Effects of moderately oxidized dietary lipid and the role of vitamin E on the stress response in Atlantic halibut (Hippoglossus hippoglossus L.)

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2007) 573–580www.elsevier.com/locate/aqua-online

Aquaculture 272 (

Effects of moderately oxidized dietary lipid and the role of vitamin Eon the stress response in Atlantic halibut

(Hippoglossus hippoglossus L.)

Dulce Alves Martins a,b, Luis O.B. Afonso b, Sho Hosoya a,c, Leah M. Lewis-McCrea a,Luisa M.P. Valente b, Santosh P. Lall a,⁎

a National Research Council Canada, Institute for Marine Biosciences, Halifax, Nova Scotia, Canada, B3H 3Z1b ICBAS—Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, and CIMAR, Rua dos Bragas, 177,

4050-123 Porto, Portugalc Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan

Received 25 April 2007; received in revised form 21 August 2007; accepted 22 August 2007

Abstract

Lipid peroxidation of marine fish diets can affect the nutritional value of the diet and consequently fish health, especially in theabsence of adequate amounts of anti-oxidants. In this study, diets with different levels of oxidized oil and dietary vitamin E werefed to juvenile Atlantic halibut for 16 weeks and the effects on the acute stress response were investigated after this period. Fishwere fed diets containing either non-oxidized (POV=0.6 meq kg−1, diet A, control) or oxidized fish oil (POV=7.5 meq kg−1, dietsB and C; 15 meq kg−1, diets D and E). Diets A, C and E were supplemented with vitamin E (300 IU kg−1). Following this period,Atlantic halibut were subjected to a 1-h heat shock (HS; from 12 to 18 °C). Plasma cortisol and glucose, and red blood cells (RBC)heat shock protein 70 (hsp70) levels were measured prior to, 0 (immediately after), 6, 12, and 24 h after stress. Two-way ANOVA,using dietary treatment and sampling point as main factors, was performed. In all experimental groups, Atlantic halibut showedincreased plasma cortisol levels immediately after (0 h) heat shock, however these returned to pre-stress levels by 6 h. Similarly,plasma glucose level increased significantly immediately after heat shock and decreased to pre-stress levels by 6 h. Dietarytreatment had a significant effect on plasma glucose levels. Fish fed the highly oxidized diet (diet E) showed lower overall plasmaglucose levels than fish fed less or non-oxidized diets (diets A, B, and C). RBC hsp70 was detected in all treatment groups.However, no significant changes in hsp70 levels were observed after exposure to heat shock. The overall results indicate thatjuvenile halibut fed diets containing oxidized fish oil up to a peroxide value of 15 meq kg−1 were able to cope with temperaturestress, regardless of dietary vitamin E content. The glucose results, however, suggest that highly oxidized diets decrease the overallglucose levels. Furthermore, plasma cortisol and glucose, but not hsp70, seemed to be adequate indicators of heat shock stress injuvenile halibut.Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved.

Keywords: Atlantic halibut; Lipid peroxidation; Cortisol; Glucose; Heat shock proteins

⁎ Corresponding author. Tel.: +1 902 426 6272; fax: +1 902 426 9413.E-mail address: [email protected] (S.P. Lall).

0044-8486/$ - see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2007.08.044

574 D. Alves Martins et al. / Aquaculture 272 (2007) 573–580

1. Introduction

Lipids are the major dietary energy source forcarnivorous fish. Marine fish diets contain high levels ofmarine fish oil rich in polyunsaturated fatty acids, such aseicosapentaenoic, docosahexaenoic and arachidonicacids, which are considered essential for these fish(NRC, 1993). These highly unsaturated fatty acids(HUFA) are precursors of prostaglandins and othercompounds and have an important role in cell membranestructure and integrity. However, polyunsaturated fattyacids are particularly prone to oxidation, which can occurduring feed processing and storage.

Lipid peroxidation occurs through a direct reaction oflipids with molecular oxygen, catalyzed by free radicals.Oxygen radicals react with the double bonds of polyunsat-urated fatty acids, generating unstable lipid radicals bothin vitro, causing rancidity in feeds, and in vivo, with detri-mental effects on tissue fatty acid composition, vitamin Econtent and sensory and nutritional attributes. Lipidperoxidation may result in damage to cellular biomem-branes, particularly to those of sub-cellular organelles, rel-atively rich in PUFA (Halliwell and Gutteridge, 1996). Infish, peroxidized lipid in diet has been shown to affectgrowth and the immune system, especially in the absenceof adequate amounts of anti-oxidants (Obach and Laur-encin, 1992; Obach et al., 1993; Baker and Davies, 1996a).

