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1 Scholrly Reserch Exchnge Volume 9 Article ID 7959 doi:1.3814/9/7959 Reserch Article Different Dietry Levels of Protein to Lipid Rtio Affected Digestive Efficiency, Skeletl Growth, nd Muscle Protein in Rinow Trout Fmilies Krisn Rungrungsk-Torrissen, 1 Lrs H. Stien, 1,, 3 Britt S. De, 1 Tone Vågseth, 1 Grethe B. Thorsheim, 1 Decln Toin, 4 nd Ossi Ritol 5 1 Institute of Mrine Reserch, Mtre Reserch Sttion, 5984 Mtredl, Norwy Deprtment of Biology, University of Bergen, P.O. Box 78, 5 Bergen, Norwy 3 Institute of Mrine Reserch, P.O. Box 187 Nordnes, 5817 Bergen, Norwy 4 School of Biologicl Sciences, Deprtment of Zoology, University of Aerdeen, Aerdeen AB4 TZ, UK 5 Finnish Gme nd Fisheries Reserch Institute, Tervo Fisheries Reserch nd Aquculture, 71 Tervo, Finlnd Correspondence should e ddressed to Krisn Rungrungsk-Torrissen, Krisnrt@imr.no Received 13 Novemer 8; Revised 31 Mrch 9; Accepted 15 April 9 Norml Protein (NP) nd High Protein (HP) diets were provided to rinow trout fmilies from juvenile to mturity. Muscle protein concentrtion incresed during growth nd the protein to lipid (P/L) rtio ws douled t lte stge with respect to the dietry P/L rtio. The fish showed higher protein deposition in ody nd white muscle, nd hd lower condition fctor due to protein deposition ssocited more with ody length thn ody weight. Fish growth rtes were decresed t mturtion, nd the fish showed lower growth rte including the ctivity rtio of trypsin to chymotrypsin (T/C rtio) nd feed efficiency. Trypsin nd chymotrypsin specific ctivities were relted to dietry protein levels, nd the T/C rtio ws relted to intestinl weight nd growth rte independent of the enzymes specific ctivity levels. The fmilies with high growth cpcity could doule increse white muscle P/L rtio levels, compred to low nd medium growth fmilies, if they were fed on. The digestive enzyme extrcts from high growth fmilies resulted in higher in vitro protein digestiility for ll diets. The effect of dietry P/L rtio on digestion, skeletl growth (length) nd the white muscle P/L rtio in fish with different growth cpcities is illustrted. Copyright 9 Krisn Rungrungsk-Torrissen et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. 1. Introduction One of the principl gols in contemporry quculture is the dul optimistion of growth efficiency nd protein content through process of selective culture. The growth efficiency of severl fish species hs een studied through vrition of dietry protein levels, nd the results hve shown not only the reltionships etween dietry protein concentrtion nd physiologicl prmeters of consumption, growth, nd protein content in ody or muscle, ut lso shown certin incresed levels of dietry protein limited consumption nd growth of the fish recently, tht is, [1 9]. Incresed growth is usully not ssocited with n increse in protein deposition rte, tht is, [1 1], ut rther n increse in lipid deposition rte, tht is, [8, 13]. Trditionlly in quculture, dietry protein nd lipid re vried throughout the life cycle to optimise the economic return. In the current work, we im to understnd sic knowledge on long-term protein growth y investigting the effects of mintining dietry protein nd lipid concentrtions throughout the fish life cycle on digestive efficiency nd protein growth using unique comintion of techniques recently descried y Rungrungsk-Torrissen s reserch tem [11, 1, 14]. In ddition, we lso im to study the potentil for mximising protein growth efficiency in rinow trout (Oncorhynchus mykiss Wlum) fmilies with differences in growth cpcity y vrying the concentrtions of dietry protein nd lipid. The current work ws prt of Europen Union project ssessing the potentil for genetic selection of protein nd growth efficiency trits in slmonids. The work ws performedin different fmilies of rinow trout divided into

2 Scholrly Reserch Exchnge Experimentl diets Tle 1: Compositions on wet weight sis of the experimentl diets, norml protein (NP) nd high protein (HP) diets. Moisture Crude protein (P) Crude lipid (L) P/L rtio Crude fire Ash Nitrogen free extrct Phosphorus Energy [kj g 1 ] NP (3 mm) NP (6 mm) NP (7 mm) HP (3 mm) HP (6 mm) HP (7 mm) two groups fed on two levels of dietry protein to lipid (P/L) rtio during two-yer life spn from juvenile to mturity, nd sudivided ccording to differences in growth cpcities. Feed intke ws mesured y X-ry method using diets lelled with rdio-opque mrkers [15]. The in vitro protein digestiility of formulted fish feeds ws determined using fish crude enzyme extrcts, s it hs een correlted with the feed efficiency [11, 16] nd the chemicl properties of dietry proteins, positively with the levels of rective sulphydryl group nd negtively with the levels of disulphide ond nd D-sprtic cid [16]. Fish digestive efficiency [trypsin specific ctivity nd the protese ctivity rtio of trypsin to chymotrypsin (T/C rtio)] ws mesured ecuse of their known positive ssocitions with fish growth rte [1 1, 14, 17, 18]. The effects of dietry protein nd P/L rtio levels on the protein nd lipid compositions s well s the P/L rtios of the ody nd white muscle were lso determined. The P/L rtio is importnt s oth protein nd lipid levels in the diets ffect the levels of oth protein nd lipid in fish where the vritions my not e oserved if either protein or lipid level is studied lone. Although oth protein nd lipid deposition re importnt ut the in vitro lipid digestiility ws not determined ecuse it hs never een shown to ssocite