitor soyen mels ffet humn duodenl proteinse tivity differently: in vivo omprison with ovine serum lumin. J. Nutr. 118, Ikenk, T., Odni, S., Koide, T., Chemil struture nd inhiitory tivities of soyen proteinse inhiitors. In: Fris, H., Tshetshe, H., Greene, L.J., Trusheit, E. Eds.., Pro. Int. Res. Conf. Proteinse Inhi., 2nd. Springer, Berlin, pp Ingh, T.S.G.A.M.v., Krogdhl, A., Olli, J.J., Hendrix, H.G.C.J.M., Koninkx, J.F.J.G., Effets of soyen-ontining diets on the proximl nd distl intestine in Atlnti slmon Slmo slr.: morphologil study. Aquulture 94, Ingh, T.S.G.A.M.v., Olli, J.J., Krogdhl, A., Alohol-solule omponents in soyens use morphologil hnges in the distl intestine of Atlnti slmon, Slmo slr, L. J. Fish Dis. 19, Iss, P.E., Lds, S., Forgs, I.S., Dowling, R.H., Ellm, S.V., Adrin, T.E., Bloom, S.R., Comprison of effets of ingested medium- nd long-hin triglyeride on gllldder volume nd relese of holeystokinin nd other gut peptides. Dig. Dis. Si. 32, Johnsen, F., Hillestd, M., Austreng, E., High energy diets for Atlnti slmon. Effets on pollution. In: Kushik, S.J., Luquet, P. Eds.., Fish Nutrition in Prtie, Proeedings of the IVth Interntionl Symposium on Fish Nutrition nd Feeding, Birritz Frne., June INRA Editions, Pris, pp Johnsen, F., Wndsvik, A., The impt of high energy diets on pollution ontrol in the fish frming industry. In: Cowey, C.B., Cho, C.Y. Eds.., Nutritionl Strtegies nd Aquulture Wste, First Interntionl Symposium on Nutritionl Strtegies in Mngement of Aquulture Wste, University of Guelph, Guelph, Ontrio, Cnd, pp. Kwi, S., Iked, S., Studies on digestive enzymes of fishes: 3. Development of the digestive enzymes of rinow trout fter hthing nd the effet of dietry hnge on the tivities of digestive enzymes in the juvenile stge. Bull. Jpn. So. Si. Fish. 39, Krogdhl, A., Nordrum, S., Brudeseth, L., Sørensen, M., Røsjø, C., Effets of diet omposition on pprent nutrient sorption long the intestinl trt nd of susequent fsting on muosl dishridse

19 ( ) S. Nordrum et l.raquulture tivities nd plsm nutrient onentrtion in Atlnti slmon Slmo slr L. Aquult. Nutr. 5, Krogdhl, A., Sundy, A., Chrteristis of pnreti funtion in fish. In: Pierzynowski, S.G., Zielski, R. Eds.., Biology of the Pnres in Growing Animls. Elsevier, Amsterdm, pp Lemieux, H., Blier, P., Dutil, J.D., Wddy, S.L., Frehette, M., Do digestive enzymes set physiologil limit upon growth rte nd onversion effiieny in Atlnti od Gdus morhu.? In: Proeedings of the ontriuted ppers, Aquulture Cnd 97, Quee, Cnd No pp Lied, E., Nj, R.L., Apprent vilility of totl nitrogen, protein nitrogen nd individul mino ids in od Gdus morhu.. Fiskeridir. Skr., Ser. Erner. 11, Moore, S., On the determintion of ystine s id. J. Biol. Chem. 238, Mustf, M.G., Nkgw, H., Ohy, S., Shimizu, T., Horikw, Y., Ymmoto, S.