Aquaculture (2012) Contents lists available at SciVerse ScienceDirect. Aquaculture

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1 Aquulture (212) Contents lists ville t SiVerse SieneDiret Aquulture journl homepge: Effets of fish size nd diet dpttion on growth performnes nd nitrogen utiliztion of rinow trout (Onorhynhus mykiss W.) juveniles given diets sed on free nd/or protein-ound mino ids Noélie Bodin, Gilles Delfosse, Trn Thi Nng Thu,, Eri Le Boulengé, Trik Aoudi, Yvn Lrondelle, Xvier Rollin, Lortoire de Pisiulture M. Huet, Life Siene Institute (ISV) nd Erth & Life Institute (ELI), Université tholique de Louvin, Route de Blory, 2, B-1348 Louvin-l-Neuve, Belgium Fulty of Animl Siene nd Aquulture, Hnoi University of Agriulture, Tru Quy, Gi Lm, Hnoi, Viet Nm rtile info strt Artile history: Reeived 19 July 211 Reeived in revised form 2 My 212 Aepted 22 My 212 Aville online 29 My 212 Keywords: Diet dpttion Free mino ids Juveniles Ontogeny Rinow trout The qulity of dietry protein is n importnt ftor influening fish growth. It is usully ssessed y mino id (AA) omposition, protein digestiility nd protein utiliztion effiieny. Here it ws investigted with rinow trout (Onorhynhus mykiss W.) juveniles 1) if the moleulr form of the ingested nitrogen (free (F) AA, peptides or proteins) my lso ffet the dietry protein qulity; 2) if this possile influene my e ffeted y juvenile size nd dpttion to the diet; nd 3) wht is the optimum syntheti FAA to protein rtio. Two experiments were rried out t 15 C (3 tnks/tretment) in whih 15 smll juveniles (.7 g) nd 45 lrge juveniles (2.85 g) were rndomly ssigned to fifteen 15 l-tnks (7 fish/tnk) nd to fifteen 45 l-tnks (3 fish/tnk), respetively. In oth experiments, fish were fed twie dy to stition for 9, 17 nd 25 feeding dys (3 periods) five isoenergeti nd isonitrogenous (412 g rude protein/kg dry mtter (DM)) diets ontining grded levels of oted FAA, repling, 25, 5, 75 or 1% (D D1) of the od musle mel, n intt protein. Compred to D, growth rte (DGC, % per dy) nd feed intke (g DM/kg.75 per dy) were signifintly redued only for smll juveniles nd lrge juveniles fed diets ontining t lest 5 nd 75% of FAA, respetively. Protein deposition (g/kg.75 per dy) ws not signifintly redued for juveniles fed diets D25 D5, ut well for juveniles fed diets D75 D1. The derese of growth rte nd protein deposition t FAA inlusion rte elow 75% ws lrgely explined y redution of voluntry feed intke while nitrogen retention effiieny ws signifintly redued only for diet D1. The mximum FAA level tht still ensured resonle growth (85% of mximum) ws 65%, t oth juvenile sizes, fter n dpttion period of 17 dys. Lrger juveniles tolerted higher FAA dietry levels thn smller juveniles, inditing ontogeny-relted hnges in FAA utiliztion effiienies. Finlly fish ustomed to FAA-rih diets were le to tolerte higher FAA dietry levels ut the mximum FAA inlusion level ws modulted y fish size. In onlusion, here we show tht the dietry protein qulity is dependent of the moleulr form of the ingested nitrogen in rinow trout juveniles nd tht this qulity is modulted y the rtio of syntheti FAA to protein (or FAA inlusion rte), the dpttion of the fish to the diet nd the fish size. 212 Elsevier B.V. All rights reserved. 1. Introdution The qulity of dietry protein is n importnt ftor influening the growth performne of fish. Dietry protein qulity is usully ssessed y mino id (AA) omposition, protein digestiility nd protein utiliztion effiieny. Some oservtions hve shown tht the moleulr form of the ingested nitrogen (free (F) AA, peptides or proteins) ffets growth rte, AA sorption kinetis, the degree of whole-ody AA oxidtion nd utiliztion for protein synthesis in fish s well s in mmmls (Collin-Vidl et l., 1994; Drowski Corresponding uthor. Tel.: ; fx: E-mil ddress: xvier.rollin@ulouvin.e (X. Rollin). et l., 23; Denzer et l., 21; Metges et l., 2; Terjesen et l., 26; Zhng et l., 26), rising the question s to whether this vrile my lso ffet protein qulity. This question is of gret eonomil nd environmentl importne in the ontext of fishmel shortge, prtiulrly when fishmel is repled y AA-defiient proteins tht need to e supplemented with free (F) indispensle (I) AA, in order to meet the requirements of the trgeted fish speies (Cheng et l., 23; Dvies nd Morris, 1997; Nng Thu et l., 27, 29; NRC, 211; Wtne et l., 1997). Severl uthors hve ttempted to define the AA requirements for different fish speies (Wilson, 23). However, in mny instnes, the oserved differenes etween studies exeeded rediility (Drowski nd Guderley, 22). For instne, tryptophne requirements of rinow trout (Onorhynhus mykiss) rnged from.5 to 1.4% nd methionine requirements in slmonids were estimted from 2.2% in /$ see front mtter 212 Elsevier B.V. All rights reserved. doi:1.116/j.quulture