with feed efficiency nd the qulity of the lipid itself [19], nd in vitro protein digestiility is the key fctor for feed qulity even in the herivore like mussel [19, ]. This work ws designed to illustrte the effects of dietry P/L rtio levels on fish digestive ility nd the qulity of fish growth performnce (condition fctor nd composition of P/L rtio in the ody nd white muscle) in fmilies of rinow trout with different growth cpcities throughout the life history. The digestive efficiency ws studied t the end of the experiment t the lte stge of life cycle. It is dvntgeous to use the different methods simultneously, s they could precisely provide prcticl importnt iologicl informtions etween fish groups with differences in genetics (growth cpcity) nd environmentl impcts (diet qulity) tht could not e explined y using growth prmeter lone, especilly when feeding regime were not completely under control see [11, 1, 14].. Mterils nd Methods.1. Experimentl Design. The experiment ws performed on rinow trout from n on-going selection progrmme t the Finnish Gme nd Fisheries Reserch Institute, Tervo, Finlnd. One-yer old trout from 45 fmilies selected were tgged with PIT-trnsponders (Trovn Inc., Germny) nd eqully distriuted into eight 3 m 3 tnks (34 fish per tnk) t the end of Jnury. Further detils of the genetic design of the experiment hve een descried [1, ]. From htching to the initition of the experiment, ll fish were fed commercil rinow trout diets, Risio Nutr Strter [5% crude protein nd % crude lipid (P/L rtio.6)] nd Risio Nutr Prr [5% crude protein nd 3% crude lipid (P/L rtio.)] (Rehurisio Inc., Finlnd) using utomtic feeders. In My, two diet tretments were initited. Four tnks (verge fish weight 141. ±.g)were provided n experimentl norml protein (NP-) diet, nd the other four (verge fish weight ±.g) were provided n experimentl high protein (HP-) diet. The experimentl NPnd s were mnufctured y Rehurisio Inc., Finlnd. The diets were formulted using stndrd ingredients for rinow trout in Finnish quculture, consisting of fish mel, fish oil, whet mel, whet strch, nd supplemented for vitmins nd minerls ccording to the Ntionl Reserch Council [3]. The compositions of the experimentl diets re shown in Tle 1. To void restricted growth, slight over-feeding regime ws pplied. In June, the fish were trnsferred to lrger tnks ( m 3 ) nd rered through until the end of the experiment in Novemer 3. Fish density ws mintined elow kg m 3. Oxygen concentrtion in the wter ws mintined ove 6 mg L 1. The fish experienced mient sesonl temperture regimes during the experimentl period, fluctuting from elow 5 C during winters to round C in summers. Individul fish weight nd fork length were recorded in My nd Septemer, nd in My, Septemer, nd Novemer 3. Individul feed consumption ws mesured using the X-ry method [15] involving oth NP- nd s lelled with rdioopque mrkers (more detils in [1]). Nine estimtes of feed intke were clculted during the three-week X-ry sessions performed during My-June, Septemer-Octoer, nd Septemer 3. Feed efficiency ws estimted, s weight gined per weight dry feed consumed in ech session, nd verged cross ll three sessions. To estimte white muscle nd whole ody compositions, smples were collected in Novemer nd Novemer 3. Of the 45 fmilies, 16 fmilies were sorted with representtives of oth diets ville for ech fmily selected. Three

3 Scholrly Reserch Exchnge 3 differentgrowthcpcitygroupsweredivided,6fmiliesfor high (H), 5 fmilies for medium (M), nd 5 fmilies for low (L) growth cpcity, sed on the ody weight mesurements recorded in Septemer 3 (ltogether 1 individuls). At the end of the experiment in Novemer 3, pyloric cec smples were collected (3 5 fish per fmily per diet). Intestinl, gond, nd crcss weights were mesured. In vitro protein digestiility of the experimentl feeds nd fish digestive efficiency were nlysed, using the pyloric cecl enzyme extrcts. Only these 1 fish were studied in the current work. As therml growth coefficient [4] did not provide different interprettions from specific growth rte (SGR) in this experiment, the simple SGR ws chosen nd clculted [5]: SGR(% dy 1 ) = 1[e g 1], (1) where g = (ln W ln W 1 )/(t t 1 ), nd W = Weight t dy t, W 1 = Weight t dy t 1. The condition fctor ws clculted [6] Condition fctor = 1 W/L 3, () where W = Live ody weight (g) nd L = Body fork length (cm)... Determintions of White Muscle nd Body Composition. Aout 1 g of epxil white muscle ws dissected from individul fish in Novemer nd Novemer 3. The reminder of the fish ws used for determintion of whole ody composition. All tissue smples were stored t 8 C nd homogenised efore nlyses. The compositions of protein, lipid, nd dry mtter were estimted for the white muscle nd whole ody. The white muscle in Novemer nd the whole ody smples in Novemer 3 were determined using FMA1 Milk Anlyser (Miris AB, Sweden) with mid-infrred trnsmittnce, fter homogenistion in stndrd solvent Mirsolve with Losmixer (Miris AB, Sweden). Due to lter technicl complictions, the ody composition in Novemer nd the white muscle composition in Novemer 3 were nlysed using n INFRATEC Food & Feed Anlyser (Tector, Sweden) with ner-infrred trnsmittnce. To clirte etween the methods, pproximtely 5 smples were conlysed y oth nlysers (more detils in [7]). The results from INFRATEC Food & Feed Anlyser were estimted using The Unscrmler, softwre for multivrite dt nlysis (CAMO Process AS, Norwy) with multiregression models stndrdised y the vlues from 5 white muscle smples nd 5 whole