Aquaculture Nutrition

Transcription

1 Aquculture Nutrition ; doi: /j x 1, ICBAS Instituto de Cieˆncis Biome dics Abel Slzr nd CIIMAR/CIMAR Centro Interdisciplinr de Investigç o Mrinh e Ambientl, Universidde do Porto, Porto, Portugl; 2 Ntionl Reserch Council of Cnd, Institute for Mrine Biosciences, Hlifx, NS, Cnd Three isonitrogenous (520 g protein kg )1 DM) nd isoenergetic (25 MJ kg )1 DM) diets contining incresing levels of flxseed oil (FxO; 0%, 40% nd 70% of totl dded oil) t the expense of fish oil (FO) were tested for 33 weeks in groups of 61 individully PIT-tgged hlibut (initil weight, 849 ± 99 g). Effects on fish growth performnce, fillet nutritionl nd sensory qulity were determined. Specific growth rte (0.2% dy )1 ), feed conversion rtio ( ) nd nitrogen nd energy retention were not ffected by dietry tretments. Dietry ftty cid composition ws reflected in ftty cid profiles of hlibut muscle, liver nd hert. Muscle of fish fed FxO diets contined higher 18:2n-6 nd 18:3n-3 concentrtions wheres 20:5n-3 nd 22:6n-3 levels were significntly reduced. However, incresing FO replcement induced preferentil retention of 22:6n-3 especilly in hert, nd trend for 20:5n-3 conservtion in hert nd muscle ws observed. FO replcement did not ffect colour, texture nd the chrcteristic fish odour nd flvour of cooked fillets. By selectively retining long-chin polyunsturted ftty cids hlibut cn dpt to lower dietry supply without dverse effects on growth, feed conversion rtio, survivl, nd fillet nutritionl nd sensory qulity. KEY WORDS: Atlntic hlibut, fish oil, flxseed oil, growth, nutritionl qulity, orgnoleptic qulity Received 30 November 2010, ccepted 11 Mrch 2011 Correspondence: L.M.P. Vlente, Centro Interdisciplinr de Investigç o Mrinh e Ambientl, Ru dos Brgs 289, Porto, Portugl. E-mil: lvlente@icbs.up.pt The production of Atlntic hlibut (Hippoglossus hippoglossus) hs incresed in recent yers in Norwy, Icelnd, Scotlnd, Chile nd Cnd, due to both consumer demnd for high qulity sefood product nd declining wild ctches. Mrine fish oils hve been the trditionl source of essentil ftty cids nd energy in diets for frmed mrine fish. The current level of mrine fish oil being used globlly by the expnding quculture industry hs led to incresed concern bout the overexploittion of mrine resources. Vegetble oils re widely vilble, hve reltively stble mrket prices nd re likely to contin lower mounts of orgnic contminnts thn lipids of niml origin. Although significnt reserch efforts hve been directed towrds the evlution of vegetble oils nd niml fts s lterntive lipid sources to fish oil in fish diets, studies on the potentil use of vegetble oils in Atlntic hlibut diets re scrce (Hugen et l. 2006; Alves Mrtins et l. 2009). Vegetble oils re well utilized by slmonids, even t high levels of fish oil replcement (up to 100%; Torstensen et l. 2005; Turchini et l. 2009; Frrell et l. 2010), nd by vrious mrine fish species (e.g. Alves Mrtins et l. 2006, 2009; Hugen et l. 2006; Piedecus et l. 2007; Wssef et l. 2009) without ny dverse effects on growth nd feed consumption. Nevertheless, chnges in muscle ftty cid composition occur s consequence of vrition in dietry ftty cid profiles (Higgs & Dong 2000). Vegetble oils re devoid of n-3 long-chin polysturted ftty cids (LC-PUFAs) bundnt in fish oils, like eicospentenoic cid (20:5n-3) nd docoshexenoic cid (22:6n-3), which mke fish unique food product tht cn ttenute the onset of crdiovsculr nd inflmmtory diseses in humns (Willims 2000). Therefore, chnges in the min dietry lipid source must be considered crefully to ensure the nutritionl qulity of the... Ó 2011 Blckwell Publishing Ltd

2 finl product. High levels of vegetble oils cn led to chnges in the orgnoleptic qulity of fish fillets lso (Wgbø et l. 1993), nd ÔfinishingÕ period with fish oil bsed diets my be required for the replenishment of both chrcteristic LC-PUFA profiles nd sensory properties (Bell et l. 2003, 2004; Regost et l. 2003; Izquierdo et l. 2005; Torstensen et l. 2005). Among plnt oils, flxseed oil is unusully rich in linolenic cid (18:3n-3), with vlues up to 60% of totl ftty cids; it is the only commercilly produced plnt oil contining higher mounts of n-3 thn n-6 ftty cids (Lnds 2005). Flxseed oil hs been climed to lower cholesterol nd blood pressure, presenting crdioprotective properties in humns (reviewed by Bssett et l. 2009). It is often used in feeds for lying hens nd hs been considered s vegetble oil of choice in fish nutrition reserch (Srgent & Tcon 1999). Reltive to mrine fish oils, flxseed oil typiclly contins higher mounts of other C18 ftty cids, such s oleic cid (18:1n-9) which is preferentilly used for b-oxidtion in fish tissues (Henderson 1996; Tocher 2003). Linoleic cid (18:2n-6) nd 18:3n-3 could be similrly utilized (Bell et l. 2001, 2003). However, due to the limited cpbility of mrine fish to desturte nd/or elongte C18 ftty cids to their longer chin more unsturted counterprts it is importnt to stisfy specific dietry requirements for essentil LC-PUFA (Srgent et l. 2002). In previous experiment with Atlntic hlibut, diets contining severl vegetble oils including flxseed oil showed high pprent digestibility coefficients for lipid nd individul ftty cids (Alves Mrtins et l. 2009). Vrious studies hve reported tht mjor proportion of fish oil cn be replced by flxseed oil in diets for severl mrine fish, such s turbot (Scophthlmus mximus, Bell et l. 1994; Regost et l. 2003), shrpsnout sebrem (Diplodus puntzzo, Piedecus et l. 2007), sebss (Dicentrrchus lbrx) nd sebrem (Sprus urt, Izquierdo et l. 2003, 2005), lthough its effects on hlibut growth performnce hve not been ssessed. This study ws conducted to evlute the efficiency of utiliztion by juvenile Atlntic hlibut of diets with prtil fish oil replcement by flxseed oil (0%, 40% nd 70%). Effects on fish growth performnce, nutritionl nd orgnoleptic qulity of the finl fillet were nlysed. In ddition, ttention