Generally, it is assumed that the nutritional state of afish can affect the animal health and possibly the way theydeal with stress. The stress response in fish is generallymediated by a neuroendocrine response, which includesthe release of stress hormones such as cortisol andcatecholamines into the circulatory system (Barton andIwama, 1991). These and possibly other hormones, elicitseveral compensatory physiological responses that helpthe fish to deal with the stressor (Mommsen et al., 1999).Glucose is one of the most important energy substratesused by fish to cope with physiological stress andtherefore plasma glucose levels have been used as a stressindicator. Moreover, heat shock protein (hsp) levels areoften used as indicators of cellular stress response in fish.Among the hsps, the 70 kDa class (hsp70) can be inducedwhen fish are exposed to temperature variation and otherenvironmental stressors (Iwama et al., 2004).

It has been shown that dietary lipid can affectresponse of fish to stress. Manipulation of dietary lipidcomposition was shown to affect the ability of larval fishto resist several stressors, hence influencing survivalfollowing stress (Izquierdo et al., 1989; Watanabe et al.,1989; Tuncer et al., 1993; Kanazawa, 1997; Gapasinet al., 1998; Koven et al., 2001, 2003; Liu et al., 2002).Likewise, in adult gilthead seabream (Sparus aurata),

high dietary intake of arachidonic acid lowered thesensitivity of fish to acute stress, as evidenced bydecreased plasma cortisol levels and plasma osmolality(Van Anholt et al., 2004).

Despite a potential relationship between dietary lipidsand resistance to stress, little is known about how oxidizeddietary lipids affect the physiological stress response infish. Furthermore, there is a lack of information withrespect to the stress response in juvenile marine fish andAtlantic halibut, in particular. This study was undertakento investigate the effects of dietary lipid oxidation and thepotential role of vitamin E on the acute heat stressresponse of Atlantic halibut. Plasma cortisol, plasmaglucose and red blood cells hsp70 levels were measuredprior to and after a 1-h heat shock.

2. Materials and methods

2.1. Experimental conditions — initial feeding trial

Atlantic halibut (Hippoglossus hippoglossus) obtainedfrom Scotian Halibut Ltd. (Clarke's Harbour, Nova Scotia,Canada) were reared at the NRC-IMBMarine Research Station(Halifax, NS, Canada). Initially, groups of 44 fish (4.9±0.09 g)were randomly distributed among 15 tanks (350-L) suppliedwith flow through seawater and continuous aeration. Followinga 15-day acclimation period, fish were fed each of theexperimental diets (Lewis-McCrea and Lall, 2007), in triplicategroups, for 14 weeks. During the experimental period, watertemperature, flow and dissolved oxygen were maintained at12.1 ± 0.03 °C, 3.0 L− 1 min and 10.3 ±0.10 mg L− 1

respectively and under constant dim lighting conditions. Fishwere hand fed to satiation twice daily (0830 and 1630).

2.2. Experimental diets

Five isonitrogenous (56%), isolipidic (18%) and isoenergetic(23 MJ kg−1) experimental diets were formulated. These diets,which differed in the oxidation level of the fish oil included: dietA (control), non-oxidized oil (peroxide value=0.6 meq kg−1);diets B and C,moderately oxidized oil (peroxide value=7.5meqkg− 1); diets D and E, highly oxidized oil (peroxidevalue=15 meq kg−1). A mixture of anchovy, herring andmackerel oil was the main lipid source in the diet. The dietary oilwas oxidized according toKoshio et al. (1994), by bubblingwithair while stirring and heating at 50 °C. Vitamin E in the form ofα-tocopheryl acetate was supplemented to diets A, C and E(356 IU kg−1), but not to diets B and D. Detailed informationregarding the formulation of the experimental diets has beenpreviously reported by Lewis-McCrea and Lall (2007).

2.3. Acute stress study and sampling procedure

At the end of the 14-wk feeding trial, fish (27.7±2.3 g) weretransferred to 15 tanks of 120 L (23 fish/tank and 3 replicate

Fig. 1. Plasma cortisol levels (ng mL−1) in juvenile Atlantic halibut fed the experimental diets and subjected to a +6 °C heat shock for 1 h. Fish weresampled before (initial) and at 0 (immediately after), 6, 12 and 24 h after heat shock. Within each diet, different letters represent significant differencesbetween sampling times (Pb0.05). Values are expressed as mean±SEM, where the mean values were obtained from the means of three replicatetanks per treatment (n=3).