ody smples nlysed chemiclly. The moisture content of the stndrds ws determined y drying known mount (out 3 g) of homogenised smple t 15 C for 4 hours to constnt weight. The lipid concentrtion ws determined from the dry mtter y extrcting with 15 ml ethylcette for 6 minutes on mixing plte, nd the lipid-ethylcette extrct ws susequently dried grdully t C, cooled in desicctor, nd then weighed [, 8 3]. The protein concentrtion of the stndrds ws determined using TRIzol regent (Life Technologies, NY, USA) to otin complete soluilistion of protein, s descried in Rungrungsk- Torrissen [11] modified from Sunde et l. [1, 18]. Protein deposition (protein growth) ws expressed s the composition rtio of protein to lipid (P/L rtio) in the ody nd white muscle..3. Determintion of Digestive Efficiency. Pyloric cec smpled from 1 individuls were nlysed for trypsin specific ctivity nd protese ctivity rtio of trypsin to chymotrypsin (T/C rtio), s indictors for digestive efficiency tht hve een relted with fish growth rte [1 1, 14, 16, 18]. All smples were kept frozen t 8 C prior to nlyses. The smples were homogenised nd the enzymes were extrcted using 1 mm HCl. After centrifugtion t 15 gfor6minutest4 C, the superntnts were kept frozen t 8 C until nlyses. Trypsin nd chymotrypsin ctivities were determined, using enzoyl-lrginine-p-nitronilide s trypsin sustrte nd N-succinyl- Al-Al-Pro-Phe-p-nitronilide s chymotrypsin sustrte, ccording to Rungrungsk-Torrissen [11] sed on Sunde et l. [1, 18] nd Rungrungsk-Torrissen et l. [16]. The concentrtion of protein in the crude enzyme extrct ws determined y the Lowry method [31] using the Bio- Rd DC (Detergent Comptile) Protein Assy (Bio-Rd Lortories, Clif, USA). The enzyme specific ctivities were expressed s μmol p-nitroniline produced h 1 mg protein 1. The protese ctivity rtio of trypsin to chymotrypsin (T/C rtio) ws directly clculted from ech individul, nd the slope T/C rtio ws otined from the regression etween trypsin (y-xis) nd chymotrypsin (x-xis) specific ctivities t ech smpling period [3]..4. In Vitro Digestiility Study. The in vitro protein digestiility of experimentl feeds ws performed ccording to Rungrungsk-Torrissen [11], modified from Rungrungsk-Torrissen et l. [16] nd Bssompierre et l. [33]. Due to limited mount of pyloric cecl smple collected from ech fish, the pyloric cecl crude enzyme extrcts were pooled per fmily per diet (16 smples for ech diet) fter determintion of the trypsin nd chymotrypsin specific ctivities in ech individul. The pooled crude enzyme extrcts were dilysed using Pierce Slide-A-Lyzer Dilysis Cssette (Pierce Chemicl Compny, Ill, USA) nd concentrted using n Amicon Ultr-15 centrifugl filter device 1 K NMWL (Millipore Corportion, Mss, USA) efore use. In vitro protein digestiility ws mesured in commercil control (C) diet [Risio Royl Response with 47% crude protein nd 3% crude lipid (P/L rtio.), Rehurisio Inc., Finlnd] nd the experimentl NP (4% crude protein with P/L rtio 1.) nd HP (49% crude protein with P/L rtio.1) diets (7 mm, see Tle 1). In vitro protein digestiility of control C-diet, nd experimentl nd were performed using crude enzyme extrcts from the 16 fmilies selected, divided into low (n = 34), medium (n = 36), nd high (n = 41) growth cpcity fmily groups. Digestion of known mount of feed (out mg in 4 ml of 5 mm phosphte uffer ph 8. nd. ml

4 4 Scholrly Reserch Exchnge Weight (g) Apr- My- Jun- Jul- Aug- Sep- Oct- Nov- Dec- Jn-3 Fe-3 Mr-3 Apr-3 My-3 Jun-3 Jul-3 Aug-3 Sep-3 Oct-3 Nov-3 Dec-3 Condition fctor Weight Figure 1: Weight nd condition fctor of rinow trout fed norml protein (NP) nd high protein (HP) diets during the entire experimentl period. The vlues with sterisk ( ) re significntly different (P <.5) etween the dietry groups. of.5% chlormphenicol) y enzyme extrct (.5 ml) ws performed in triplicte t 15 Cfor4hours[11] forech diet nd ech dilysed pooled crude enzyme extrct. The rective mino groups of peptides produced fter digestion were determined using the trinitroenzene sulphonic cid (TNBS) ssy nd clculted using DL-lnine s stndrd, s descried in Rungrungsk-Torrissen [11], modified from Bssompierre et l. [33] sed on Ihekoronye [34]. The in vitro protein digestiility vlue ws expressed s μmol DLlnine equivlent lierted rective mino group of cleved peptides per mg feed smple fter stndrdising trypsin ctivity in ech dilysed pooled crude enzyme extrct to 3 μmol p-nitroniline produced h 1 ml Sttisticl Anlysis. The sttisticl procedures were performed using the SAS softwre pckge (v. 8. for Windows, SAS Institute Inc., NC, USA). Reltionships, on n individul sis, etween different diets, fmilies, nd growth cpcity groups were studied using the generl liner model (glm) procedure. This nlysis included individul continuous vriles (covrites: e.g., white muscle P/L rtio or SGR). In cses where clss-y-covrite interction ws significnt, the nlysis ws roken down nd performed per clss (e.g., per diet or per growth cpcity group). All results re given s the lest squres mens±stndrd error. Differences etween mens were tested y t-test. The difference ws set t 95% significnce level. 