ws given to the ftty cid composition of liver, nd hert. The ftty cid profiles of the polr lipid frctions in muscle nd liver were lso determined s importnt shifts in LC-PUFA levels could be relted to poorer growth performnce nd helth sttus of the fish t the end of the experiment. This experiment ws conducted t the Mrine Reserch Sttion of the Ntionl Reserch Council of Cnd, Institute for Mrine Biosciences (Hlifx, NS, Cnd). Atlntic hlibut juveniles weighing 849 ± 99 g were obtined from Scotin Hlibut Ltd (ClrkÕs Hrbour, NS, Cnd). Fish were individully weighed, PIT-tgged, nd 61 fish were rndomly distributed into ech of three, 7000 L tnks. Averge wter temperture rnged between 6 nd 12 C, ccording to sesonl environmentl chnges. Dissolved oxygen ws mintined t round 100% sturtion, wter flow t 30 L min )1 in flow through system, nd fish were kept under constnt dim lighting conditions. Fish were fed to pprent stition twice dily on weekdys (10:00 h nd 16:00 h) nd once on weekends (10:00 h) over 33 weeks, nd feed consumption ws monitored weekly. The experimentl diets were prepred t the Mrine Reserch Sttion (MRS) initilly, nd lter on by Shur-Gin Aquculture (Truro, NS, Cnd) to obtin lrger sized pellets (>7 mm), nd both btches presented similr composition. Three experimentl, isonitrogenous diets (520 g kg )1 DM) were formulted to include three fish oil (FO) replcement levels, 0%, 40% nd 70% (Tble 1). The control diet (0% FxO) contined herring oil s the min lipid source, wheres in 40% FxO nd 70% FxO diets 40% nd 70% of the herring oil ws replced by flxseed oil (FxO), respectively. In ll diets, herring mel ws used s the min protein source. The test oil(s) were dded fter mixing the dry ingredients in HOBART mixer (Model H600T, Rpids Mchinery Co., Troy, OH, USA). The diets were pelleted to the required size for the fish through lbortory stem-pellet mill (Cliforni Pellet Mills Co., Sn Frncisco, CA, USA) nd dried in forced ir convection drier. At Shur-Gin Aquculture, the oil ws spryed onto the extruded feed in vcuum infusion system, nd well bsorbed into the pellets. Diets were sieved, bgged nd stored t )20 C until utilized. All fish were individully weighed t the strt nd end of the experiment. At the initil smpling, six fish were killed with n over-dose of TMS (tricine methnesulfonte, Syndel Lbortories Ltd, Vncouver, BC, Cnd) for determintion... Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

3 Tble 1 Formultion nd proximte composition 1 of the experimentl diets Diets 0% FxO 40% FxO 70% FxO Ingredients (g kg )1 ) Herring mel Krill hydrolyzte CPSP-G Corn gluten mel Herring oil Flxseed oil Whet middlings Whey Strch, pre-geltinized Vitmin mixture Minerl mixture Proximte composition Moisture Protein (g kg )1 DM) Lipid (g kg )1 DM) Energy (MJ kg )1 DM) Ash (g kg )1 DM) Crbohydrtes 13 (g kg )1 DM) Averge vlues of four replicte nlyses of ech diet. 2 Corey Feed Mills Ltd, Fredericton, NB. 3 Specilty Mrine Products Ltd, West Vncouver, BC. 4 Fish protein hydrolyste, Sopropêche, Frnce. 5 Corey Feed Mills Ltd, Fredericton, NB. 6 Stbilized with 0.06% ethoxyquin. Commeu Sefood, Sulnierville, NS. 7 Bioriginl Food nd Science Corp., Ssktoon, Ssktchewn. 8 Wlker s Livestock Feeds, Drtmouth, NS. 9 Frmer s Co-opertive Diry Ltd, Truro, NS. 10 Ntionl Strch nd Chemicl Co., Bridgewter, NJ, USA. 11 Vitmin dded to supply the following (per kg diet): vitmin A, 8000 IU; vitmin D 3, 4500 IU; vitmin E, 300 IU; vitmin K 3, 40 mg; thimine HCl, 50 mg; riboflvin, 70 mg; d-c pntothente, 200 mg; biotin, 1.5 mg; folic cid, 20 mg; vitmin B 12, 0.15 mg; nicin, 300 mg; pyridoxine HCl, 20 mg; scorbic cid, 300 mg; inositol, 400 mg; choline chloride, 3000 mg; butylted hydroxy toluene, 15 mg; butylted hydroxy nisole, 15 mg. 12 Minerl dded to supply the following (per kg diet): clcium phosphte (mono), 6000 mg; mngnous sulphte (32.5% Mn), 40 mg; ferrous sulphte (20.1% Fe), 30 mg; copper sulphte (25.4% Cu), 5 mg; zinc sulphte (22.7% Zn), 75 mg; sodium selenite (45.6% Se), 1 mg; coblt chloride (24.8% Co), 2.5 mg; sodium fluoride (42.5% F), 4 mg. 13 Crbohydrtes = 1000 ) (protein + lipid + sh). for rigor mortis resolution before filleting. After filleting specific portion behind the pelvic fin ner the dorsl line (fillet A), fillets were bgged, lbelled nd stored in ice for one dy until orgnoleptic qulity tests were performed. Further eight fish per tretment were killed with n overdose of TMS, nd stored t )30 C for whole body composition studies. Another eight fish per diet were killed by the sme method nd liver, hert, nd fillet A smples collected for determintion of lipid content nd ftty cid composition. These smples were frozen t )80 C until nlysed. Fish lengths were mesured for clcultion of condition fctor nd liver weights recorded for heptosomtic index determintion. Sensory qulity testing ws conducted t the Deprtment of Food Science nd Technology, Dlhousie University (Hlifx, NS, Cnd). The skinned fillets were cut into smll portions before cooking. Ech smple (2 cm thickness) ws plced into Petri dish coded with three digit number, nd bked t 204 C (internl temperture) for 2 min with 3 ml of wter. The smples were covered nd served hot in trys, to nine non-trined pnelists who were sked to compre ech of three unknown smples (one from ech dietry tretment) ginst reference smple (ÔRÕ; multiple comprison test). The ÔRÕ smple presented in ll trys corresponded to smple from one single fish fed 0% FxO (control), wheres the other three coded smples belonged to different individuls from ech experimentl group. The pnel ws given instructions regrding the process involved nd questionnire ws delivered to ech of the pnelists. The coded smples were evluted ccording to their bright white colour, firmness of the flesh, nd fishy odour nd flvour, using scle from 1 to 9 s follows: smples would be scored Ô5Õ if no difference ws detected in reltion to the reference smple; less thn Ô5Õ if the intensity