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tanks per treatment) and fed with the same diets as previously,once daily (0900), for an additional 2 weeks. During this periodwater temperature, water flow and dissolved oxygen weremaintained at 12.1±0.03 °C, 5.0 L−1 min and 8.9±0.3 mg L−1,respectively, under a 16 h light:8 h dark photoperiod.

Following this 2-week acclimation period, all fish weresubjected to a heat shock by increasing the water temperaturefrom 12 to 18 °C within 5 min. After a 1-h exposure, the watertemperature was returned to 12 °C. Three fish per tank (9 fishper treatment) were sampled prior to the heat shock (initial),and immediately after (0 h) and 6, 12, and 24 h after heat stress.Fish were quickly captured, and placed in a bucket containing alethal dose (400 mg L−1) of tricaine methanesulfonate (Syndel

Fig. 2. Plasma glucose levels (mg dL−1) of juvenile Atlantic halibut fed the exsampled before (initial) and at 0 (immediately after), 6, 12 and 24 h after heat sbetween sampling times (Pb0.05). Symbols represent significant differences (SEM, where the mean values were obtained from the means of three replica

Laboratories Ltd., Vancouver, BC, Canada) prior to sampling.Blood was collected within 2 min from the caudal vessel, usingheparinized syringes and placed in Eppendorf tubes on ice untilcentrifugation (1000 g for 10 min at 4 °C). Plasma wasseparated for subsequent analysis of glucose and cortisol,whereas red blood cells were aliquoted in 1.5 mL Eppendorftubes for determination of heat shock protein 70 (hsp70) levels.All samples were frozen at −80 °C until further analysis.

2.4. Determination of plasma cortisol and glucose

Plasma glucose levels were measured (in duplicate) using amodified Trinder (1969) enzymatic assay (Diagnostic

perimental diets and subjected to a +6 °C heat shock for 1 h. Fish werehock. Within each diet, different letters represent significant differencesPb0.05) between experimental groups. Values are expressed as mean±te tanks per treatment (n=3).

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Chemicals Ltd. Charlottetown, PEI, Canada). Plates were readat 505 nm on a Molecular Devices VERSAmax microplatereader (Molecular Devices Corporation, Sunnyvale, CA, USA)and intra- and inter-assay coefficients of variation were alwaysless than 5%. Total plasma cortisol levels were determinedusing an enzyme-linked immunosorbent assay (ELISA) kit(Neogen Corp., Lexington, KY, USA), read at 650 nm (Basuet al., 2001) in the Molecular Device VersaMax microplatereader. Intra- and inter-assay variation did not exceed 10%.

2.5. Determination of red blood cells hsp70 by SDS-PAGEand immunodetection

Red blood cell samples were homogenized in ice-cold lysisbuffer (1:10 w:v) containing: 100 mM Tris–HCl (pH 7.5),0.1% SDS, 1 mM ethylenediaminetetraacetic (EDTA), 1 μMpepstatin A, 1 mM phenylmethylsulfonyl fluoride (PMSF),1 μM leupeptin, and 0.01 μM aprotinin. Lysates werecentrifuged at 10,000 g for 3 min at room temperature and10 μL of supernatant were removed for protein determinationusing the bicinchoninic acid (BCA) assay (Smith et al., 1985)and bovine serum albumin (BSA) as a standard. A 1:1 solutionof supernatant and SDS-sample dilution buffer preparedaccording to Laemmli (1970) was boiled for 3 min and frozenat −80 °C until hsp70 analysis. Samples were subjected todiscontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) following the method of Laemmli (1970). Proteinswere resolved with a 4% stacking and 12% resolving gel on aMini-Protean II electrophoresis cell (Bio-Rad Laboratories,

Fig. 3. A. Representative autoradiograph from western blot analysis of RBCperiods after a 1-h heat shock. B. Red blood cell (RBC) heat shock protein 70fed the experimental diets and subjected to a +6 °C heat shock for 1 h. Fish wafter heat shock. Absence of letters and symbols indicates no significantrespectively. Values are expressed as mean±SEM, where the mean values we

Hercules, CA, USA). Molecular weight markers (Magic-Mark™ XP Western Protein Standards, Invitrogen, Carlsbad,CA, USA) were loaded onto every gel. Each lane was loadedwith approximately 20 μg of protein.