3. Results 3.1. Fish Growth nd Feed Efficiency. Due to some uncertin lelling, only 111 out of 1 fish were used for the nlyses. Pired comprison indicted tht the fish hd lower condition fctor thn the fish during the entire experimentl period (P <.5; Figure 1). The differences in condition fctor were enhnced during utumn to winter, especilly the lst period from Septemer to Novemer Condition fctor (g/cm 3 ) (P <.1; Figure 1 nd Tle ).This ws due to differences in ody length etween the diet tretments (P <.3; Tle ). There were no significnt differences in ody weight etween the two dietry groups lthough the fish seemed to hve reltively higher ody weight thn the fish efore mturtion (P >.5). During mturtion in the lst two months, fish growth rtes were reduced in oth dietry groups, nd the fish showed higher decrese in SGR (P <.1) withtheody weight chnged (P >.5) from reltively higher in Septemer 3 to reltively lower in Novemer 3, compred to the fish (Figure 1 nd Tle ). The fish lso hd lower feed efficiency (FE) thn the fish (P <.5; Tle ). Individul feed intke in Septemer 3 ws not correlted with FE ut showed positive correltion with SGR during the lst two months (R =.11, P<.5). There were reltionships etween ody weight nd crcss weight, nd etween ody weight nd intestinl weight (R =.49.98, P <.1). The reltionships etween SGR with crcss weight nd etween SGR with intestinl weight were lso oserved (R =.8.9, P<.1). This ws consistent for oth dietry tretments. However, intestinl weight ws lower in the fish (P <.1; Tle ), due to reduction in lipid deposits round the intestine (oserved visully). No differences in crcss nd gond weights (Tle ) were oserved etween the two dietry groups. Mturtion rte seemed to e higher in the thn the fish in ll growth cpcity groups, vried from % in the fish compred to % in the fish. 3.. Digestive Proteses in the Pyloric Cec. At mturity in Novemer 3, rinow trout fed on hd higher specific ctivities of the protese enzymes trypsin (P <.) nd chymotrypsin (P <.1),nd lower T/C rtio(p <.3), thn those fed on (Tle ). When the fish were grouped ccording to fmily growth cpcity, the sme trends were lso oserved within ech growth cpcity group, ut the difference ws only significnt in the chymotrypsin specific ctivity etween the fish fed HP- nd s in the high growth cpcity fmilies (P <.5; Tle ). Trypsin (P<.; Figure ()) nd chymotrypsin (P <.3; Figure ()) specific ctivities were inversely correlted with the intestinl weight. In contrst, the T/C rtio ws positively correlted with the intestinl weight (P <.3; Figure (c)). Similrly, the specific ctivities of trypsin (R =.5, P <.) nd chymotrypsin (R =.4, P <.5) were inversely correlted with ody weight, nd the T/C rtio showed trend of positive correltion with fish ody weight lthough insignificnt (dt not shown). Among the protese prmeters mesured, only the specific ctivity of trypsin ws inversely correlted with SGR (R =.4, P<.4) during mturtion, regrdless of dietry type (dt not shown). Within ech dietry group, these reltionships were not significnt, except for the negtive correltion in the group etween intestinl weight nd trypsin specific ctivity

5 Scholrly Reserch Exchnge 5 Tle : Different iologicl prmeters in rinow trout fed the experimentl norml protein (NP) nd high protein (HP) diets, in vitro protein digestiility ws expressed s μmol DL-lnine equivlent lierted rective mino group of cleved peptides per mg feed fter stndrdising trypsin ctivity in ech dilysed pooled crude enzyme extrct to 3 μmol p-nitroniline produced h 1 ml 1, nd the vlues with sterisk ( ) re significntly different (P <.5). Prmeters group (n = 56) group (n = 55) Initil weight in My [g] 14 ± ± 5 Septemer 3 Weight [g] 1 ± ± 44 Length [cm] 47.9 ± ±.34 Condition fctor [g cm 3 ] 1.91 ±. 1.8 ±. Novemer 3 Finl weight [g] 6 ± ± 6 Finl length [cm] 51. ± ±.4 Finl condition fctor [gcm 3 ] 1.96 ± ±.3 Specific growth rte (Sept Nov 3) [%dy 1 ].5 ±..4 ±. Feed efficiency [dily-gin in g per g feed-intke] Averge of ll three X-ry sessions 1.73 ± ±.9 Trypsin specific ctivity [μmol p-nitroniline h 1 mg protein 1 ] 8.7 ±. 41. ± 4.5 low growth cpcity group 3.1 ± 4.5(n = 17) 39.3 ± 7.9 (n = 17) medium growth cpcity group 7.7 ± 3.3(n = 17) 4. ± 7. (n = 19) high growth cpcity group 8.3 ± 3.7(n = ) 43.4 ± 8.8 (n = 19) Chymotrypsin specific ctivity [μmol p-nitroniline h 1 mg protein 1 ] 1 ± ± 1 Low growth cpcity group 18 ± 19 (n = 17) 139 ± (n = 17) Medium growth cpcity group 13 ± 17 (n = 17) 138 ± 15 (n = 19) High growth cpcity group 96 ± 13 (n = ) 151 ± 4 (n = 19) Protese ctivity rtio of trypsin to chymotrypsin (T/C rtio).3 ±..7 ±.1 Low growth cpcity group.31 ±. (n = 17).5 ±. (n = 17) Medium growth cpcity group.33 ±.4 (n = 17).8 ±.3 (n = 19) High growth cpcity group.3 ±.3 (n = ).9 ±.3 (n = 19) In vitro digestiility [μmol DL-lnine equivlent mg feed 1 ] Control diet 4.5 ±.45 (n = 16) 4.61 ±.69 (n = 16) NP diet 4.4 ±.45 (n = 16) 4.8 ±.75 (n = 16) HP diet 4.9 ±.48 (n = 16) 5.49 ±.8 (n = 16) In vitro digestiility regrdless of diet type Low growth cpcity group 4.3 ±.44 (n = 15) 3.85 ±.48 (n = 15) Medium growth cpcity group 4.17 ±.39 (n = 15) 3.91 ±.43 (n = 15) High growth cpcity group (P <.6) 4.85 ±.37 (n = 18) 6.77 ±.49 (n = 18) Intestinl weight [g] 34 ± 1 79 ± 9 Gond weight [g] 6 ± 7 7± 7 Crcss weight [g] 191 ± ± 5 Proximte composition [% on wet weight sis] Muscle protein.9 ± ±.4 Muscle lipid 8.6 ± ±.3 Muscle rtio of protein to lipid (P/L rtio).9 ± ±. Body protein 15.7 ± ±.7 Body lipid.1 ±.17. ±.19 Body rtio of protein to lipid (P/L rtio).71 ±.1.8 ±.1 (P <.3; Figure ()) nd etween finl weight nd trypsin specific ctivity (R =.9, P<.3). The oservtions of similrity in slope T/C rtio (Figure 3) etween the (.) nd the (.1) groups, nd the lck of the reltionship etween trypsin specific ctivity nd the T/C rtio (see detils in [1, 14]) indicted tht the fish were t similr stedy growth phse t smpling t the end of the experiment.