of the prmeter ws lower in the coded smple (Ô1Õ representing extreme difference) nd more thn Ô5Õ if the intensity of the prmeter ws stronger in the coded smple thn in ÔRÕ (Ô9Õ stnding for extreme difference). of whole body composition. In the finl smpling, nine fish from ech of the FxO diets fed groups nd 10 from the control group (one fish from this group served s reference smple, ÔRÕ), weighing t lest 2 kg (to eliminte possible sensory qulity differences due to size vrition) were collected for the sensory qulity studies. These fish were killed by quick blow to the hed, bled in icy wter, gutted, clened in icy wter nd kept in crushed ice for 3 dys, to llow... The experimentl diets nd freeze-dried whole body smples were nlysed for chemicl composition following AOAC (1990) methods: dry mtter by weight loss fter 24 h in n oven t 105 C; crude sh by incinertion in muffle furnce t 550 C for 24 h; crude protein (%N 6.25) s mesured using LECO NITROGEN DETERMINATOR (Model FP-528, Leco Corportion, St. Joseph, MI, USA); energy Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

4 content of the smples determined with n dibtic bomb clorimeter (Model 1261, Prr Instruments, Moline, IL, USA); lipid extrcted by the method of Bligh & Dyer (1959), nd determined grvimetriclly fter vcuum drying. Ftty cid methyl esters (FAMEs) were prepred ccording to Kirsch et l. (1998). FAMEs were seprted by gs chromtogrph (Hewlett Pckrd 6890 GC SYSTEM, Wilmington, DE, USA) equipped with flme-ioniztion detector (260 C), on n OMEGAWAX 320 fused silic cpillry column (30 m 0.32 mm, 0.25 lm film thickness; Supelco, Bellefonte, PA, USA), using helium s crrier gs t 15 psi constnt pressure (HP 6890 series split splitless injector, t 250 C injection temperture). A therml grdient ws progrmmed to hold t 185 C for 8 min nd rise to 230 C, rmping t 3 C min )1. The finl temperture ws mintined for 10 min. FAMEs were identified by comprison of retention times with those of known stndrds (SUPELCO 37 nd menhden oil, Supelco, Bellefonte, PA, USA). Also, lipid extrcts from liver nd fillet smples were seprted into polr nd neutrl frctions by dsorption chromtogrphy on silic column (Juned & Rocquelin 1985) nd their ftty cid composition nlysed s previously described. Individul fish (PIT-tgged) were used s repliction units in this experiment due to fesibility constrints regrding the vilbility of tnks of n pproprite volume for the size of hlibut under study. In generl, dt re presented s men ± SD nd were tested for equlity of vrinces (LeveneÕs test) followed by one-wy ANOVA with SIGMAS- TAT, STAT32, softwre pckge. A pir wise comprisons test (TukeyÕs HSD) ws conducted when significnt differences were detected between mens, except for the sensory qulity nlysis in which plnned comprisons between 0% FxO group nd the other experimentl groups were performed in cse of significnt differences (P < 0.05). Dt regrding feed conversion rtio, protein efficiency rtio, nd voluntry feed intke were determined on the bsis of the overll feed consumption per tnk (i.e. per tretment); hence only verge vlues re displyed nd no sttisticl nlyses could be conducted. Tble 2 Selected ftty cid composition of the experimentl diets 1 Diets 0% FxO 40% FxO 70% FxO Ftty cid 14: : :1n :2n :3n :4n : :1n :1n :2n :3n :4n :1n :4n :3n :5n :1n :1n :4n :5n :5n :6n :1n P SAFA P MUFA P PUFA P LC-PUFA P n-3 PUFA P n-3 LC-PUFA P n-6 PUFA P n-9 FA P P n-3 PUFA/ n-6 PUFA P P n-3 PUFA/ n-9 FA Dt expressed s percentge of totl ftty cid methyl esters. Averge vlues of four replicte nlyses of ech diet. Sums of PUFA include LC-PUFA g kg )1 (DM) nd gross energy MJ kg )1 (DM; Tble 1). The ftty cid composition of the diets (Tble 2) showed grdul increse in 18:3n-3 from 0.9% to 29.8% with FxO inclusion. LC-PUFAs such s 20:5n-3, 22:5n-3 (docospentenoic cid; DPA), nd 22:6n-3, decresed with FO replcement. Grdul FO substitution by FxO resulted in higher n-3 nd n-6 polyunsturted ftty cid levels, s well s lower sturtes in the diets. The lipid level of the experimentl diets ws between 232 nd 244 g kg )1 (dry mtter bsis), protein content bout Lrger inter-individul vribility within tretments ws observed for finl fish weight (coefficient of vrition, 24 30%) thn for initil fish weight vlues (CV, 11 13%).... Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

5 Tble 3 Growth performnce of Atlntic hlibut fed the experimentl diets 1 Diets 0% FxO 40% FxO 70% FxO Initil body weight (g) ± ± ± 92.5 Finl body weight (g) ± ± ± Finl body length (cm) * 51.7 ± ± ± 3.0 Condition fctor 2,* 1.1 ± ± ± 0.1 HSI 3,** (%) 1.6 ± ± ± 0.4 SGR (% dy )1 ) ± ± ± 0.1 FCR PER VFI 7 (%) Vlues re men ± SD, except for FCR, PER, nd VFI (men vlues only); n = 61, except when indicted otherwise; bsence of superscripts indictes no significnt differences between tretments (one-wy ANOVA, P 0.05). 2 Condition fctor = 100 body weight/(body length^3). 3 HSI, heptosomtic index = 100 liver weight/body weight. 4 SGR, specific growth rte = 100 (ln(finl body weight) ) ln(initil body weight))/dys. 5 FCR, feed conversion rtio = dry feed intke/weight gin. 6 PER, protein efficiency rtio = weight gin/protein intke (dry mtter). 