2 16 N. Bodin et l. / Aquulture (212) rinow trout to 4.% in hinook slmon (O. tshwytsh) (Akiym et l., 1997). Severl ftors hve een proposed to explin these lrge vritions, eing either nutritionl or methodologil (Bodin et l., 27, 29; Rollin et l., 23). Aording to Drowski nd Guderley (22), the questionle formultion of test diets to seure eptility nd mximize growth of fish is the single, most importnt ftor resulting in disrepnies oserved in pulished IAA requirements in fish. Diet formultion for IAA requirement re sed entirely, or in vrile proportions, on syntheti FAA (Bureu nd Enrnção, 26; Drowski nd Guderley, 22). This pproh, presuming tht these FAA re le to sustin lose to optiml growth nd re utilized with the sme effiieny s the AA derived from dietry proteins, hs een questioned y severl uthors (Drowski nd Guderley, 22; Nolles et l., 28; Rollin, 1999). Therefore, it seems tht more studies re required to test this hypothesis. In 197, Aoe nd ollegues strted still ongoing disussion s to whether the nutritionl vlue of FAA nd protein of similr AA omposition in the diet of rinow trout is equivlent (Aoudi et l., 26; Aoe et l., 197; Brroso et l., 1999; Cowey, 1992, 1994; Cowey nd Luquet, 1983; Cowey nd Wlton, 1988; Drowski nd Guderley, 22; Drowski et l., 23; Espe nd Nj, 1991; Kim et l., 1991; Rodehutsord et l., 1995; Rollin et l., 23, 26; Terjesen et l., 26). Some uthors onluded tht FAA n reple protein-ound AA in diets of rinow trout (Muri, 1992; Rodehutsord et l., 1995) while other otined sometimes drmtilly redued growth performnes when intt protein soures were sustituted y FAA mixtures equivlent to their AA omposition (Brroso et l., 1999; Cowey nd Luquet, 1983; Cowey nd Wlton, 1988; Drowski et l., 23; Terjesen et l., 26). These ontrsting results ould possily e relted to differenes of dietry FAA level, fish size nd development stge s well s diet dpttion to these FAA-rih diets. Here we propose to investigte the effet of those three vriles nd to optimize the rtio of FAA to protein in experimentl diets imed t exmining requirements for individul AA in rinow trout juveniles. The im of this work ws to study the effet of totl or prtil replement of od musle mel protein, essentilly omposed of protein-ound AA, y mixtures of FAA of equivlent AA omposition on growth performnes nd nitrogen deposition nd utiliztion in rinow trout t two juvenile sizes during three feeding periods. The mximum sustitution levels tht still support resonle growth or nitrogen utiliztion effiieny ws evluted for oth juvenile sizes t eh period. 2. Mterils nd methods 2.1. Experimentl diets Five isonitrogenous (64 g N/kg DM) nd isoenergeti (±18.75 MJ kg/dm) diets (Tle 1) were formulted. The dietry N ws supplied y geltin nd y od musle mel nd/or FAA. The od musle mel, otined from ommeril soure (Toro Food Division, Rieer nd Søn AS, Bergen, Norwy), ws otined y freeze-drying musle of Atlnti od (Gdus morhu). Its nitrogen ontent is high (14.7% DM) nd ws reported erlier to e highly digestile (pprent digestiility of 94.5%) nd to provoke high growth rtes in slmonids (Aoudi et l., 29; Rollin et l., 23). It ontins mostly intt protein, FAA nd peptide AA representing only.3% nd 4.3% of the totl AA, respetively (Espe, 1993). Diet D ws the ontrol diet nd ll the AA were in the form of od musle mel (4,5% DM) nd geltin (3% DM). The od musle mel ws repled t levels of 25, 5, 75, nd 1% y mixture of rystlline FAA (Tle 2) of similr AA omposition to produe diets D25, D5, D75 nd D1. Sine this sustitution led to progressive derese of the dietry N ontent, the diets were mde isonitrogenous y the ddition of some more rystlline dispensle L-AA in the AA mixtures (2.1, 4.2, 6.3 nd 8.3% DM in diets D25, D5, D75 nd D1, respetively) in the following Tle 1 The sustitution of od musle mel y mixture of rystlline free mino ids of similr omposition for rinow trout (Onorhynhus mykiss) juveniles: ingredients nd hemil omposition (g/kg dry mtter (DM) 1 ) for the five experimentl diets. Diet D D25 D5 D75 D1 Components (g/kg DM) Cod musle mel Free mino ids mixture Geltin Cod liver oil d Modified strh e Shrose f Soy leithin g Vitmin premix f,h Minerl premix f,i f CHPO Agr f NHCO Croxymethylellulose j Chemil omposition Gross energy (MJ/kg DM) Crude ft (%) Crude protein (%) ph Toro Food Division, Rieer nd Søn AS (Bergen, Norwy). Ajinomoto Ltd., Tokyo, Jpn. See Tle 2 for mino ids mixtures omposition. Geltin Sigm (St. Louis, MO, USA), G-6144; gr, Sigm A-536. d Feder, Brussels, Belgium. e Merigum, AMYLUM, Alost, Belgium. f Fluk Chemik, Buhs, Switzerlnd. g Cerel, Beerzel, Belgium. h The vitmin omplex (per kg dry mtter): vit. A 1, IU; vit. D3 4 IU; vit. E 342 IU; vit. K3 22 mg; vit. B1 56 mg; vit. B2 12 mg; vit. B6 45 mg; vit. B12.3 mg; niine 3 mg; iotine 1 mg; foli id 15 mg; inositol 5 mg; pntoteni id 141 mg; holine hloride 3 mg; vit. C 1 mg; nthxntin 1 mg; utylted hydroxytoluene (BHT) 15 mg; α-ellulose 3238 mg. i The minerl omplex (g/kg mixture): AlKO 8 S 2 (1.5) H 2 O 1.39, CCO ; C(H 2 PO 4 ) 2 H 2 O , CoCl 2 6 H 2 O 1.4, CuSO 4 5H 2 O.6, KCl 132.5, KI.11, MgSO 4 7H 2 O 48.89; MnSO 4 H 2 O.4; HN 2 O 4 P , N 2 O 3 Se 5H 2 O.5, ZnCO , C 6 H 5 FeO 7 H 2 O j Croxymethylellulose, Merk (Drmstdt, Germny) proportions (modified from Kim et l., 1991; g/kg): sprti id 191.7, sprgine 95.9, lnine 157.4, glutmi id 183.7, glutmine 91.9, glyine 54.1, proline 12.6 nd serine The nlytil indispensle AA omposition of the five diets ws equivlent mong diets (Tle 3). Sodium ironte ws dded in the FAA mixtures t similr proportion (out 3% y weight of the FAA mixture) with Tle 2 Amino ids supplied y inresing mounts of the free L-mino id mixture in diets of smll nd lrge rinow trout juveniles (g/kg diet). Diet D D25 D5 D75 D1 Arginine Histidine Isoleuine Leuine Lysine HCl Methionine Cystine Phenyllnine Tyrosine Threonine Tryptophne Vline Alnine Asprti id Asprgine H 2 O Glutmi id Glutmine Glyine Proline Serine Σ For the detils of diets nd proedures, see Tle 1.