6 6 Scholrly Reserch Exchnge Trypsin specific ctivity Chymotrypsin specific ctivity T/C rtio Intestinl weight (g) () y (totl) =.979x R =.98, n = 111, P<. y (HP) =.159x R =.95, n = 55, P<.3 y (NP) =.41x R =.5, n = 56, ns y (totl) =.3x +.4 R =.48, n = 111, P<.3 y (HP) = 1E 4x +.43 R =.3, n = 55, ns y (NP) =.x +.5 R =.37, n = 56, ns Intestinl weight (g) () y (totl) =.3574x R =.789, n = 111, P<.3 y (HP) =.45x R =.51, n = 55, ns y (NP) =.165x R =.46, n = 56, ns Intestinl weight (g) (c) Liner () Liner () Figure : Reltionships etween intestinl weight with trypsin specific ctivity [], chymotrypsin specific ctivity [], nd protese ctivity rtio of trypsin to chymotrypsin (T/C rtio) [c]. The protese specific ctivities were expressed s μmol p-nitroniline produced h 1 mg protein White Muscle nd Body Compositions. SGR correlted positively with lipid concentrtion (P <.1) in the white muscle, nd negtively with moisture (P <.1) nd protein (P <.) concentrtions (Figure 4). This resulted in n inverse reltionship etween SGR nd the composition rtio of protein to lipid (P/L rtio) in the white muscle (P < Trypsin specific ctivity y =.x R =.73, n = 55, P<.1 y =.1x R =.785, n = 56, P< Liner () Chymotrypsin specific ctivity Liner () Figure 3: Reltionships etween trypsin (T) nd chymotrypsin (C) specific ctivities showing the slope T/C rtios (old vlues) of rinow trout fed norml protein (NP) nd high protein (HP) diets t smpling t the end of the experiment in Novemer 3..1; Figure 4). Within ech dietry group, the reltionships were significnt only in the fish (P <.), except for protein concentrtion tht no correltions were oserved in oth dietry groups (Figure 4). At successive mesurements, protein concentrtion incresed significntly in the white muscle (P <.1) from Novemer (.5 ±.8%) to Novemer 3 (4.34 ±.1%). Lipid concentrtion (7.48 ±.19 vs 7.71 ±.8%), P/L rtio (3.53 ±.9 vs 3.6 ±.5) lso incresed, leit insignificnt. Moisture concentrtion, on the other hnd, decresed (P <.1) from Novemer (7.1 ±.1%) to Novemer 3 (7.14 ±.5%). At the end of the experiment, the moisture concentrtion in the white muscle ws significntly higher in fish fed on thn those fed on (71. ±. vs69.7 ±.%, P <.1). The concentrtion of protein (P <.4) nd the P/L rtio (P <.1) in the white muscle were lso higher nd the lipid concentrtion ws lower (P <.1) in the fish fed the HPdiet thn the (Tle ). According to the dietry P/L rtio levels in the HP (.1.7) nd the NP (1. 1.5) diets (Tle 1), rinow trout t lte stge seemed to doule the P/L rtios in the white muscle ( fish: 4.44 ±.; NPdiet fish:.9 ±.14) (Tle ) with respect to the P/L rtio of the diet consumed. For whole ody composition, SGR lso showed positive correltion with lipid concentrtion (R =.9, P<.), nd negtive correltion with protein concentrtion (R =.1, P <.1), resulting in n inverse reltionship with the P/L rtio (R =.11, P <.1). Similr to white muscle composition, whole ody concentrtions of protein (Novemer : 9.94 ±.14%; Novemer 3: 16.4 ±.6%), lipid (17.98 ±.8 vs 1.14 ±.16%), nd P/L rtio (.56 ±.1 vs.77 ±.1) incresed etween successive mesurement periods (P <.1). At the end of the experiment, fish fed on hd higher concentrtions of whole ody protein (16.44 ±.7 vs ±.7%) nd P/L

7 Scholrly Reserch Exchnge 7 White muscle composition (%) Moisture P/L rtio Protein Lipid R =.8, ns Liner () Liner () SGR (%/dy) Liner () Liner () y = 1.4x R =.1, ns y = 3.76x R =.136, P<.1 y = 1.878x R =., ns y =.381x R =.115, P<. y =.467x R =.8, ns y = 4.715x +5.8 R =.48, ns y = 5.595x R =.18, P<.1 y = 1.647x Figure 4: Reltionships etween specific growth rte (SGR) with white muscle composition nd white muscle rtio of protein to lipid (P/L rtio). Regrdless of dietry group, the regressions re y = 4.641x (R =.1579, n = 111, P <.1 for moisture), y = 5.95x (R =.553, n = 111, P<. for protein), y = x (R =.1444, n = 111, P<.1 for lipid) nd y = x (R =.153, n = 111, P<.1 for P/L rtio). rtio (.8 ±.1 vs.71 ±.1), nd lower concentrtion of whole ody lipid (.15 ±.19 vs.1 ±.17%) thn the fish (P <.1; Tle ). Moreover, there ws significnt inverse reltionship etween ody P/L rtio nd the condition fctor, regrdless of dietry group (P <.3; Figure 5). Within the dietry groups, the reltionship ws significnt only in the fish (P <.4; Figure 5). Among different composition prmeters studied, white muscle protein concentrtion ws the only fctor tht significntly correlted with feed efficiency, with negtive reltionship (R =.11, P<.5), regrdless of dietry type In Vitro Digestiility nd Comprisons etween Different Growth Cpcity Groups. Low growth cpcity fmilies hd lower finl weight (53 ± 63 g) thn those from medium (684 ± 63 g) nd high (791 ± 85 g) growth cpcities (P <.1). Within ech growth cpcity group, no differences in weight (Figure 6()) ndthet/crtio(figure 6()) were oserved etween the fish fed nd. Feed efficiency ws significntly different etween the two dietry tretments only in the medium growth cpcity fmilies (P <.1; Figure 6(c)).TheP/Lrtiointhewhitemuscle ws higher in the thn the fish in ll growth groups (P <.; Figure 6(d)). Interestingly in the high growth fmilies, the white muscle P/L rtio of the Muscle rtio of protein to lipid (P/L rtio) Condition fctor (g/cm 3 ) y (totl) =.793x +.58 R =.15, n = 111, P< Body P/L rtio Liner () Liner () y =.476x +.46 R =.37, n = 55, ns y = 1.336x R =.155, n = 56, P<.4 Figure 5: Reltionship etween ody P/L rtio nd condition fctor. fish incresed 8% more thn the fish, wheres the increse ws only 4% in the low nd medium growth fmilies (P <.3; Figure 6(d)). The digestive ility ws different mong different growth cpcity groups (Figure 7). The enzyme extrcts from fmilies with high growth cpcity resulted in reltively higher in vitro protein digestiility vlues for C-diet (P <.5), (P <.5), nd (P >.5) thn those from low nd medium growth cpcity fmilies (Figure 7). Pired comprison indicted significntly higher in vitro protein digestiility of the thn the C-diet nd the, regrdless of the enzyme extrcts used (P <.1). Within ech growth cpcity fmily, tendency of higher in vitro protein digestiility ws oserved in the enzyme extrcts from fish fed, leit insignificnt (Tle ). A significnt inverse reltionship etween in vitro protein digestiility vlues nd SGR (R =.18, P <.) ws oserved t mturity in Novemer 3, regrdless of dietry type Anlysis of Covrince. Thecontinuousvrilewhite muscle P/L rtio ws included s covrite in the models in ordertostudyitseffect on growth together with fish growth cpcity group or fmily fed on the different diets. Due to clss-y-covrite interction eing significnt (P <.5), the sttisticl nlysis ws performed seprtely per clss. The effects on the growth prmeters (finl weight, condition fctor, nd SGR) y growth cpcity group nd muscle P/Lrtio, nd y fmily nd muscle P/L-rtio, were studied seprtely for ech dietry group (Tle 3). Differences in fish growth cpcity significntly ffected finl ody weight for oth diets ut did not ffect condition fctor nd SGR. Fmily influenced fish SGR for oth diets, nd the growth of the fish fed on ws highly ffected y fmily nd muscle P/L rtio compred to the fish. The results clerly indicted tht the differences in fish growth cpcity mong different fmilies nd the level of P/L rtio in the whitemuscleffected fish growth, nd their effects vried ccording to the dietry P/L rtio.