-i., Effets of vrious level of dietry medium hin triglyerides on growth nd lipid reservtion in yu. Nippon Suisn Gkkishi 57, Olli, J.J., Krogdhl, A., Venoe, A., Dehulled solvent-extrted soyen mel s protein soure in diets for Atlnti slmon, Slmo slr L. Aquult. Res. 26, Opstvedt, J., Miller, R., Hrdy, R.W., Spinelli, J., Het-indued hnges in sulfhydryl groups nd disulfide onds in fish protein nd their effet on protein nd mino id digestiility in rinow trout Slmo girdneri.. J. Agri. Food Chem. 32, Ppmndjris, A.A., MDougll, D.E., Jones, P.J.H., Medium hin ftty id metolism nd energy expenditure: oesity tretment implitions. Life Si. 62, Pike, I.H., Andorsdottir, G., Mundheim, H., The role of fish mel in diets for slmonids. Teh. Bull. Int. Asso. Fish Mel Mnge., 1 35, 24 Mrh.. Refstie, S., Hellnd, S.J., Storekken, T., Adpttion to soyen mel in diets for rinow trout, Onorhynhus mykiss. Aquulture 153, Ringø, E., Hthery-rered lndloked Arti hr, SlÕelinus lpinus L.., from Lke Tkvtn rered in fresh nd se wter: effet of slinity on digestiility of protein nd individul mino ids in pelin roe diet nd ommeril feed. Aquult. Res. 26, Røsjø, C., Berg, T., Mnum, K., Gjøen, T., Mgnusson, S., Thomssen, M.S., Effets of temperture nd dietry ny3 nd ny6 ftty-ids on endoyti proesses in isolted rinow-trout Onorhynhus mykiss, Wlum. heptoytes. Fish Physiol. Biohem. 13, Skguhi, M., Murt, M., Dikoku, T., Ari, S., Effets of dietry turine on tissue turine nd free mino id levels of the hum slmon, Onorhynhus ket, rered in fresh wter nd se wter environment. Comp. Biohem. Physiol. 89A, SAS Institute, SASrSTAT Users s Guide. SAS Institute, Cry, NC, 846 pp. Smith, L.S., Digestive funtions in teleost fishes. In: Hlver, J. Ed.., Fish Nutrition. 2nd edn. Ademi Press, Sn Diego, pp Spkmn, D.H., Stein, W.H., Moore, S., Automti reording pprtus for use in the hromtogrphy of mino ids. Anl. Chem. 30, Stryer, L., Biohemistry. Freemn, New York, 1089 pp. Stus, R.S., Stile, B.E., Role of holeystokinin in pnreti exorine response to intrluminl mino ids nd ft. Am. J. Physiol. 11, G347 G352. Tjim, K., Tkd, R., Itshi, H., Kmeok, K.I., Sugimur, K.I., Effets of long-hin nd medium-hin triglyerides on mino-id-uptke in rt intestinl rush-order memrne-vesiles. Comp. Biohem. Physiol. 106A, Tkd, R., Kji, Y., Sitoh, M., Mori, T., Effets of dietry medium nd long-hin triglyerides on the ilel digestiilities of mino ids in pigs. Anim. Feed Si. Tehnol. 65, Tntihedhyngkul, P., Hshim, S.A., Medium-hin triglyeride feeding in premture infnts: effets on ft nd nitrogen sorption. Peditris 55, Teo, T.C., DeMihele, S.J., Sellek, K.M., Btn, V.K., Blkurn, G.L., Bistrin, B.R., Administrtion of strutured lipid omposed of MCT nd fish oil redues the protein tolism in enterlly urned rts. Ann. Surg. 210, Wlton, M.J., Cowey, C.B., Adron, J.W., Methionine metolism in rinow trout fed diets of differing methionine nd ystine ontent. J. Nutr. 112, Wng, H., Dudley, A.W., Dupont, J., Reeds, P.J., Hhey, D.L., Dudley, M.A., The durtion of

20 360 ( ) S. Nordrum et l.raquulture medium-hin triglyeride feeding determines rush order memrne lipid omposition nd hydrolse tivity in newly wened rts. J. Nutr. 126, Wilson, R.P., Amino ids nd proteins. In: Hlver, J.E. Ed.., Fish Nutrition. 2nd edn. Ademi Press, New York, pp Yokoym, M., Nkzoe, J., Utilistion of methionine supplemented to diet in rinow trout. Nippon Suisn Gkkishi 58, Yokoym, M., Nkzoe, J., Aumultion nd exretion of turine in rinow trout Onorhynhus mykiss. fed diets supplemented with methionine, ystine nd turine. Comp. Biohem. Physiol. 102A, Yokoym, M., Udgw, M., Nkzoe, J., Influene of dietry-protein levels on hepti ysteine dioxygense tivity in rinow-trout. Fish. Si. 60,