3 N. Bodin et l. / Aquulture (212) tht reported y Espe nd Lied (1994). The experimentl diets were produed (pellets of 1 nd 1.6 mm) s previously reported (Rollin et l., 23) nd more speifilly, the rystlline-aa mixtures were oted with 1% gr, to hinder lekge, dely their digestive sorption nd optimize their use for protein gin (Mmrini nd Kushik, 1994). After freeze-drying, the diets were stored t 2 C until feeding or nlysis Animls nd feeding The experiment ws pproved y the Animl Welfre Commission of the Université tholique de Louvin in ordne with the EU Diretive onerning verterte lortory nimls. Rinow trout (Onorhynhus mykiss) of domesti origin were supplied to our lortory hthery (M. Huet Fish Culture Lortory, Université tholique de Louvin, Louvin-l-Neuve, Belgium) s eyed diploid eggs y ommeril fish frm (L Fontine ux Truites, Gerouville, Belgium). The juveniles were rered in our lortory hthery, from eggs to the eginning of the experiment, ording to Rollin et l. (23). They were fed the Trouvit,, nd ommeril diets (Trouw Frne SA, Fontine-les-Vervins, Frne) up to the strt of the experiment. Fifty-one dys fter hthing, 15 smll rinow trout juveniles (initil men odyweight±stndrd devition (SD):.7±.1 g) were rndomly distriuted to groups of 7 fish in 15 quri ( m) of 15 l eh. For the lrge juveniles, 94 dys fter hthing, 45 rinow trout juveniles (initil men odyweight± SD: 2.86±.2 g) were hosen. They were rndomly distriuted to groups of 3 fish in 15 quri of ( m) 45 l eh. For oth sizes, temperture nd wter flow were mintined onstnt during the experiment, nd were respetively 15±1 (SD) C nd 2 l min 1. An ertion system mintined the dissolved oxygen levels lose to sturtion (±1 mg l 1 ) nd the light intensity ws onstnt (±15 lux). Eh test diet ws rndomly lloted to the quri (three quri per diet nd per size). In ddition, three quri per size served s initil smples. These initil juveniles were weighed, killed with exess ethylene glyol monophenyl ether, nd kept frozen ( 2 C) until hemil nlysis. The experiment ws of twenty-five feeding dys for the smll juveniles nd the lrge juveniles. Eh group ws fed mnully twie dy (1 h nd 18 h) to pprent stition. The mount of feed distriuted to eh qurium ws reorded fter eh mel. During the mels, it ws ensured tht the pellets were eten y the juveniles within mximum of 15 s of ontt with the wter in order to minimize the lehing of rystlline AA into the wter. Mortlity ws reorded dily. After 36 h of fsting, the juveniles were weighed y Tle 3 Indispensle mino id omposition of the experimentl diets (g/kg dry mtter; men±sem (n=5)), long with dt on reltive indispensle mino id requirements for optimum growth of rinow trout ording to the Ntionl Reserh Counil (211). Diets D D1 Requirement Arginine 22.49± Histidine 7.4 ±.22 8 Isoleuine 16.74± Leuine 29.17±.3 15 Lysine 32.74± Methionine 11.71± Cystine 4.45 ±.14 Phenyllnine 15.21± Tyrosine 12.13±.17 Threonine 16.81± Tryptophne Vline 16.59± Σ Vlue lulted on the sis of ingredient mino id omposition (Rollin, 1999). Methionine+ystine. Phenyllnine +tyrosine. groups nd ounted every nine, eight nd eight feeding dys orresponding to three suessive experimentl periods. At the end of the experiment, nd fter 72 h of fsting, the fish were weighed y groups nd ounted. The finl men odyweight ws lulted. All fish were killed with exess ethylene glyol monophenyl nd kept frozen ( 2 C) for further determintion of the finl whole ody hemil omposition Smpling nd hemil nlysis Initil nd finl fish whole odies were freeze-dried, pulverized (prtile dimeter1 mm) nd homogenized (Grindomix GM 2, Retsh, Hn, Germny), nd finlly kept frozen ( 2 C) until hemil nlysis. The diets were nlyzed for ph, DM, rude protein (N 6.25), rude lipid, gross energy nd the mino id ontents. The juveniles whole odies were nlyzed for the mount of DM, rude protein (N 6.25) nd rude sh. Proximte nlyses of smples were onduted using the following onventionl proedures (AOAC, 25): dry mtter y drying t 15 C for 24 h, sh y ininertion t 55 C for 12 h, rude protein (N 6.25) y the Kjeldhl method fter id digestion, rude lipid using the id-hydrolysis Soxhlet method. The gross energy of the diets ws determined with n IKA-C-4 diti lorimeter (Ik-Werk, Breisgu, Germny). The mino id nlysis of diets nd fish smples ws rried out using Biotronik LC3 mino id nlyzer s previously reported (Rollin et l., 26). Tryptophn nnot e mesured with this proedure, ut ws estimted on the sis of the mino id omposition of the ingredients (Rollin, 1999) Clultions The vriles tht were otined or mesured diretly during nd fter the experiment were the following: DI is the dry diet intke per fish (g DM/fish) during the experimentl period; NI is the N intke per fish (g N/fish); n is the men numer of fish per qurium, otined y summing the numer of fish per qurium t the eginning nd t the end of the experiment nd then dividing y 2; W f nd W i re the verge finl nd initil fresh ody weights of fish (g/fish); feeding d is the numer of feeding dys; N f nd N i re the men N ontents of the whole ody juveniles t the end nd t the eginning of the experimentl period (g N/g). Men metoli ody weight (MBW in kg/fish) ws lulted s ((W f /1).75 +W i /1).75 )/2. The following response riteri were used to evlute fish growth, feed intke nd nutrient utiliztion: Body weight gin ðg=fishþ ¼ W f W i ; Dily growth oeffiient;. DGC ð%per dþ ¼ 1 W 1=3 f W 1=3 i feeding d ; Protein gin ðg=kg MBW per dyþ ¼ 6:25 ðw f N f W i N i Þ= ðmbw feeding d Þ; Ft gin ðg=kg MBW per dyþ ¼ ðw f LIP f W i LIP i Þ= ðmbw feeding d Þ; where LIP is the lipid ontent in g/g ody weight nd it ws lulted s: 1 wter ontent (g/g ody weight) sh (g/g ody weight) protein ontent (g N 6.25/g ody weight);

4 18 N. Bodin et l. / Aquulture (212) Voluntry feed intke; VFI ðg DM=kg MBW per dyþ ¼ DI= ðmbw feeding d Þ; Nitrogen intke ðmg=kg MBW per dyþ ¼ NI= ðmbw feeding d Þ; Protein in weight gin ðg=kgþ ¼ ð1 protein ginþ ðmbw feeding d Þ= ðw f W i Þ; Ft in weight gin ðg=kgþ ¼ ð1 ft ginþ ðmbw feeding d Þ= ðw f W i Þ; Feed effiieny; FE ðg=gdm Protein effiieny rtio; PER Protein produtive vlue; PPV ð% Ft produtive vlue; FPV ð% where FI 2.5. Dt nlysis Þ ¼ ðw f W i Þ=DI; ðg=g proteinþ ¼ ðw f W i Þ= ðni 6:25Þ; Þ ¼ 1 ðw f N f W i N i Þ=NI; Þ ¼ 1 ðw f LIP f W i LIP i Þ=FI; is the lipid intke per fish (g ft/fish). All dt were sujeted to one-wy nlysis of vrine. Signifint differenes etween tretments were tested using the Tukey's multiple rnge test nd results of P.5 were deemed sttistilly signifint. The following full ANOVA model ws used to nlyze simultneously the results for the three periods (diet dpttion or time effet), the two initil juvenile sizes nd the five sustitution levels (diets): response prmeter=dpttion effet+juvenile size effet+diet effet+(dpttion juvenile size)+(dpttion diet) +(juvenile size diet) +(dpttion juvenile size diet) +qurium [nested to (juvenile size diet)] +qurium [nested to (juvenile size diet)] dpttion+ε, whereε is the error term. Signifint effets were tested with the Fisher F-test. The following simple ANOVA model ws pplied to eh diet seprtely to nlyze further the effets of the dpttion nd juvenile size on the response riteri: dpttion effet+juvenile size effet+(dpttion juvenile size)+ε. To determine the suitle sustitution level nd dpttion period for resonle growth, seond degree polynomil model ws fit to the dt for eh juvenile size nd eh period. Next, the mximum vlue of the response riterion of these polynomil urves, 85% of this vlue, nd then the orresponding x vlues, were lulted. The highest of these x vlues gve n estimtion of the sustitution level tht should llow 85% of the mximum of the hosen response riterion. The prmeters of the polynomil models were estimted y the lest-squres method for eh response riterion. All lultions were rried out with the SAS/JMP sttistil softwre (SAS Institute, Cry, NC, USA). 