8 8 Scholrly Reserch Exchnge Finl weight (g) T/C rtio Low growth Medium growth High growth Low growth Medium growth High growth () () Feed efficiency Muscle P/L rtio c.5 1 Low growth Medium growth High growth Low growth Medium growth High growth (c) (d) Figure 6: Finl ody weight [], protese ctivity rtio of trypsin to chymotrypsin (T/C rtio) [], feed efficiency [c], nd white muscle composition rtio of protein to lipid (P/L rtio) [d], in groups of rinow trout with low, medium, nd high growth cpcity, sudivided ccording to feeding norml protein (NP) or high protein (HP) diet. The vlues with different superscripts re significntly different (P <.5). In vitro digestiility C-diet Low growth Medium growth High growth Figure 7: In vitro protein digestiility of control (C), norml protein (NP), nd high protein (HP) diets, mong rinow trout with low, medium, nd high growth cpcities. The vlues with different superscripts re significntly different (P <.5). In order to study the ssocition etween SGR, dietry qulity, nd digestive ilities of different growth cpcity groups, the continuous vrile SGR ws included s covrite in the models. Similrly, the clss-y-covrite interction ws significnt (P <.5); thus, the sttisticl nlysis ws lso performed seprtely per clss. The effects on specific ctivities of trypsin nd chymotrypsin, T/C rtio, nd in vitro protein digestiility of diet, y dietry P/L rtio nd SGR were studied for ech growth cpcity group (Tle 4). The results indicted n effect of the diet on the T/C rtio of the low growth cpcity group. There ws n indiction of the ssocitions etween SGR nd the specific ctivities of trypsin nd chymotrypsin. Trypsin specific ctivity ws ffected in the high growth cpcity group, wheres chymotrypsin specific ctivity ws ffected in the low growth cpcity group. There ws n ssocition etween SGR nd in vitro protein digestiility of the experimentl diets in the high growth cpcity group. The nlyses of covrince (Tles 3 nd 4) indicted the ssocitions etween dietry P/L rtio, fish digestive ility (trypsin nd chymotrypsin specific ctivity, T/C rtio, in vitro protein digestiility), white muscle P/L rtio, growth prmeters (fish weight, condition fctor, SGR), nd vritions in growth cpcity of different fmily memerships.

9 Scholrly Reserch Exchnge 9 Tle 3: Anlysis of covrince showing the effects of fish growth cpcity (Group) or fmily nd white muscle rtio of protein to lipid (P/L rtio) s covrite, on finl weight, condition fctor, nd specific growth rte (SGR) during Septemer Novemer 3. Due to clss-ycovrite interction ws significnt (P <.5); the sttisticl nlysis ws performed seprtely for the experimentl norml protein (NP) nd high protein (HP) diets. Proilities with significnt effects re shown y the P vlues <.5 (old vlues). () Covrince effect df Finl weight Condition fctor SGR NP HP NP HP NP HP Group Muscle P/L rtio Fmily < Muscle P/L rtio Group Fmily () NP HP Finl weight R =.389 (P <.1) R =.4 (P <.5) Condition fctor R =.186 (P <.15) R =.13 (P =.883) SGR R =.117 (P =.89) R =.66 (P =.31) Finl weight R =.66 (P <.5) R =.47 (P =.17) Condition fctor R =.75 (P <.1) R =.37 (P =.195) SGR R =.56 (P <.5) R =.467 (P <.35) 4. Discussion Fish growth ws ffected y dietry P/L rtio nd y the inherent growth cpcity of the fish s well s y white muscle P/L rtio (Tle 3) nd y genetic differences in protese digestive ility (Tle 4). At lte stge, rinow trout seemed to doule their white muscle P/L rtio with respect to the dietry P/L rtio (Tles 1 nd ). Incresed dietry protein concentrtion resulted in elevted protein nd reduced lipid concentrtions in fish ody ([1, 6, 35], current work). In the white muscle, incresed dietry protein resulted in elevtion of oth protein nd moisture contents [current work]. Dietry protein concentrtion is lso inversely correlted with protein efficiency rtio nd productive lipid vlue [3]. Incresing dietry protein concentrtion hs een ssocited with incresed muscle protein in slmonids ([35], current work), chnnel ctfish Ictlurus puncttus [] nd grss crp Ctenophryngodon idell [4], nd incresed fillet mss nd decresed liver nd dipose tissue mss in the dominl cvity in Africn ctfish Clris griepinus [7]. In ddition, fish fed high protein diets tend to hve lower totl lipids, triglycerides, nd cholesterol in fillets, with higher percentge of polyunsturted ftty cids (PUFAs) nd lower sturted ftty cids [7]. Thus, incresing protein in the diet could result in improved fillet qulity. Moreover, ppered to promote n increse in length rther thn weight during growth (Figures 1 nd 5). Our work illustrtes the importnce of dietry protein level not only on fillet qulity ut lso on skeletl growth. Deformities in juvenile tench (Tinc tinc L.) with significntly elevted whole ody lipid hve een reported [36]. Incresed growth in Atlntic slmon (Slmo slr L.) stimulted y continuous light, which is not cused y incresed protein deposition [1] ut y incresed lipid deposition [13], results in reduced mechnicl strength nd growth of the vertere in Atlntic slmon postsmolts [37]. Thus, stimulting fish growth without concerning with pproprite dietry P/L rtio nd P/L rtio deposition in muscle or ody my ffect the strength of skeletl growth nd cuse deformities. Fish growth could e explined not only y ody weight ut lso y ody length illustrted s condition fctor ([6], current work). Therefore, the conventionl study for dietry protein requirement through optiml weight gin in fish (recently, i.e., [1, 3, 5, 6, 8]) should proly insted indicte the effectof optiml dietry protein level on the chnge of fish growth performnce to greter increse in length thn weight. It is interesting to note tht fish with different growth cpcities [current work] nd trypsin phenotypes [14, 38] hve different protese digestive ilities. Fish from high growth group hd reltively higher digestive ility for ll feed types, compred to those from low nd medium growth groups (Figure 7). There were ssocitions etween growth nd protein digestiility (Tle 4)s well s etween growth nd muscle P/L rtio (Tle 3). The high growth cpcity fmilies hd higher cpcity to increse their growth through increse in white muscle protein deposition (Figure 6(d)). In ddition, white muscle fire size ws lso significntly lrger in the high growth cpcity fish compred to the medium nd low growth cpcity groups (pooled dt, P <.1), irrespective of the diet (unpulished dt). Furthermore, genetic correltions etween percent white muscle re nd protein percent in rinow trout cutlets were very high on oth diets,.76 in nd.9 in groups [39]. These results indicte the dvntge of high growth cpcity in protein growth performnce qulity. For more understnding on the effect of dietry P/L