7 VFI, voluntry feed intke = 100 dry feed intke/(finl body weight + initil body weight)/ 2)/dys. * n = 25. ** n =8. However, finl weight per fish did not differ significntly between tretments (Tble 3). All groups displyed similr specific growth rtes (SGR; 0.2% dily) nd feed conversion rtios (FCR; ). Protein efficiency rtio rnged between 1.5 nd 1.6 nd voluntry feed intke ws similr mong dietry groups. No sttisticl differences were found for other biologicl prmeters such s totl body length, heptosomtic index, nd condition fctor. Whole body composition is presented in Tble 4. Lipid content showed 20 g kg )1 increse from the initil stge, but remined similr between dietry groups ( g kg )1 ). Protein content decresed slightly in 40% FxO Tble 4 Whole body composition (g kg )1 wet weight) nd utiliztion of nitrogen (N), lipid nd energy (E) in Atlntic hlibut fed the experimentl diets 1 Initil Diets 0% FxO 40% FxO 70% FxO Whole body composition Dry mtter (g kg )1 ) ± ± ± 19.0 Protein (g kg )1 ) ± ± 6.0 b ± 7.0 b Lipid (g kg )1 ) ± ± ± 21.0 Energy (MJ kg )1 ) ± ± ± 0.8 Ash (g kg )1 ) ± ± ± 2.0 Nitrogen utiliztion N intke 2 (g kg )1 gin) N retention 3 (%) 23.8 ± ± ± 2.6 Lipid utiliztion Lipid intke 2 (g kg )1 gin) Lipid retention 3 (%) 43.4 ± ± ± 17.5 Energy utiliztion E intke 2 (MJ kg )1 gin) E retention 3 (%) 29.7 ± ± ± Vlues re men ± SD, except for nutrient nd energy intke (men vlues only; n = 8); different superscripts in the sme row indicte significnt differences (one-wy ANOVA, P < 0.05 followed by Tukey s HSD); bsence of superscripts indictes no significnt differences between tretments (one-wy ANOVA, P 0.05). 2 Nutrient or energy intke = (feed intke nutrient or energy content of feed)/weight gin. 3 Nutrient or energy retention = 100 nutrient or energy gin/nutrient or energy intke. Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

6 fed fish, wheres energy nd dry mtter vlues were kept the sme mong groups. At the end of the experiment, no significnt differences were observed for nitrogen, lipid or energy utiliztion mong dietry tretments. Nitrogen retention rnged from 22.7% to 24.6%, energy retention from 29.7% to 31.9%, wheres lipid retention ws between 43.4 nd 46.8%. Results concerning the orgnoleptic qulity of fish fed the experimentl diets re presented in Tble 5. All fish were well ccepted by the sensory pnel. Comprisons mong the vrious coded smples in reltion to the ÔRÕ smple (0% FxO fed fish) suggested tht differences within tretments were more importnt thn those between dietry groups. Given the consistency in muscle ftty cid composition vlues mong sme tretment fish (Tble 6) it is likely tht the vribility in sensory qulity test results ws due to differences in perception mong vrious pnelists combined with the effect of fillet lipid content which vried considerbly mong fish from sme tretment. Nonetheless, different dietry tretment smples showed close scores for ll tested prmeters nd no extreme differences reltive to the ÔRÕ smple were identified. Replcement of FO with FxO did not result in significnt chnges in colour, texture or the ÔfishyÕ odour nd flvour of the fillets. 30%). Totl ftty cid composition generlly mirrored dietry profile. The inclusion of FxO in the diet resulted in significnt decrese of both sturtes nd monounsturtes, nd in incresed polyunsturtes. Both n-3 nd n-6 series ftty cid levels incresed with FO replcement, nd the rtio P n-3/ P n-6 ws significntly higher in fish fed the control diet. Fish fed 0% FxO exhibited the highest plmitic cid (PA; 16:0), 20:5n-3 nd 22:6n-3 percentges in the fillets wheres the 70% FxO group fillets showed the highest 18:2n-6 nd 18:3n-3 levels, the ltter being the most bundnt ftty cid in the muscle of 70% FxO fed hlibut. Incresed steric (SA; 18:0) nd eicostrienoic (ETA; 20:3n-3) cid concentrtions with dietry FxO inclusion were lso found in muscle, despite similr dietry levels. Lipid recovery following column frctiontion ws reltively high (96 99%). Neutrl lipid (82 85% of totl lipid) ftty cid profiles followed similr trends to those observed cross tretments for totl lipid. LC-PUFA levels in the polr frction of muscle lipid were more conserved nd totl sturted, monounsturted nd polyunsturted ftty cids exhibited similr levels cross experimentl groups. Prticulrly, 16:0, 18:1n-9, 20:5n-3 nd 22:6n-3 remined the most bundnt ftty cids in polr lipid in ll experimentl groups. In ddition, smll decrese in n-3 ftty cids nd in the rtio P n-3 FA/ P n-9 FA with dietry vegetble oil inclusion ws ctully observed. Lipid content in fillet A ws g kg )1 in ll groups (Tble 6) nd lower inter-individul vrition ws observed in 70% FxO fed fish (CV, 16%) thn in the other groups (CV, Tble 5 Sensory ttributes of fillets from Atlntic hlibut fed the experimentl diets 1 Diets 0% FxO 40% FxO 70% FxO Attributes Odour fishiness 3.4 ± ± ± 1.6 Colour whiteness 6.3 ± ± ± 2.0 Flvour fishiness 4.6 ± ± ± 1.8 Texture firmness 4.5 ± ± ± Sensory evlution of cooked hlibut fillets using scle from 1 (lower intensity compred to the reference smple) to 9 (higher intensity compred to the reference smple); smples scored Ô5Õ when no difference ws detected reltive to the reference smple. Vlues re men ± SD, (n = 9); bsence of superscripts indictes no significnt differences between tretments (one-wy ANOVA, P 0.05). Liver lipid content showed high vrition mong fish (CV, 26 37%) nd verge vlues rnging from 355 to 401 g kg )1 (Tble 7). Totl lipid ftty cid profiles generlly reflected dietry ftty cid composition. Dietry FxO inclusion resulted in significnt decrese of sturtes, wheres the sum of n-3 nd n-6 series polyunsturtes incresed significntly. Monounsturted ftty cid concentrtions did not vry significntly. As in muscle, 70% FxO fish livers exhibited the highest 18:3n-3 nd 20:3n-3 levels. Other LC-PUFAs of the n-3 series decresed significntly with dietry FxO inclusion. Recovery of lipids following seprtion of polr nd neutrl frctions ws 98 99%, with neutrl lipid levels representing 92 94% of liver totl lipid. As in fillet, the ftty cid composition of liver polr lipid seemed more conserved thn tht of neutrl lipid. Also, s in muscle, 16:0, 18:1n-9, 20:5n-3 nd 22:6n-3 were kept t higher percentges thn other ftty cids. Reduced rchidonic cid (AA; 20:4n-6) nd 22:6n-3 levels were observed in hlibut fed the highest FxO diet, wheres 20:5n-3 showed the highest vlues in the control fish. Significntly higher levels of 18:2n-6, 18:3n-3 nd 20:3n-3 were observed with dietry FxO.... Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