3. Results 3.1. Growth performnes nd voluntry feed intke During the feeding tril, ll diets were well epted y the fish. Men mortlity rte ws less thn.2% per dy nd ws not relted to dietry tretments. Fig. 1A shows the responses for the whole experimentl durtion of the dily growth oeffiient (DGC, % per dy) to dietry tretments. Signifint differenes were oserved etween diets D nd D5, D5 nd D75 for the lrger juveniles nd etween diets D75 nd D1 for oth sizes of juvenile. Body weight gin (g/fish) ws redued y 71% nd 48% from diets D to D1 (P.5), ut only y 8% nd 6% from diets D to D5 (P>.5) for the smller juveniles nd the lrger juveniles, respetively (Tle 4, Fig. 1A). The drmti redution of A DGC (% per dy) B Protein gin (g/kg MBW per dy) g 2.86 g g 2.86 g Sustitution level (%) growth in the smller juveniles fed on D1 ( 71%) ws prtly explined y redution of voluntry feed intke ( 34%) nd y derese of feed effiieny ( 35%). By ontrst, the redution of growth oserved in the lrger juveniles fed on D1 ( 48%) ws mostly explined y derese of feed effiieny ( 31%; P.5) nd moderte redution of voluntry feed intke ( 12%; P>.5; Tle 4). When the dt ws nlyzed with the full ANOVA model, tking into ount the DGC performnes for the three periods (r²=.94), the only signifint effet ws the diet effet (P.1), s well s the diet dpttion intertion (P=.5). This ment tht for oth juvenile sizes, the sustitution level of intt od musle mel y FAA hd n effet on their growth, nd tht this diet effet signifintly hnged with time (dpttion effet). A simple ANOVA model ws used to nlyze further the effet of the dpttion period on DGC (% per dy). The three effets studied were dpttion time, juvenile size nd dpttion time juvenile size nd the model ws pplied seprtely for eh diet (Tle 5). Adpttion time ws signifint for diets D75 nd D1. Indeed, the growth performnes of juveniles fed D75 nd D1 improved s the experiment progressed. Juvenile size ws signifint for ll diets, from D to D1, mening tht for ll diets, the growth performne of the smller juveniles differed from tht of the lrger juveniles. Finlly, the dpttion time juvenile size intertion ws signifint only for D nd D25, mening tht the evolution of juveniles' growth performne s the Sustitution level (%) Fig. 1. (A) Dily growth oeffiient (DGC) or protein gin (B) in rinow trout of two different initil ody weights (.7 nd 2.86 g), fed five diets (4.5% rude protein) with inresing levels of sustitution of od musle mel y rystlline mino ids of similr omposition (25, 5 75 nd 1%). Brs represent mens±stndrd errors (n=3). Within size, mens with different letters re signifintly different (P.5). d

5 N. Bodin et l. / Aquulture (212) Tle 4 Initil nd finl ody weight, ody weight gin, dily growth oeffiient (DGC; g 1/3 per dy), voluntry feed intke (mg/kg metoli ody weight (MBW) per dy), feed effiieny (FE; wet weight gin/dry feed intke), nitrogen intke (mg/kg MBW per dy), protein effiieny rtio (PER; wet weight gin/protein intke), ft nd protein (N 6.25) depositions (g/kg MBW per dy) nd retention effiienies (ft retention effiieny (FRE), ft gin/ft intke; nitrogen retention effiieny (NRE), nitrogen gin/nitrogen intke) of rinow trout juveniles fed diets with inresing sustitution of od musle mel y rystlline mino ids of similr omposition showing men vlues of three groups of 3 (2.86 g) or 7 (.7 g) fish (SE, stndrd errors). e. Diets Initil fish size (g) D D25 D5 D75 D1 men SE men SE men SE men SE men SE Finl weight f (g/fish) ,y (.7) 3.52,y (.13) 3.33,y (.5) 2.4,y (.13) 1.57 d,y (.3) ,x (.48) 9.56,x (.45) 9.12,x (.5) 8.3,x (.28) 6.41,x (.48) Body weight gin g (g/fish).7 3.3,y (.7) 2.83,y (.14) 2.63,y (.4) 1.71,y (.12).87 d,y (.4) ,x (.46) 6.69,x (.5) 6.22,x (.49) 5.13,x (.25) 3.57,x (.42) DGC f (% per dy) ,x (.4) 2.54,y (.9) 2.43,x (.3) 1.81,y (.1) 1.11 d,y (.4) ,x (.14) 2.81,x (.13) 2.68,x (.16) 2.33,x (.9) 1.75,x (.18) Protein deposition h (g/kg MBW per dy) ,y (.15) 1.55,x (.7) 1.4,y (.3) 1.4,y (.3).63,y (.4) ,x (.14) 1.69,x (.7) 1.69,x (.2) 1.46,x (.4).91,x (.16) Ft deposition i (g/kg MBW per dy) ,y (.5) 1.6,x (.5) 1.45,x (.) 1.9,y (.9).62 d,y (.4) ,x (.11) 1.58,x (.6) 1.53,x (.12) 1.34,x (.11) 1.1,x (.6) Feed intke i (g/kg MBW per dy) ,y (.36) 8.78,y (.7) 8.3,y (.1) 7.29,y (.39) 6.24 d,y (.13) ,x (.8) 11.89,x (.45) 1.88,x (.98) 9.37,x (.31) 9.82,x (.3) Nitrogen intke i (mg/kg MBW per dy) ,y (23.56) ,y (4.53) 56.29,y (.6) ,y (25.18) d,y (8.7) ,x (5.6) ,x (29.83) 734.1,x (65.83) 65.72,x (19.75) ,x (19.98) Protein in weight gin j (g/kg) ,y (13.8) ,x (5.55) 12.35,y (2.16) ,y (2.72) 19.17,x (6.36) ,x (6.4) 135.7,x (5.43) 142.2,x (6.24) ,x (1.58) ,x (1.9) Ft in weight gin i (g/kg) ,x (4.46) 132.1,x (2.39) ,x (1.34) 12.76,x (4.4) 18.62,y (4.4) ,x (11.1) ,x (2.78) ,x (5.93) 127.5,x (6.5) ,x (6.55) FE i (g/g dry mtter) ,x (.4) 1.39,x (.4) 1.41,x (.2) 1.23,x (.2).89,x (.4) ,y (.6) 1.11,y (.6) 1.13,y (.13) 1.13,y (.4).84,x (.6) PER i (wet weight gin/rude protein intke) ,x (.1) 3.3,x (.7) 3.32,x (.4) 3.6,x (.6) 2.22,x (.11) ,y (.14) 2.51,y (.14) 2.61,y (.3) 2.77,y (.3) 2.,x (.11) NRE k (%) ,x (5.5) 42.45,x (1.78) 39.92,x (.92) 35.49,y (1.5) 24.3,x (2.12) ,x (3.27) 34.12,y (2.51) 37.,x (3.54) 38.65,x (.33) 22.31,x (3.25) FRE k (%) ,x (1.97) 82.65,x (2.52) 79.23,x (.19) 67.85,x (2.51) 45.42,x (3.2) ,x (4.37) 6.58,y (2.11) 64.23,y (6.18) 64.84,x (3.29) 5.78,x (1.18),,,d Men vlues within row with different supersript letters were signifintly different (one-wy ANOVA nd Tukey's multiple rnge test; P.5). e For the detils of diets nd proedures, see Tle 1. f ANOVA test (diet, size, diet size): diet nd size effets were highly signifint (P.1), nd diet size effet ws signifint (P.5). g ANOVA test (diet, size, diet size): diet nd diet size effets were highly signifint (P.1), nd size effet ws signifint (P.5). h ANOVA test (diet, size, diet size): diet