10 1 Scholrly Reserch Exchnge Tle 4: Anlysis of covrince showing the effects of dietry P/L rtio level (Diet) nd specific growth rte (SGR) during Septemer Novemer 3 s covrite, on the expression of trypsin nd chymotrypsin (expressed s μmol p-nitroniline h 1 mg protein 1 ), the ctivity rtio of trypsin to chymotrypsin (T/C rtio), nd in vitro protein digestiility (expressed s μmol DL-lnine equivlent per mg feed fter stndrdising trypsin ctivity to 3 μmol p-nitroniline produced h 1 ml 1 ). Due to clss-y-covrite interction ws significnt (P <.5); the sttisticl nlysis ws performed seprtely for low (L), medium (M), nd high (H) growth cpcity groups. Proilities with significnt effects re shown y the P vlues <.5 (old vlues). Covrince effect df () Trypsin Chymotrypsin T/C rtio In vitro digestiility L M H L M H L M H L M H Diet SGR () L M H Trypsin R =.111 (P =.163) R =.139 (P =.85) R =.173 (P <.3) Chymotrypsin R =.5 (P <.35) R =.147 (P =.78) R =.169 (P <.3) T/C rtio R =.131 (P =.13) R =.9 (P =.13) R =.15 (P =.746) In vitro digestiility R =.41 (P =.53) R =.16 (P =.764) R =.163 (P <.35) rtio on muscle composition nd growth, if possile, other prmeters such s cellulr growth should e investigted in the future in order to study the insight effect of genetics nd nutrition on fish growth. Growth rte ws correlted with feed intke ut ws not correlted with feed efficiency (FE), s FE reduces when consumption increses eyond threshold level [11, 1]. The inverse reltionship oserved etween FE nd white muscle protein concentrtion (not with ody protein concentrtion) ws proly due to limited cpcity to deposit muscle protein. Differences in FE hve een ssocited with vritions in the levels of totl free mino cids in the plsm nd white muscle s well s in the rtio of essentil to nonessentil free mino cids, nd with vritions in free hydroxyproline nd RNA concentrtions in the white muscle [18]. In ddition, reltionships hve een oserved etween T/C rtio in the pyloric cec nd concentrtion rtios of essentil to nonessentil free mino cids in plsm nd white muscle s well s etween T/C rtio with free hydroxyproline nd RNA concentrtions in the white muscle [1]. Moreover, significnt positive reltionship etween FE nd T/C rtio hs een oserved in fish fed diets vrying in dietry qulity [11, 16]. The current work further emphsises these ssocitions etween FE, T/C rtio, nd levels of protein nd lipid utilistion for deposition in the white muscle. High protein concentrtion in the white muscle of mturing rinow trout, s high s 3% with 4 ±.1% on totl verge oserved in the current study (Tle ) compred to norml vlues of 15 % reported [4], ws proly due to complete soluilistion of protein y using the newly developed method, s higher protein soluility resulted in higher percentge protein mesurements. The high white muscle protein concentrtion might s well e chrcteristic of mturing slmonids, s it hs lso een oserved in mturing Atlntic slmon [11], which is proly due to moilistion of muscle lipid s energy for reproduction. The highest muscle protein concentrtion oserved in mturing Atlntic mckerel (Scomer scomrus L.) ws only 4% with 14 ± 1% on verge [1]. Whilst it ws % with 17 ±.1% on verge in Atlntic slmon of out 5 g [11], nd 5% with 1 ±.1% on verge in rinow trout of out 1 kg [current work]. During the finl yer of the experiment, the composition of ody nd white muscle successively chnged with concentrtion of protein incresing nd moisture concentrtion decresing. Higher dietry protein induced increses in protese specific ctivities of trypsin nd chymotrypsin in the pyloric cec (Tle ). In previous experiment using the sme feed ut vrying rering conditions, oth trypsin specific ctivity nd T/C rtio were relted to growth rte [1], wheres in the current work vrying in feed qulity, only the T/C rtio ws positively ssocited with growth rte (Tle ). Thus, the T/C rtio hs n effect on growth efficiency independent of the specific ctivity level of the proteses. This hs lso een oserved in Atlntic slmon [3] nd gene mnipulted fish [1, 41].Areduction in T/C rtio in the fish,reltive to fish, cused y greter increse in the ctivity of chymotrypsin over trypsin confirmed reduction in growth rte (Tle ).Thishseenreportedinseverlworks[11, 1, 14, 18, 3]. The confirmtion is lso strengthened y the oserved ssocitions etween trypsin specific ctivity nd high growth nd etween chymotrypsin specific ctivity nd low growth (Tle 4). In vitro protein digestiility vlues of different diets (Figure 7 nd Tle 4) were influenced y fish growth cpcity nd the levels of protese expression in fish enzyme extrcts. The in vitro protein digestiility of diets hs een ffected y genetic differences in trypsin phenotypes [33, 38], nddifferent trypsin phenotypes ffectedvritions in the protese ctivity expression levels [14] nd FE[4]. At smpling in Novemer 3, the fish were t similr stedy growth phse showing y similr slope T/C rtio (Figure 3) (detils in [3]) nd no correltion etween trypsin specific ctivity nd the T/C rtio (dt not shown) (detils in [1, 14]). At stedy growth phse, utiliztion of protein for deposition nd growth would e reduced