7 Tble 6 Fillet lipid content (g kg )1 wet weight) nd selected ftty cid composition of totl, neutrl nd polr lipid (% of totl ftty cid methyl esters) 1 Lipid frction Totl lipid Neutrl lipid Polr lipid Diets 0% FxO 40% FxO 70% FxO 0% FxO 40% FxO 70% FxO 0% FxO 40% FxO 70% FxO Lipid content 60.0 ± ± ± 9.0 Ftty cid 14:0 5.1 ± ± 0.3 b 2.8 ± 0.3 c 5.7 ± ± 0.3 b 3.0 ± 0.4 c nd nd nd 16: ± ± 0.4 b 11.6 ± 0.5 c 14.5 ± ± 0.3 b 10.7 ± 0.4 c 19.4 ± ± 0.5 b 17.8 ± 0.7 b 16:1n ± ± 0.5 b 4.6 ± 0.4 c 9.3 ± ± 0.5 b 5.1 ± 0.5 c nd nd nd 18:0 2.8 ± 0.2 b 2.9 ± 0.2 b 3.0 ± ± ± ± ± ± ± :1n ± 0.6 b 14.3 ± ± ± 0.6 c 15.2 ± 0.8 b 16.3 ± ± 1.3 b 6.9 ± ± :1n ± ± 0.1 b 2.1 ± 0.1 c 3.3 ± ± 0.1 b 2.2 ± 0.1 c nd nd nd 18:2n ± 0.1 c 6.8 ± 0.4 b 9.1 ± ± 0.2 c 7.4 ± 0.4 b 9.6 ± ± 0.1 c 3.9 ± 0.6 b 6.1 ± :3n ± 0.1 c 12.3 ± 1.5 b 20.6 ± ± 0.1 c 14.1 ± 1.4 b 22.9 ± ± 0.0 c 2.2 ± 0.4 b 3.9 ± :4n ± ± 0.1 b 0.7 ± 0.1 c 1.6 ± ± 0.1 b 0.8 ± 0.1 c nd nd nd 20:1n ± ± 0.6 b 4.0 ± 0.6 b 5.5 ± ± 1.0 b 4.5 ± 0.8 c 1.0 ± ± ± :4n ± ± 0.1 b 0.7 ± 0.1 c 0.9 ± ± ± ± ± 0.1 b 2.5 ± 0.2 c 20:3n ± 0.0 c 1.0 ± 0.1 b 1.6 ± ± 0.0 c 1.1 ± 0.1 b 1.6 ± ± 0.0 c 0.6 ± 0.1 b 1.2 ± :5n ± ± 0.4 b 5.1 ± 0.5 c 9.7 ± ± 0.6 b 4.0 ± 0.3 c 15.2 ± ± ± 1.0 b 22:1n ± ± 0.6 b 3.9 ± 0.6 b 4.8 ± ± ± 0.8 nd nd nd 22:5n ± ± 0.0 b 0.2 ± 0.0 c nd nd nd 1.0 ± ± 0.1 b 0.8 ± 0.1 c 22:5n ± ± 0.1 b 1.0 ± 0.1 c 2.1 ± ± 0.1 b 0.9 ± 0.1 c 2.4 ± ± ± 0.1 b 22:6n ± ± 1.8 b 7.6 ± 1.5 b 8.0 ± ± 0.6 b 3.9 ± 0.5 c 37.8 ± ± 1.5 b 32.4 ± 3.2 b 24:1n-9 P nd nd nd nd nd nd 1.1 ± ± ± 0.2 SAFA P 24.2 ± ± 0.6 b 18.2 ± 0.9 c 25.0 ± ± 1.5 b 17.3 ± 0.8 c 27.8 ± ± ± 1.2 MUFA P 36.3 ± ± 1.9 b 30.7 ± 1.8 c 39.4 ± ± 3.4 b 34.5 ± 2.2 b 9.1 ± ± ± 0.9 PUFA P 37.5 ± 2.6 c 44.3 ± 2.3 b 49.6 ± ± 2.2 c 41.8 ± 2.2 b 47.2 ± ± ± ± 1.6 LC-PUFA 26.9 ± ± 2.2 b 16.9 ± 2.0 c 22.6 ± ± 1.1 b 11.7 ± 0.9 c 60.4 ± ± 1.4 b 53.1 ± 3.0 c P n-3 PUFA 26.3 ± 2.2 c 31.8 ± 1.9 b 36.6 ± ± 2.2 c 29.4 ± 2.2 b 34.1 ± ± ± 1.2 b 53.7 ± 2.2 b P n-3 LC-PUFA 25.0 ± ± 2.1 b 15.9 ± 1.9 c 21.2 ± ± 1.2 b 11.0 ± 0.9 c 56.0 ± ± 1.4 b 49.6 ± 2.9 c P n-6 PUFA 6.1 ± 0.1 c 8.9 ± 0.5 b 10.7 ± ± 0.2 c 8.8 ± 0.5 b 10.7 ± ± 0.3 c 8.2 ± 0.7 b 10.0 ± 1.1 P n-9 FA P P 19.7 ± ± ± ± ± ± ± ± ± 0.9 n-3 PUFA/ n-6 PUFA 4.3 ± ± 0.2 b 3.4 ± 0.2 b 3.9 ± ± 0.2 b 3.2 ± 0.1 b 9.2 ± ± 0.6 b 5.4 ± 0.7 c P P n-3 PUFA/ n-9 FA 1.4 ± 0.2 c 1.6 ± 0.2 b 1.8 ± ± 0.1 b 1.4 ± ± ± ± 0.7 b 6.0 ± 0.8 b 1 Vlues re men ± SD (n = 8); different superscripts within rows for ech lipid frction indicte significnt differences between tretments (one-wy ANOVA, P < 0.05); bsence of superscripts indictes no significnt differences between tretments (one-wy ANOVA, P 0.05). Sums of PUFA include LC-PUFA. nd, not detected.... Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

8 Tble 7 Liver lipid content (g kg )1 wet weight) nd selected ftty cid composition of totl, neutrl nd polr lipid (% of totl ftty cid methyl esters) 1 Lipid frction Totl lipid Neutrl lipid Polr lipid Diets 0% FxO 40% FxO 70% FxO 0% FxO 40% FxO 70% FxO 0% FxO 40% FxO 70% FxO Lipid content ± ± ± Ftty cid 14:0 4.0 ± ± 0.4 b 2.4 ± 0.2 c 4.1 ± ± 0.4 b 2.5 ± 0.2 c nd nd nd 16: ± ± 0.6 b 10.7 ± 0.6 c 12.5 ± ± ± 0.6 b 18.8 ± ± ± 0.7 b 16:1n ± ± 1.1 b 5.2 ± 0.9 c 9.6 ± ± 1.1 b 5.4 ± 0.8 c nd nd nd 18:0 1.5 ± ± ± ± ± ± ± ± ± :1n ± ± ± ± ± ± ± ± ± :1n ± ± 0.3 b 2.1 ± 0.2 c 3.2 ± ± 0.3 b 1.9 ± 0.8 c nd nd nd 18:2n ± 0.6 c 6.5 ± 1.2 b 9.9 ± ± 0.6 c 6.7 ± 1.4 b 10.3 ± ± 0.3 c 2.6 ± 0.4 b 4.6 ± :3n ± 0.1 c 9.6 ± 2.0 b 18.3 ± ± 0.2 c 10.0 ± 2.3 b 19.0 ± ± 0.0 c 3.0 ± 0.7 b 6.8 ± :1n ± ± ± ± ± 0.5 b 2.4 ± 0.4 b 1.5 ± ± ± :4n ± ± 0.2 b 0.6 ± 0.2 c 1.2 ± ± 0.1 b 0.4 ± 0.1 c 4.5 ± ± ± 0.1 b 20:3n ± 0.0 c 3.0 ± 0.6 b 4.9 ± ± 0.1 c 3.0 ± 0.6 b 5.0 ± ± 0.0 c 2.1 ± 0.4 b 3.6 ± :4n ± ± 0.8 b 0.5 ± 0.2 b 1.7 ± ± ± 0.2 nd nd nd 20:5n ± ± 0.7 b 4.9 ± 0.3 c 11.4 ± ± 0.5 b 4.5 ± 0.3 c 14.1 ± ± 1.1 b 11.6 ± 0.6 b 22:1n ± ± ± ± ± ± 0.2 nd nd nd 22:5n ± ± 0.0 b 0.2 ± 0.0 c 0.4 ± ± 0.0 b 0.1 ± 0.0 c nd nd nd 22:5n ± ± 0.2 b 1.6 ± 0.1 c 3.5 ± ± 0.3 b 1.6 ± 0.1 c 2.3 ± ± 0.2 b 1.9 ± 0.2b 22:6n ± ± 0.7 b 7.1 ± 1.0 c 12.1 ± ± 1.1 b 5.8 ± 1.2 c 35.8 ± ± ± 2.6 b 24:1n-9 P nd nd nd nd nd nd 2.5 ± ± 0.3 b 2.0 ± 0.4 b SAFA 18.9 ± ± 0.9 b 15.1 ± 0.8 c 18.7 ± ± 1.1 b 14.7 ± 0.8 c 26.5 ± ± 0.6 b 23.2 ± 0.9 c P MUFA P 36.8 ± ± ± ± ± 5.7 b 31.4 ± 4.4 b 13.8 ± ± ± 1.5 PUFA 41.4 ± 4.4 b 45.5 ± 6.1 b 51.4 ± ± 4.4 b 44.8 ± 5.9 b 51.0 ± ± 1.1 c 61.3 ± 1.5 b 63.1 ± 1.6 P LC-PUFA 33.8 ± ± 3.2 b 20.6 ± 1.4 c 32.8 ± ± 2.2 b 19.0 ± 1.2 c 57.9 ± ± 1.1 b 50.9 ± 2.6 c P n-3 PUFA P 31.7 ± 2.7 b 33.9 ± 3.6 b 37.7 ± ± 2.8 b 33.0 ± 3.2 b 37.0 ± ± ± ± 2.2 n-3 LC-PUFA 30.7 ± ± 2.2 b 19.2 ± 1.2 c 29.8 ± ± 1.4 b 17.8 ± 1.2 c 52.7 ± ± ± 2.7 b P n-6 PUFA 7.0 ± 2.0 c 9.9 ± 2.4 b 12.6 ± ± 2.2 b 10.0 ± ± ± 0.7 c 7.7 ± 0.7 b 8.8 ± 1.0 P n-9 FA P P 21.5 ± ± ± ± ± ± ± ± 0.3 b 3.2 ± 0.5 b n-3 PUFA/ n-6 PUFA 4.8 ± ± 0.5 b 3.0 ± 0.2 b 4.6 ± ± 0.6 b 2.9 ± 0.3 b 8.2 ± ± 0.8 b 6.3 ± 0.9 b P P n-3 PUFA/ n-9 FA 1.5 ± ± ± ± ± ± ± 1.7 b 15.3 ± 1.7 b 17.4 ± Vlues re men ± SD (n = 8); different superscripts within rows for ech lipid frction indicte significnt differences between tretments (one-wy ANOVA, P < 0.05); bsence of superscripts indictes no significnt differences between tretments (one-wy ANOVA, P 0.05). Sums of PUFA include LC-PUFA. nd, not detected.... Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