nd size effets were highly signifint (P.1), nd diet size effet ws not signifint (P=.8). i ANOVA test (diet, size, diet size): ll effets were highly signifint (P.1). j ANOVA test (diet, size, diet size): size effet ws highly signifint (P.1), nd diet nd diet size effets were signifint (P.5). k ANOVA test (diet, size, diet size): diet nd diet size effets were highly signifint (P.1), nd size effet ws not signifint (P >.5). x,y Men vlues within olumn (etween the two sizes for given response riterion), with different supersript letters were signifintly different (one-wy ANOVA nd Tukey's multiple rnge test; P.5). experiment progressed ws not the sme for the smll juveniles s for the lrge juveniles for those diets. A similr pttern ws oserved for protein deposition s response riterion (Tle 5). When ANOVA type III model ws pplied to feed intke expressed in g DM/kg MBW per dy (r²=.98) the diet effet (P=.2) ws the only signifint effet. Tking into ount the whole experimentl durtion fish ingested reltively similr mounts of diets D nd D25 for the smller juveniles nd diets D to D5 for the lrger juveniles (P>.5), ut lower quntities of the diets D5 to D1 for the smller juveniles nd D75 nd D1 for the lrger juveniles (Tle 4). When voluntry feed intke ws expressed in perent of feed intke oserved for the protein-ound AA-diet D (=1) (Fig. 2), signifint dpttion time effet ws oserved (P.5) for the lrge juveniles fed diets D75 nd D1. Indeed, ontrry to the lrge juveniles fed the other diets, lrge juveniles fed diets R75 nd 1 inresed their voluntry feed intke s the experiment progressed. This phenomenon ws not oserved in the smller juveniles (Fig. 2). When ANOVA type III model ws pplied to FE (r²=.92) the diet effet (P=.4) ws lso signifint. In ddition for FE, the diet dpttion effet ws lso signifint (P=.5) inditing tht dpttion time to the diet hd n effet on FE ut tht this effet ws different from diet to diet (Fig. 3). In prtiulr, FE signifintly deresed with time in juveniles fed diets D D5 s the experiment progressed in oth juvenile size. In juveniles fed D75 nd D1, FE inresed signifintly with time in the smller juveniles, ut not in the lrger juveniles. Tle 5 P-vlues of the two-ftor, rossed ANOVA model (see Fig. 1) for the dily growth oeffiient (DGC, % per dy), voluntry feed intke (VFI, % VFI of the ontrol-diet, D) nd protein gin (PG; g/kg metoli ody weight per dy). Signifint vlues (P.5) re in old. Effet Adpttion time Initil size Adpttion time initil size DGC VFI PG DGC VFI PG DGC VFI PG Sustitution level % % % % %

6 11 N. Bodin et l. / Aquulture (212) VFI (% of protein-ound AA diet, D) VFI (% of VFI of protein-ound AA diet, D) A. IBW.7 g Sustitution level (%) B. IBW 2.86 g Sustitution level (%) Fig. 2. Voluntry feed intke (VFI, % of the VFI oserved with the od musle mel protein-ound mino id (AA)-sed diet (% sustitution level, D)) in rinow trout of two different initil ody weights (A..7 g nd B g). Five diets (4.5% rude protein) were fed under the form of od musle mel (% sustitution level) or inresing levels of sustitution of od musle mel y rystlline mino ids of similr omposition (25, 5, 75 nd 1%) Protein deposition nd utiliztion effiieny Compred to the D ontrol diet, protein gin (g/kg MBW per dy) ws not signifintly redued for juveniles of oth sizes fed diets D25 nd D5 (Fig. 1B). The redution of protein deposition in juveniles fed on diet D75 ( 25%, P.5, nd 18%, P>.5, ompred to D in the smll juveniles nd the lrge juveniles, respetively), ws entirely explined y derese of nitrogen intke ( 24% nd 17%, respetively) sine no signifint redution of nitrogen utiliztion effiieny (PPV) ws oserved for this tretment. By ontrst, the signifint redution of protein gin in juveniles fed D1 ( 54% nd 49%, respetively) ws explined y oth redution of nitrogen intke ( 33%, P.5, nd 11%, P>.5, respetively) nd nitrogen utiliztion effiieny (PPV; 32% nd 43%, respetively; P.5). Ft gin followed similr pttern s protein gin (Tle 4). Fig. 4 illustrtes the effet of dpttion time on protein deposition expressed in perent of protein deposition oserved with the protein-ound AA-diet D. It n e seen tht the effet tends to inrese with the dietry FAA level in oth juvenile size, eing smll (P>.5) for diet D25, intermediry (P>.5) for D5 nd high (P.5) for diets D75 nd D1 (Tle 5). The size effet ws signifint for diets D5 to D1 (Tle 5), protein deposition eing signifintly higher in the lrge juveniles ompred to the smller ones for these diets (Tle 4). Finlly, the dpttion time size effet ws signifint for ll diets, mening tht the dpttion effet ws juvenile size-dependent (Tle 5) Sustitution level nd dpttion period for resonle growth When the two initil juvenile sizes were studied seprtely nd over eh period, smll differenes in the influene of the FAA Feed effiieny (g/g DM) Feed effiieny (g/g DM) sustitution level were oserved. Six polynomil models were fit to the DGC nd the sustitution level tht should llow for 85% of the mximum DGC or of the mximum nitrogen retention effiieny ws estimted. The resulting sustitution vlues were represented grphilly for eh initil juvenile size nd eh period (Fig. 5). After 17 feeding dys (fter period two), the sustitution level tht llowed for 85% of the mximum DGC for oth initil juvenile sizes ws of out 65%. Fig. 5A shows tht the juveniles of oth sizes needed 17 dys to dpt to the diets ontining etween 5% nd 75% of FAA. It lso shows tht diets with higher FAA ontent required longer dpttion period of t lest 25 dys to reh 85% of the mximum DGC performne. Very similr results were otined for protein deposition (g/kg MBW per dy, Tle 6). Fig. 5B showed tht the sustitution level tht llowed 85% of the mximum nitrogen retention effiieny inresed with dpttion time to the diet, rehing 1% fter 25 feeding dys of dpttion. It n lso e seen tht the lrger juveniles need muh less dpttion to the diet for given performne thn the smller ones nd tht sustitution level of 1% n e rehed for oth juvenile sizes fter 25 feeding dys of dpttion for this speifi response riterion. 4. Disussion A. IBW.7 g Sustitution level (%) B. IBW 2.86 g Sustitution level (%) The nutritionl vlue of FAA s ompred to protein-ound AA is still ontroversil in fish nutrition for mny speies (for review, see Drowski nd Guderley, 22) nd rinow trout is not n exeption. Some uthors onluded tht FAA n reple proteinound AA in experimentl diets of rinow trout (Kim, 1997; Kim Fig. 3. Feed effiieny (g fresh ody weight gin/g dry diet intke) oserved with the od musle mel protein-ound mino id (AA)-sed diet (% sustitution level, D) in rinow trout of two different initil ody weights (A..7 g nd B g). Five diets (4.5% rude protein) were fed under the form of od musle mel (% sustitution level) or inresing levels of sustitution of od musle mel y rystlline mino ids of similr omposition (25, 5, 75 nd 1%).