11 Scholrly Reserch Exchnge 11 to mintennce level tht should e stedy irrespective of diet. This is confirmed y the oserved higher rte of nitrogen excretion in the fish thn the fish with similr solute nitrogen retention etween the two dietry groups t the end of the experiment (D. Toin, personl communiction). If the fish hd een t growing phse, lower nitrogen excretion per kg of fish growth in rinow trout fed diet with higher protein content [43] nd incresed nitrogen retention with incresed protein deposition [35] would hve een oserved in the HPdiet fish. Growth sttus of the fish is very importnt for interpreting iologicl prmeters. At present, the slope T/C rtio nd the reltionship etween trypsin specific ctivity nd the T/C rtio in the pyloric cec seem to e the only indictors ville for predicting reltive growth level nd growth sttus of the fish t smpling, respectively, without knowing fish history see [1, 14, 3]. Our results suggest the ssocition etween the diet dependent in vitro protein digestiility vlue nd protein growth in fish, wheres the T/C rtio is ssocited with fish growth regrdless of protein or lipid growth. In vitro digestiility y protein digestion is the key fctor for diet qulity, not only in crnivores ut lso in herivores where crohydrte digestion (the second importnt digestion fctor in herivores fter protein digestion) is lso relted to dietry protein levels [19, ] while lipid digestion is not relted to ny diet qulity [19]. A high protein diet my limit feed efficiency, weight, nd growth rte of the fish, s they ecome mture. Anlyses of covrince lso indicte the ssocitions mong genetic differences in protese digestive efficiency, the dietry P/L rtio, nd fish growth (Tle 4), nd showing the influence of white muscle P/L rtio on the SGR (Tle 3). The T/C rtio (directly clculted from the enzyme ctivities) nd the slope T/C rtio (otined s shown in Figure 3) re the suitle fctors for comprison of growth efficiency etween fish groups over period of 1- months nd t smpling, respectively, regrdless of either protein or lipid growth or the protese specific ctivity levels ([1 1, 14, 18, 3], current work). It is importnt to clculte the T/C rtio nd slope T/C rtio in the pyloric cec for comprison insted of using trypsin specific ctivity lone. If fish re needed live, iopsy of pyloric cec could e performed [44], or the T/C rtio in feces could lso e used for comprison of digestive nd growth efficiency ut not for predicting growth sttus [11]. Most oserved reltionships etween key fctors hve very low correltion coefficients [1, 14, 45], current work). This is due to their indirect interction in series of iologicl mechnisms. The phenomenon is common, s mny fctors eing involved in iologicl processes. The dietry protein concentrtion of 5% used in this work is not ctully prcticl in the production of lrge fish due to high cost. It ws purely intended to ct s chllenge environment to optimising the genetic response of trits such s protein nd growth efficiency in slmonids [1,, 7]. However, s long-term study, it hs enled us to investigte the effect of dietry protein on skeletl growth (length), which is importnt knowledge for future production of fish with high growth performnce qulity [3] s well s for future growth study in nturl ecosystems where protein deposition is the min cuse [1]. 5. Conclusions Dietry P/L rtio ffected the level of digestion nd utilistion of protein nd lipid for deposition in the white muscle tht influenced feed efficiency nd growth of the fish. Muscle protein concentrtion incresed etween successive mesurement periods, nd older lrger fish hd higher concentrtion thn younger smller fish. Fish with higher growth cpcity hd etter chnce to deposit higher protein. Protein deposition ws ssocited more with ody length thn ody weight, resulting in lower condition fctor. Fish with higher protein growth hd lower feed efficiency nd SGR during mturtion. Genetic effect of protese digestive efficiency on fish growth is evident ([14, 38], current work). In vitro protein digestiility of experimentl diets using fish crude enzyme extrct nd stndrdised y trypsin ctivity is useful not only for comprison nd prediction of iologicl qulity of different diets [11, 16], ut lso for comprison of genetic differences in fish digestive efficiency ([33], current work) nd fish growth cpcity [current work]. Specific ctivity levels of trypsin nd chymotrypsin were relted to dietry protein concentrtions. The T/C rtio nd slope T/C rtio were relted to growth efficiency over period of 1- months nd t smpling, respectively, regrdless of protein or lipid growth or the specific ctivity levels of the two enzymes ([1, 3], current work). A reltively higher increse in chymotrypsin over trypsin ctivity, resulting in lower T/C rtio, ffected reduction in growth rte nd indicted the ssocitions etween trypsin specific ctivity nd high growth nd etween chymotrypsin specific ctivity nd low growth ([1, 14, 3], current work). The ssocitions of ll prmeters studied y the nlyses of covrince (Tles 3 nd 4) indicte the influences of oth externl (dietry P/L rtio) nd internl (vritions in inherent growth cpcity of different fmily memerships) fctors on fish growth, through iologicl chnges in digestive ility (trypsin nd chymotrypsin specific ctivity, T/C rtio, in vitro protein digestiility) nd white muscle P/L rtio. High dietry protein results in similr weight, ut higher growth in length (skeletl growth) nd etter fillet qulity tht would nutritionlly enefit to consumers, compred to high dietry lipid. The work shows the dvntge of using the unique comintion of the prcticl methods for growth performnce qulity study tht could e performed in ny uncontrolled condition, such s in the nturl ecosystem where protein deposition is importnt nd study on iologicl responses to environmentl chnges for survivl hs een ignored. It lso provides more knowledge on dietry protein ffecting skeletl growth s well s the interction etween genetics nd nutrition ffecting digestive ility nd growth performnce qulity of the niml. Acknowledgments The uthors thnk T. Pnnen nd K. Mnninen, the stff t the Finnish Gme nd Fisheries Reserch Institute (Tervo,

12 1 Scholrly Reserch Exchnge Finlnd), H. Whlroos (then t MTT Agrifood Reserch Finlnd) for their contriutions nd tking cre of the fish, nd L. Dyrhovden (Institute of Mrine Reserch, Norwy) for ssisting in collecting smples. The work ws finncilly supported y the Commission of the Europen Communities, Qulity of Life nd Mngement of Living Resources Progrmme. It ws prt of the EU-project Q5RS-1-994, protein nd growth efficiency in slmonid selection (PROGRESS), where the uthors would like to thnk K. Ruohonen (then t Finnish Gme nd Fisheries Reserch Institute, Turku, Finlnd), E. Mäntysri nd A. Kuse (MTT Agrifood Reserch Finlnd, Jokioinen, Finlnd), D. F. Houlihn nd S.A.M. Mrtin (University of Aerdeen, UK), nd A. Kiessling (then t the Institute of Mrine Reserch, Norwy) for their coopertions. The project ws coordinted y the Finnish Gme nd Fisheries Reserch Institute, Finlnd. The University of Bergen, Norwy, ws the sucontrctor of the Institute of Mrine Reserch, Norwy. 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