9 Hert lipid content (20 24 g kg )1 ) did not differ significntly mong fish fed the experimentl diets (Tble 8). As hert is not typiclly lipid storge orgn, totl lipid extrcts were not seprted into polr nd neutrl frctions for ftty cid nlysis. Hert ftty cid composition ws generlly more stble mong experimentl groups thn tht of muscle nd liver. Wheres sturtes decresed slightly with dietry FxO inclusion, monounsturtes showed no significnt differences nd polyunsturtes were bout 4% higher in 70% FxO fish compred to control individuls. Ftty cids of the n-3 series corresponded to bout 50% of totl ftty cids nd 22:6n-3 in prticulr represented % of totl ftty cids, the mjor ftty cid in this tissue, regrdless of the diet. Lower 20:4n-6 nd 22:5n-3 levels were Tble 8 Hert lipid content (g kg )1 wet weight) nd selected ftty cid composition of totl lipid (% of totl ftty cid methyl esters) 1 Diets 0% FxO 40% FxO 70% FxO Lipid content 22.0 ± ± ± 3.0 Ftty cid 14:0 1.2 ± ± 0.2 b 0.8 ± 0.2 b Iso 16:0 1.5 ± ± ± : ± ± 0.8 b 15.6 ± 0.6 b 16:1n ± ± ± 0.3 b 18:0 4.5 ± ± ± :1n ± ± ± :1n ± ± 0.1 b 2.3 ± 0.1 c 18:2n ± 0.1 c 4.3 ± 0.2 b 5.8 ± :3n ± 0.0 c 3.5 ± 0.5 b 5.4 ± :1n ± ± ± :4n ± ± 0.4 b 3.2 ± 0.4 b 20:3n ± 0.0 c 0.8 ± 0.2 b 1.2 ± :5n ± ± 0.6 b 6.7 ± 0.4 b 22:1n ± ± ± :5n ± ± ± :5n ± ± 0.1 b 1.6 ± 0.2 b 22:6n ± ± ± 1.4 P SAFA 25.4 ± ± 0.6 b 22.9 ± 0.6 c P MUFA 14.5 ± ± ± 1.0 P PUFA 58.9 ± 1.9 b 59.8 ± 2.3 b 63.0 ± 0.7 P LC-PUFA 54.3 ± ± 2.8 b 49.9 ± 1.1 b P n-3 PUFA 49.4 ± 1.6 b 48.8 ± 2.5 b 51.2 ± 0.9 P n-3 LC-PUFA 49.0 ± ± 2.9 b 45.6 ± 1.2 b P n-6 PUFA 7.7 ± 0.5 b 9.7 ± ± 0.8 P n-9 FA 8.1 ± ± ± 0.8 P P n-3 PUFA/ n-6 PUFA 6.4 ± ± 0.5 b 4.9 ± 0.4 b P P n-3 PUFA/ n-9 FA 6.2 ± ± ± Vlues re men ± SD (n = 8); different superscripts within rows indicte significnt differences between tretments (one-wy ANOVA, P < 0.05); bsence of superscripts indictes no significnt differences between tretments (one-wy ANOVA, P 0.05). Sums of PUFA include LC-PUFA.... shown in the hert of fish fed FxO diets. As observed in the polr frction of liver nd muscle lipid, 16:0, 18:1n-9, 20:5n-3 nd 22:6n-3 were the most bundnt ftty cids in hert totl lipid. Also, significnt increse in 20:3n-3 ws observed with dietry FxO level. In this study, sesonl wter temperture fluctution led to low feed consumption nd hence low SGR vlues ( % dy )1 ), s previously reported in kg hlibut rered under mbient wter temperture (Berge & Storebkken 1991; Nortvedt 1997). During the winter months hlibut growth cn be miniml (Hug et l. 1989; Hugen et l. 2006). Lrger size hlibut ( kg), rered t 4 9 C, presented similr growth rtes ( %; Bjo rnsson & Tryggvdo ttir 1996; Hellnd et l. 1997). In hlibut there is evidence for cler size-dependent growth rtes nd effectiveness of feed utiliztion (Bjo rnsson & Tryggvdo ttir 1996; Nortvedt 1997) which cn be influenced by both temperture (Bjo rnsson & Tryggvdo ttir 1996; Hugen et l. 2006) nd photoperiod (Imslnd et l. 2009). The individul vrition in weight gin observed in this experiment is common chrcteristic of wild nd frmed hlibut (Hug 1990; Berge & Storebkken 1991; Nortvedt 1997) possibly explined by feed intke, differences in metbolic energy loss (Tuene & Nortvedt 1995) nd different degrees of interctions mong individuls (Imslnd et l. 2009b). The inclusion of up to 70% FxO in the diets hd no effect on condition fctor, heptosomtic index, nd protein efficiency rtio further suggesting tht FxO ws well utilized by hlibut. Generlly, PUFA digestibility is prticulrly high in hlibut (Alves Mrtins et l. 2009). Essentil ftty cid levels were mintined bove those recommended for mrine fish in ll the diets (NRC 1993), which did not induce dverse effects on survivl, growth performnce or pprent generl helth sttus of hlibut. Reltively good growth performnce results were reported when FxO diets were fed to slmonids (e.g. Rosenlund et l. 2001; Bell et l. 2004; Turchini & Frncis 2009), turbot (Bell et l. 1994; Regost et l. 2003), sebss nd sebrem (Izquierdo et l. 2003, 2005). Feed ccounts for mjor proportion of fish production costs nd the utiliztion of vegetble oils could be economiclly dvntgeous. However, s FxO is widely used s n industril product, its prices re determined by those of crude oil, rther thn vegetble oils destined for humn use. Chnges in lipid source my influence ft content in both muscle (Greene & Selivonchick 1990; Bell et l. 2001; Rosenlund et l. 2001; Turchini et l. 2003) nd liver (Bell Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

10 et l. 1994, 2001; Tocher et l. 2003); however, such effects were not observed. Muscle lipid levels were within vlues previously reported for frmed hlibut (Nortvedt & Tuene 1998; Olsson et l. 2003). In comprison to the reltively low lipid storge cpcity in fltfish muscle, hlibut showed high lipid deposition in liver which ws previously reported s n importnt lipid storge orgn in this (Alves Mrtins et l. 2007) nd other fltfish species like turbot (Regost et l. 2001) nd Seneglese sole (Sole seneglensis, Borges et l. 2009). Dietry ftty cid composition clerly influenced ftty cid deposition in hlibut tissues. In generl, dietry FxO cused decrese in sturtes nd n increse in n-3 nd n-6 series polyunsturtes which is linked to feeding vegetble oil diets. Monounsturted ftty cids in hlibut tissues reflected dietry profile lso, decresing in muscle nd liver neutrl lipid with FO replcement. Chnges induced by the experimentl diets on polr lipid ftty cid composition of muscle nd liver were less mrked thn on the neutrl lipid frction, similr to previous findings (Menoyo et l. 2002; Regost et l. 2003). Thus, totl sturted, monounsturted nd polyunsturted ftty cid levels of the muscle polr frction remined similr cross tretments. In liver polr lipid, neither monounsturtes nor totl n-3 ftty cid depositions were ffected. Since dietry ftty cids re efficiently bsorbed by hlibut (Alves Mrtins et l. 2009), direct comprison between ftty cid concentrtions in fish nd diets (% totl ftty cids) cn provide indictions regrding their preferentil metbolic fte. Figure 1 shows tht liner correltions between the concentrtions of selected dietry ftty cids nd their tissue levels could be estblished. Furthermore, line of equlity inserted to ech figure cn lso suggest whether prticulr ftty cid is preferentilly retined in tissue (dots bove the correltion line) or otherwise preferentilly metbolized (dots below the correltion line). Preferentil utiliztion of monounsturtes in tissues of higher energy Tissue 18:2n-6 (% totl FA) 11.5 R 2 = R 2 = R 2 = Diet 18:2n-6 (% totl FA) Tissue 18:3n-3 (% totl FA) R 2 = R 2 = R 2 = Diet 18:3n-3 (% totl FA) Tissue 20:5n-3 (% totl FA) Tissue 22:5n-3 (% totl FA) 12.5 R 2 = R 2 = R 2 = Diet 20:5n-3 (% totl FA) 3.7 R 2 = R 2 = R 2 = Diet 22:5n-3 (% totl FA) Tissue 20:4n-6 (% totl FA) Tissue 22:6n-3 (% totl FA) R 2 = R 2 = R 2 = Diet 22:6n-3 (% totl FA) R 2 = R 2 = 1 R 2 = Diet 20:4n-6 (% totl FA) Figure 1 Reltionship between dietry nd muscle (squres), hert (tringles) or liver (circles) ftty cid concentrtions for linoleic (18:2n-6), linolenic (18:3n-3), eicospentenoic (20:5n-3), rchidonic (20:4n-6), docospentenoic (22:5n-3), nd docoshexenoic (22:6n-3) cids, in Atlntic hlibut fed the experimentl diets. A line of equlity is represented (dshed). Dt re men ± sd. Sttisticl differences between tretments re not represented (see Tbles 6 8).... Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