7 N. Bodin et l. / Aquulture (212) Protein gin (% of PGmx) Protein gin (% of PGmx) A. IBW.7 g Sustitution level (%) B. IBW 2.86 g Sustitution level (%) et l., 1991; Rodehutsord et l., 1995) while others onluded to sometimes drmtilly inferior performnes for FAA-rih diets (Aoe et l., 197; Brroso et l., 1999; Cowey nd Luquet, 1983; Cowey nd Wlton, 1988; Drowski et l., 23; Zhng et l., 26). These ontrsting results need lrifition. Here we studied (1) the effets of dietry FAA level, fish size nd diet dpttion to these FAA-rih diets in rinow trout juveniles nd (2) the optimum rtio of FAA to od musle mel, musle protein essentilly devoid of FAA, in experimentl diets imed t exmining requirements for individul AA in rinow trout juveniles. In the present study, inresed sustitution of od musle mel y mixtures of FAA of similr AA omposition led to signifint derese in growth, protein deposition nd feed intke when sustitution rtes were higher thn 5% (D75 nd D1). From polynomil regressions (Tle 6) we evluted tht sustitution rte of 65% of od mel intt protein y n equivlent FAA mixture llowed for 85% of the mximum DGC for oth juvenile sizes fter 17 dys of dpttion to the diet. A higher FAA inlusion rte deresed drmtilly the growth performnes, exept if longer dpttion period ws pplied in the lrger juveniles only. The dietry FAA level seems to e mjor ftor le to explin some differenes etween studies in slmonids. Hlver (1957) first reported on the growth of juvenile slmonid fed FAA-sed diets. The growth rtes were sid to e very slow euse the fish were rered t very low wter temperture (8 C) (Drowski et l., 23), ut proly lso euse of the high dietry FAA level in the diets. Lter, Aoe et l. (197) onluded tht rinow trout Fig. 4. Protein deposition (% of the mximum protein deposition (PGmx) oserved with the od musle mel protein-ound mino id (AA)-sed diet (% sustitution level, D)) in rinow trout of two different initil ody weights (A..7 g nd B g) Five diets (4.5% rude protein) were fed under the form of od musle mel (% sustitution level) or inresing levels of sustitution of od musle mel y rystlline mino ids of similr omposition (25, 5, 75 nd 1%). A. DGC Sustitution level (%) Sustitution level (%) B. NRE g 2.68 g.7 g 2.86 g 17 Feeding dys 17 Feeding dys Fig. 5. Estimted sustitution rtes llowing for 85% of the mximum dily growth oeffiient (A. DGC, % per dy) or of the mximum nitrogen retention effiieny (B. NRE, %) for two sizes of juvenile,.7 g nd 2.86 g, in rinow trout. See Setion 2.5 (Dt nlysis) for explntions. (IBW 8.3 g) rered t 15 C exhiited norml or only slightly retrded growth when fed on FAA-sed diet. In ft, ody weight gin otined with the FAA-sed diet ws 67% of tht of the sein/geltin-sed diet (no replites), nd the mximum oserved growth rtes for oth diets were rther low (relulted DGC of % per dy) ompred to the optiml (relulted) rtes pulished for rinow trout of similr size nd rised t similr temperture (Austreng et l., 1987). On their side, Rodehutsord et l. (1995) otined similr weight gins in 55 g-trout rered t 16.5 C nd fed diets either ontining 15% whet gluten plus 22% FAA or 27% whet gluten plus 6.9% FAA, nd onluded tht dietry FAA do not negtively ffet the effiieny of utiliztion of AA for growth in omprison to protein-ound AA. However, ording to Drowski nd Guderley (22) their onlusions do not seem to e wrrnted euse of the low growth rtes oserved (speifi growth rte of 1.7% Tle 6 Prmeters of the polynomil models (Y=x²+x+) fit to the dily growth oeffiient (DGC, % per dy) or the protein gin (g/kg metoli ody weight per dy) s funtion of sustitution level (x, %) nd x-vlue t 85% of the mximum vlue of the response riterion. Response riterion Prmeter Initil weight.7 g Initil weight 2.86 g DGC x-vlue t 85% Protein gin x-vlue t 85%

8 112 N. Bodin et l. / Aquulture (212) versus 4.1% in norml onditions) nd low dietry protein ontent. Our view is tht the results of Rodehutsord et l. (1995) n e explined y omintion of ftors, i.e. the reltively low mximum dietry FAA level tested (out 63% y weight), the reltively long durtion of the study (61 feeding dys) nd the rther high trout size used (see the diet dpttion effet nd the size effet disussed elow). In the present study, the dietry protein level ws onsidered to e optiml for rinow trout (Sti, 1974) nd the DGC vlues of % per dy otined in the present study for the juveniles fed D re lose to the optiml (relulted) rtes ( % per dy) pulished for rinow trout juveniles of similr size nd rised t similr tempertures (Austreng et l., 1987). Therefore, the onlusions of the present study n e generlized, t lest for rinow trout t the juvenile stge, nd re useful in experimentl diet formultions. Severl explntions hve een proposed to explin the inferior performnes of fish (nd other nimls) fed FAA-sed diet ompred to diet ontining protein-ound AA. The most populr one is sed on the ft tht FAA do not hve to e relesed from protein y intestinl digestion nd n therefore e immeditely sored in the first prt of the intestine (Cowey nd Wlton, 1988) nd only y AA trnsporters (Espe nd Lied, 1994). By ontrst, protein-ound AA hve to e digested first nd thus will eome ville lter for sorption, t lower rte, y wider vriety of trnsporters (peptides, FAA, proteins) nd in more distl prt of the intestine. After sorption, s the size of the FAA pool remins reltively onstnt during the postprndil phse, the protein synthesis pity my e exeeded y the high AA pperne rte in the FAA pool of fish fed FAA-rih diets. In this se, the surplus in FAA, potentilly toxi for the ody t high levels (Nolles et l., 28), is removed y hepti oxidtion (Bos et l., 23; Morens et l., 2, 23; Nolles et l., 29; Tome nd Bos, 2), leding to n imlned AA profile t the tissue level (Espe nd Nj, 1991) nd provoking higher mmoni nd/or ure exretion (Drowski et l., 23; Kushik nd Drowski, 1983; Shuhmher et l., 1997), or y diret AA exretion (Muri et l., 1984; Ng et l., 1996) or fel egestion (Drowski nd Drowsk, 1981; Kushik nd Drowski, 1983). Surplus AA my lso e metolized into ft (Mmrini nd Kushik, 1994; Ymmoto et l., 22) nd glyogen (Kushik nd Drowski, 1983; Wlton et l., 1986). All these proess led to n inferior nitrogen utiliztion effiieny. Drowski et l. (23) oserved mrked inresed demintion of dietry FAA in rinow trout levins ompred with fish fed sein-sed diet, ut not in juveniles. Furthermore, ure exretion ws two-fold higher nd ontinued for more thn 24 h post-feeding in trout juveniles fed n FAA-sed diet, ompred to the fstergrowing juveniles fed sein-sed diet, leding to inferior growth in the former. These oservtions onfirms the higher postprndil oxidtive losses oserved in rts nd humns fed FAA ompred to protein-ound AA (Denzer et l., 21; Metges et l., 2; Nolles et l., 29). Reent results of Nolles et l. (29) further suggested tht postprndil AA oxidtions of free nd protein-derived AA re independent of eh other, in ordne with the ide tht AA sorption in the intestine is lso seprted in time nd ple. No indition for n intertion etween the oxidtion of protein-ound AA nd FAA ws oserved, even when these two different soures were given together in the sme diet. In the present study, we gve FAA nd protein-ound AA lone (D1 nd D) or in vrious omintions (D25 D75). We oserved higher reltive losses of nitrogen (=1-NRE, % of nitrogen intke) in juveniles of oth sizes fed the FAA-sed diet ompred to the protein-ound AA-sed diet, supporting the previous oservtions. However, it is diffiult to mke sfe omprison etween tretments euse nitrogen intke were not the sme in ll groups. Overll, these oservtions indite mjor differenes in digestive physiology nd metolism of FAA versus protein-ound AA nd in their development etween the levin nd juvenile stges in trout fed FAA-sed diet (Drowski et l., 23). The present study shows tht rinow trout juveniles fed diet in whih the AA soure is entirely supplied in the form of rystlline FAA (D1 diet) hs positive ody weight gin, equivlent to 29 52% (.7 nd 2.86 g IBW, respetively) of the one of juveniles fed highqulity protein-sed ontrol diet (D diet). Moreover there ws size effet, growth rte eing signifintly higher with the former diet in the lrger juveniles ompred to the smller ones (+58%). This is in greement with some uthors tht otined positive growth rte nd high survivl in rinow trout juveniles (.8 g IBW) fter 2 weeks when fed FAA-sed diet (Drowski et l., 23), ut no growth nd low survivl in first feeding levins (Drowski et l., 23; Terjesen et l., 26). This possile size effet on FAA utiliztion needs further nlysis of the literture. Therefore, we ompiled our dt with some dt ville in the rinow trout literture. Keeping in mind tht these dt were not otined in the sme onditions (intt protein soure used s ontrol, temperture, et.), whih n possily led to invlid extrpoltion, we otined n interesting logrithmi reltionship (Fig. 6, Y=16.67 LnX+32.59, n=7, r 2 =.99) etween the reltive ody weight gin otined in rinow trout fed FAA-sed diet (Y, expressed in perent of the ody weight gin otined in protein-ound AA-sed diet of equivlent AA omposition) nd the trout initil ody weight (X, g/fish), onfirming the suggested size effet on FAA utiliztion for trout growth. From this eqution, it n e predited tht trout of out 57 g should perform s well with FAA-sed diet thn with protein-ound AA diet. Interestingly Rodehutsord et l. (1995) used rinow trout of 55 g (IBW) in n experiment from whih they onluded tht FAA n reple protein-ound AA in test diets for studies in rinow trout. Proly, this reltively high fish IBW my hve ontriuted to this ility. Overll, these results suggest tht the utiliztion of FAA for growth in slmonids is size-dependent or ge-speifi t the juvenile stge, levins eing unle to utilize FAA for growth t first feeding, ut quiring progressively this ility in logrithmi wy in their further development. This size effet n proly explin prt of the vrition of performnes reported in the pisine literture with rinow trout fed FAA-rih diet. Further studies will hve to nlyze if this ftor resulted in disrepnies oserved in pulished IAA requirements. Ontogeny involves the formtion nd development of systems nd funtions (Drowski, 1986), nd in fish, ontogeny onerns mostly the lrvl life stges nd somewht the juvenile life stges. For exmple, mrine fish lrve initilly ssimilted simple forms of AA (FAA) more effiiently nd 3.5 times fster thn omplex forms (proteinound) (Ronnestd et l., 2) ut these differenes in ssimiltion rtes deresed s the lrve grew (Rust, 1995). Though the digestive Body weigh gin (% of proteinound AA diet) Trout size (g) Fig. 6. Logrithmi reltionship (Y=16.67 LnX , n=7, R 2 =.99) etween the reltive ody weight gin otined in rinow trout fed free mino id (AA)-sed diet (Y, expressed in perent of the ody weight gin otined in protein-ound AAsed diet of equivlent AA omposition) nd the trout initil ody weight (X, g/fish). Dt soures: Aoe et l. (197); Drowski et l. (23); Terjesen et l. (26); nd present study.