11 requirement (liver, hert) versus muscle (predominntly white muscle) is expected in reltively sedentry bottom-dwelling species (Henderson 1996) like hlibut. In muscle, 18:1n-9 ws preferbly retined, in greement with previous dt for slmon (Rosenlund et l. 2001; Bell et l. 2004), sebss nd sebrem (Izquierdo et l. 2003). Although mintined t similr levels in hert of fish in vrious experimentl groups, 18:1n-9 ws preferentilly metbolized, most likely for energy production in the mitochondril system (Henderson 1996; Tocher 2003; Fig. 1). Despite similr levels of 20:1n-9 nd 22:1n-11 in liver nd hert mong experimentl groups these ftty cids were preferentilly metbolized, in greement with observtions by Suontm et l. (2007). Indeed, 22:1 hs been referred to s preferentil substrte for energy production (Tocher 2003; Bell et l. 2004) in liver peroxisomes (Henderson & Srgent 1984). Levels of 18:2n-6 nd 18:3n-3 incresed in ll tissues with FO replcement s observed in slmon (Bell et l. 2003), sebrem (Izquierdo et l. 2003), sebss (Izquierdo et l. 2003), nd turbot (Bell et l. 1999; Regost et l. 2003) fed vegetble oil diets. In the hert of FxO fed hlibut, both ppered to be preferentilly metbolized in ll tissues (Fig. 1). Severl studies hve reported similr trend for C18 PUFA metbolism (Rosenlund et l. 2001; Suontm et l. 2007; Turchini et l. 2009) through either desturtion (Bell et l. 1997) or b-oxidtion (Bell et l. 2001, 2004; Izquierdo et l. 2003; Tocher et l. 2003). In muscle polr lipid nd crdic tissue 18:2n-6 remined higher thn 18:3n-3 in ll tretment groups, likely due to higher ffinity of cyltrnsferses for the former in phospholipid synthesis (Cbllero et l. 2002). Preferentil utiliztion of 18:3n-3 for either energy production purposes (Ruyter et l. 2003) or ftty cid elongtion ws suggested by the overll increse in 20:3n-3 with dietry FxO inclusion in ll tissues, especilly liver (Fig. 1), despite its low dietry supply. This hs been noted lso in brown trout, Slmo trutt (Greene & Selivonchick 1990) nd turbot (Bell et l. 1994; Regost et l. 2003) fed FxO diets. The bsence of 18:2n-6 elongtion products in hlibut tissues suggested the preference of the ftty cid elongses for 18:3n-3, s observed in severl species (Agb et l. 2005). The block in the desturtion/elongtion pthwy in mrine fish is generlly ttributed either to deficiency of C18 20 elongse or D5 desturse (Tocher et l. 2003b). It ppers tht in hlibut, the limiting step must be t the desturtion level, s reported for turbot (Bell et l. 1994; Regost et l. 2003). Indeed, s dietry FxO incresed, most LC-PUFAs decresed in liver, which could reflect lower dietry supply but lso mobiliztion into other tissues, such s hert, where chnges induced by dietry FxO in LC-PUFA levels were slighter.... The high content in 16:0, 18:1n-9, 22:6n-3 nd 20:5n-3 ftty cids in muscle nd liver polr lipid, nd hert totl lipid supports erlier observtions (Hug et l. 1988) nd ccounts for the min differences in ftty cid composition between polr nd neutrl lipids (Higgs & Dong 2000; Srgent et l. 2002). Despite reduced levels with FxO diets, these ftty cids remined t concentrtions bove those of C18 polyunsturtes. Together, 22:6n-3 nd 20:5n-3 typiclly ccount for 35 40% of phospholipid ftty cids (Polvi & Ackmn 1992). It ppers tht the LC-PUFA levels re conserved when their supply is reltively low from the diet (Hugen et l. 2006). However, differences in 20:5n-3 concentrtion between diets nd fish hert suggested shift in preferentil metbolic fte from metbolism to retention s its dietry supply decresed (Fig. 1). Incresed polyunsturtes nd n-3 ftty cid levels (prticulrly 18:3n-3) in hlibut muscle with FxO diets cn benefit the nutritionl vlue of fillets (Sinclir et l. 2002). Nevertheless, reduced n-3 LC-PUFA levels re undesirble from humn nutrition perspective. Muscle 20:5n-3 nd 22:6n-3 levels (10% nd 12% of totl ftty cids, respectively, in control fish) decresed 49% nd 34%, respectively, with dietry 70% FxO inclusion reltive to control fish. However, 22:6n-3 seemed to ttin minimum threshold t 40% FxO, which could be ttributed to the specificity of the ftty cyltrnsferses for this ftty cid nd/or to its reltive resistnce to b-oxidtion stemming from the complex ctbolic pthwy for this ftty cid (Bell et l. 2001; Tocher 2003). As good substrte for mitochondril oxidtion (Tocher 2003), 20:5n-3 ws metbolized in hlibut muscle, lthough to smller degree in fish fed 70% FxO (Fig. 1). Higher FO replcement levels could perhps led to its preferentil ccumultion in the muscle, s reported by Suontm et l. (2007). Although seldom mentioned, the importnce of 22:5n-3 in humn helth hs been demonstrted, s n inducer of cell migrtion, criticl step in tissue repir nd rtery mintennce (Knysu-Toyod et l. 1996). This ftty cid ws lso preferentilly retined in hlibut flesh. The fillet portion chosen for the sensory qulity ssessment ws representtive of the edible portion of the fish. Lipids hve mjor influence on the sensory chrcteristics of fish flesh s the mount of ft cn ffect muscle texture nd flvour (Ackmn 1990; Andersen et l. 1997) nd fish rom ssocited orgnic compounds in fresh fish re derived from PUFAs (Josephson & Lindsy 1986). In this experiment, no differences were found regrding the fish-like chrcter of flvour or odour of hlibut fillets, nd both firmness nd colour, two importnt chrcteristics in hlibut flesh, remined unltered by dietry FxO inclusion, substntiting Aquculture Nutrition 17; Ó 2011 Blckwell Publishing Ltd