9 N. Bodin et l. / Aquulture (212) trt of slmonids lredy resemles the dult form t the eginning of feeding (Drowski, 1986), it is prole tht ll of the digestive nd sorptive funtions re not yet fully opertionl. For instne, in rinow trout, protese tivities in the stomh nd intestine rehed their highest levels only t size of round 9 g live weight (Kitmikdo nd Thino, 196). Also in rinow trout levin, dipeptide trnsporters nd hydrolyzing enzymes my hve limited the utiliztion for growth of diet ontining 1% sustitution level of intt protein y syntheti dipeptides (Terjesen et l., 26). In the present study it is suggested tht the inferior growth performnes oserved in smller juveniles with high FAA-rih diets ( 75% inlusion rte) ompred to the lrger juveniles ws relted to the inferior pility of the digestive trt of the former to sor (not enough AA trnsporters) nd/or regulte sorption of this high flux of AA, leding to higher FAA tolism nd lower feed intke. Further studies will hve to test this hypothesis. In this study, the size effet showed some limittions in trout juveniles. Indeed, when the juveniles of oth sizes were fed FAAsed diet (D1), nitrogen utiliztion effiieny ws impired (out one third) ontrry to ll other diets (D D75), suggesting n intrinsi indequy of this type of diet in slmonids. However, sine voluntry feed intke ws mrkedly lower in fish fed D1 ompred to the others, it is not possile to onlude from the present results the inility of rinow trout juveniles to utilize effiiently FAA-sed diets. The redution of protein deposition in juveniles fed on diets D75 nd D1 ws lrgely explined y derese of voluntry feed intke. Aording to Hrper nd Rogers (1965), redution in feed intke with suh diets ould e explined y n AA imlne. Indeed, feeding nimls dietry FAA levels would led to n imlned pttern of plsm FAA nd to higher hepti uptke for FAA. This would, in turn, led to hnge in the FAA plsm levels nd would indue metoli signl to n ppetite-regulting enter, whih would negtively impt feed intke. This type of response hs een referred to s the neurologil pthwy response (Drowski et l., 27). Another possile feed intke regultion mehnism is the olftory response pthwy (Drowski et l., 27; Ksumyn nd Doving, 23), or pltility. Tste version n use redued voluntry feed intke nd growth (Gtlin et l., 27; Glenross et l., 26; Opstvedt et l., 2) while ttrtnts n improve feed intke (Tiril et l., 28). Poor diet pltility hs een evoked s possile reson for redued growth in FAA-rih diets (Peres nd Oliv-Teles, 27). In the present study, the redued feed intke oserved for diets D75 nd D1 for oth juvenile sizes n e explined y either the olftory or the neurologil pthwy response hypotheses, ut this is ertinly size-dependent dynmi proess. Indeed, the time effet ws signifint in the lrger juveniles ut not in the smller one for voluntry feed intke, inditing differentil dpttion of juveniles to FAA-rih diets. Mrked dpttion to FAA-rih diets ws oserved in the present study for growth rte nd protein deposition in oth juvenile sizes, ut lso for voluntry feed intke in the lrger juveniles nd for FE for the smller juveniles. This time effet ws lredy reported with similr diets in oth rinow trout levins nd juveniles (Drowski et l., 23), ut lso in other fish speies (Gye-Siessegger et l., 27). Reently, Nolles et l. (29) oserved in rts tht the higher oxidtion losses of dietry FAA oserved during the postprndil phse fter 5 dys of dpttion deresed with time. After n dpttion of 2 dys, the mximl oxidtion rte ws lower nd the pek vlue of oxidtion ws delyed for 2 3 h, suggesting tht the hndling of FAA ws slowed down, most proly in the gstrointestinl trt, thus reduing the pperne rte of the FAA in the ody pool nd the oxidtion losses. In the present study, nitrogen retention effiieny ws oserved to inrese with dpttion time to the diet (Fig. 5B), suggesting tht ertin pity of the ody to dpt to the presene of FAA in the diet does lso exist in rinow trout juveniles. Furthermore, this pity my e dependent of the developmentl stge. Indeed, the juveniles' dpttion seemed dependent of their size (Fig. 5). In prtiulr, lrger juveniles were le to ontinue to dpt (>17 dys) to higher sustitution levels (going from 65% to lmost 9%) whilst smller juveniles, despite dditionl exposure time (25 dys totl) were not le to dpt to higher levels nd stgnted round 65% sustitution level, for the growth response (DGC). Another possile reson of redued AA utiliztion for growth in FAA-rih diets in this study is lehing. With lrvl miro-diets, the lehing of solule nutrients suh s FAA is mjor prolem euse they re omposed of muh smller prtiles (5 25 μm) thn juvenile fish diets (severl millimeters in dimeter), nd the surfe to volume rtio is very high in omprison (Lopezlvrdo et l., 1994). In the present study, prtile size ws of 1 nd 1.6 mm, n intermedite size etween lrvl mirodiets nd juvenile fish diets. Furthermore, the FAA were oted with gr to prevent lehing. Coting or enpsulting the FAA hs een shown to redue soluility nd sorption rtes of FAA therey improving the effiieny of utiliztion of these AA (Chen et l., 1992; Segovi-Quintero nd Reigh, 24; Teshim et l., 1992; Zhou et l., 27). Cho et l. (1992) were the first to ot the FAA omponent of the diet with solution of queous gr prior to mixing it with the other dietry omponents. Lter, Mmrini nd Kushik (1994) dded the step of heting the gr nd the wter to 1 C nd then dding the FAA only when the mixture ooled to 4 C. Mny susequent studies used this gr oting method for FAA nd otined resonle growth rtes (Aoudi et l., 27; Nng Thu et l., 27, 29; Peres nd Oliv-Teles, 25; Rollin et l., 23). Finlly, in the present study, during feeding phse, it ws ensured tht the juveniles te the prtiles within 15 s of ontt with the wter. These preutions were elieved to redue FAA lehing to minimum in the present study nd to wrrnt our onlusions. In onlusion, these trils showed tht the dietry protein qulity depends on the moleulr form of the ingested nitrogen (t lest for rinow trout juveniles) nd tht this qulity is modulted y the rtio of syntheti FAA to protein, the dpttion to the diet nd juvenile size. In prtiulr, our results showed tht 1) the growth performnes of juveniles fed inresing dietry FAA levels follow qudrti funtion of FAA inlusion rte, eing mximum t 5% nd then deresing rpidly; 2) mximum FAA level tht still ensures resonle growth (85% of mximum) fter n dpttion period of 17 dys minimum is 65% t oth juvenile sizes; 3) the derese of growth rte nd protein deposition t FAA inlusion rte elow 75% is lrgely explined y redution of voluntry feed intke while nitrogen utiliztion effiieny remins essentilly unhnged in this rnge; 4) lrger juveniles tolerte higher FAA dietry levels thn smller juveniles inditing ontogeny-relted hnges in FAA utiliztion effiienies; nd 5) fish ustomed to FAA-rih diets efore the strt of the experiment n tolerte higher FAA dietry levels ut the mximum sustitution level is modulted y fish size. Further investigtions re needed, though, in order to determine if the low AA utiliztion effiienies of very high FAA sustituted diets (>75%) in juveniles re result of deresed voluntry feed intke, indequte intestinl sorption, inresed AA oxidtion, or other ftors, nd if the fish ody shows pity to dpt to the presene of FAA in the diet nd if this pity is dependent of the developmentl stge, s suggested y the present study. Aknowledgements The uthors thnk Mr Mihotte for expert tehnil ssistne nd the Université tholique de Louvin for funding. The uthors wish to thnk the two referees for their onstrutive omments tht improved the present mnusript.