12 the fesibility of FO replcement by FxO in Atlntic hlibut diets. Regost et l. (2006) nd Hugen et l. (2006) showed tht 50% fish oil replcement with soyben oil hd no effect on qulity ttributes like freshness, texture, ft content, colour, or liquid-holding cpcity of hlibut fillets. In turbot fed with FxO diets, Regost et l. (2003b) registered smll decrese in fillet texture ttributed to the lower muscle ft content. Also, except for lower hrdness in sebrem flesh (ttributed to lower muscle sturte levels), diets contining vrious vegetble oils, such s up to 60% FxO, gve promising results in sebrem nd sebss (Izquierdo et l. 2003). Therefore, s in other mrine fish, inclusion of FxO in hlibut diets seems possible without mjor negtive consequences in the orgnoleptic prmeters of the fillet. The overll results suggest flxseed oil hs good potentil to prtilly replce mrine fish oil in commercil grow-out hlibut diets. Atlntic hlibut could tolerte FO replcement levels up to 70% with FxO without detrimentl effects on growth performnce, nutritionl qulity of the fillets or sensory prmeters perceived by the consumers. Ftty cid nlysis of tissues suggested hlibut cn efficiently utilize C18 ftty cids present in high mounts in vegetble oils for metbolic purposes, lrgely for energy production. Also, by selectively retining highly unsturted ftty cids, especilly in muscle nd hert, hlibut could dpt to lower dietry supply of LC-PUFAs during the experimentl period without dverse effects on growth, feed utiliztion nd survivl. This work ws finnced by AquNet CndÕs Reserch Network in Aquculture, project AP33, ÔNutritionl Strtegies to Improve Lipid Utiliztion in Diets for Commercilly Importnt Cndin Finfish SpeciesÕ. Dulce Alves Mrtins ws funded by grnt SFRH/BD/9301/2002 from ÔFundc ão pr Cieˆ nci e TecnologiÕ (Portugl). The uthors thnk the technicl support provided by Joyce Milley, Sen Tibbetts, Ronld Melnson, Crl Wlbourne nd Leh Lewis- McCre. We lso cknowledge Kris Nichols (Shur-Gin Aquculture, Truro, N.S., Cnd), Dr. Tom Gill nd Kthy Spencer (Deprtment of Food Science nd Technology, Dlhousie University, Hlifx, N.S., Cnd), Bioriginl Food nd Science Corportion (Ssktoon, Ssktchewn, Cnd) nd ll the prticipnts in the sensory qulity tste pnel. Ackmn, R.G. (1990) Sefood lipids nd ftty cids. Food Rev. Int., 6, Agb, M.K., Tocher, D.R., Dickson, C.A., Zheng, X., Dick, J.R. & Tele, A.J. (2005) Cloning nd functionl chrcteristion of polyunsturted ftty cid elongses from mrine nd freshwter teleost fish. Comp. Biochem. Physiol. B, 142, Alves Mrtins, D., Gomes, E., Rem, P., Dis, J., Ozo rio, R.O.A. & Vlente, L.M.P. (2006) Growth, digestibility nd nutrient utiliztion of rinbow trout (Oncorhynchus mykiss) nd Europen se bss (Dicentrrchus lbrx) juveniles fed different dietry soyben oil levels. Aqucult. Int., 14, Alves Mrtins, D., Vlente, L.M.P. & Lll, S.P. (2007) Effects of dietry lipid level on growth nd lipid utiliztion by juvenile Atlntic hlibut (Hippoglossus hippoglossus L.). Aquculture, 263, Alves Mrtins, D., Vlente, L.M.P. & Lll, S.P. (2009) Apprent digestibility of lipid nd ftty cids in fish oil, poultry ft nd vegetble oil diets by Atlntic hlibut, Hippoglossus hippoglossus L. Aquculture, 294, Andersen, U.B., Thomssen, M.S. & Rørå, A.M. (1997) Texture properties of frmed rinbow trout effects of diet, muscle (Oncorhynchus mykiss): ft content nd time of storge on ice. J. Sci. Food Agricult., 74, AOAC (1990) Officil Methods of Anlysis, 15th edn. Assocition of Officil Anlyticl Chemists, Wshington, DC, USA. Bssett, C.M.C., Rodriguez-Leyv, D. & Pierce, G.N. (2009) Experimentl nd clinicl reserch findings on the crdiovsculr benefits of consuming flxseed. Appl. Physiol. Nutr. Metb., 34, Bell, J.G., Tocher, D.R., McDonld, F.M. & Srgent, J.R. (1994) Effects of diets rich in linoleic (18:2n-6) nd lph-linolenic (18:3n- 3) cids on the growth, lipid clss nd ftty cid compositions nd eicosnoid production in juvenile turbot (Scophthlmus mximus L.). Fish Physiol. Biochem., 13, Bell, J.G., Tocher, D.R., Frndle, B.M., Cox, D.I., McKinney, R.W. & Srgent, J.R. (1997) The effect of dietry lipid on polyunsturted ftty cid metbolism in Atlntic slmon (Slmo slr) undergoing prr-smolt trnsformtion. Lipids, 32, Bell, J.G., Tocher, D.R., Frndle, B.M., McVicr, A.H. & Srgent, J.R. (1999) Effects of essentil ftty cid-deficient diets on growth, mortlity, tissue histopthology nd ftty cid compositions in juvenile turbot (Scophthlmus mximus). Fish Physiol. Biochem., 20, Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Cmpbell, P.J. & Srgent, J.R. (2001) Replcement of fish oil with rpeseed oil in diets of Atlntic slmon (Slmo slr) ffects tissue lipid compositions nd heptocyte ftty cid metbolism. J. Nutr., 131, Bell, J.G., Tocher, D.R., Henderson, R.J., Dick, J.R. & Crmpton, V.O. (2003) Altered ftty cid compositions in Atlntic slmon (Slmo slr) fed diets contining linseed nd rpeseed oils cn be prtilly restored by subsequent fish oil finishing diet. J. Nutr., 133, Bell, J.G., Henderson, R.J., Tocher, D.R. & Srgent, J.R. (2004) Replcement of dietry fish oil with incresing levels of linseed oil: modifiction of flesh ftty cid compositions in Atlntic slmon (Slmo slr) using fish oil finishing diet. Lipids, 39, Berge, G.M. & Storebkken, T. (1991) Effect of dietry ft level on weight gin, digestibility, nd fillet composition of Atlntic hlibut. Aquculture, 99, Bjo rnsson, B. & Tryggvdóttir, S.V. (1996) Effects of size on optiml temperture for growth nd growth efficiency of immture Atlntic hlibut (Hippoglossus hippoglossus L.). Aquculture, 142, Bligh, E.C. & Dyer, W.J. (1959) A rpid method of totl lipid extrction nd purifiction. Cn. J. Biochem. 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