Following electrophoretic separation, levels of hsp70 weredetermined according to the method of Basu et al. (2001).Briefly, proteins were transferred onto nitrocellulose mem-branes (at 100 V for 60 min). After blotting, membranes wereincubated for 1 h with a monoclonal anti-hsp70 antibodyproduced in mouse (Sigma-Aldrich, St. Louis, MO, USA).This antibody recognizes both the constitutive and inducibleforms of hsp70. Liver, gill and red blood cell samples wereused to optimize the concentration of the antibodies used forimmunodetection, and concentrations of 1:2000 and 1:10,000for the primary and secondary antibodies, respectively,produced the best results. During the optimization procedure,it was noted that red blood cell tissue samples displayed themost consistent bands. Therefore, only this tissue wasexamined in the present study. Following incubation with theprimary antibody, membranes were incubated for 1 h with ananti-mouse IgG peroxidase antibody (1:10,000) produced ingoat (Sigma-Aldrich, St. Louis, MO, USA). Both antibodieswere diluted with 2% skim milk powder in TBS (20 mM Tris,500 mMNaCl, 0.05% Tween-20, pH 7.5). Hsp70 was detectedby chemiluminescence (ECL™ Western Blotting AnalysisSystem, Amersham Biosciences UK Ltd., Buckinghamshire,UK) on Kodak BioMax Light film (Eastman Kodak Company,Rochester, NY, USA), and bands were scanned using an HPScanjet 4570C (Hewlett–Packard, Palo Alto, CA., USA) and

hsp70 expression, in Atlantic halibut, before (initial) and at different(hsp70) levels (relative to positive control), in juvenile Atlantic halibutere sampled before (initial) and at 0 (immediately after), 6, 12 and 24 hdifferences (PN0.05) between sampling times or dietary treatments,re obtained from the means of three replicate tanks per treatment (n=3).

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quantified using Quantity One software (Version 4.5.1, Bio-Rad Laboratories, Hercules, CA., USA). All values werenormalized using band intensity of positive controls.

2.6. Statistical analysis

Statistical analyses followed methods outlined by Zar(1996). Data were submitted to a two-way ANOVA withdietary treatment and sampling time as main effects usingSPSS 14 for windows (SPSS Inc., Chicago, IL, USA). When Fvalues showed significance, individual means were comparedusing the Tukey's HSD test. Significant differences wereconsidered when Pb0.05. All data in the Results sectionrepresent mean values of 3 tanks per treatment and they arereported as mean±SE.

3. Results

3.1. Plasma cortisol and glucose levels

Cortisol levels were significantly affected by sampling time(P=0.000), but no effect of dietary treatments was observed(P=0.916). Initial plasma cortisol levels ranged from 3.3 to24.6 ng mL−1, in all groups of fish (Fig. 1). Plasma cortisollevels, in all treatments, increased significantly (4–29 fold)immediately after (0 h) the heat shock, but returned to valuesno longer different from pre-stress levels by 6 h.

Plasma glucose levels (Fig. 2) showed significant effects ofboth dietary treatment (P=0.003) and sampling time(P=0.000), however no effect of the interaction betweenthese factors was detected (P=0.567). Prior to stress, plasmaglucose levels ranged from 22.0 to 29.0 mg dL−1. Plasmaglucose levels increased significantly in all treatmentsimmediately after HS (0 h), but returned to values no longerdifferent from pre-stress levels by 6 h.

Throughout the study, the highly oxidized diet supplemen-ted with vitamin E (diet E) presented significantly lowerplasma glucose levels than the non-oxidized or moderatelyoxidized diets (A, B and C). Plasma glucose levels in alltreatments were further decreased by 12 and 24 h.

3.2. Red blood cells hsp70 levels

Using a mouse monoclonal hsp70 antibody, whichrecognizes both the constitutive and inducible forms of thisprotein, bands of approximately 70 kDa were detected(Fig. 3A). The 2-way analysis of variance showed that neitherdietary treatment nor sampling point had significant effects onred blood cell hsp70 levels (Fig. 3B). High inter-individualvariation in the hsp response was observed in all groups.

4. Discussion

To our knowledge, this is the first study that providesinformation on plasma cortisol and red blood cells hsp70levels in juvenile Atlantic halibut. It also demonstrates

that diets containing up to 15 meq kg−1 oxidized fish oil,whether supplemented with vitamin E or not had no effecton the ability of juvenile halibut to respond to an acutethermal stress.

Atlantic halibut showed a large inter-individualvariation in the acute physiological response to a heatstress. In all groups, both plasma cortisol and glucoselevels peaked immediately after the heat shock (0 h) andreturned to pre-stress levels after 6 h. Glucose levelsshowed a further decrease after 12 h in all treatments.The profiles of the cortisol and glucose response prior toand after stress were similar to that observed in otherteleosts (Barton and Iwama, 1991). In addition, thebaseline and maximum plasma cortisol levels (3.3–24.6 ng mL−1 and 78.1–103.4 ng mL−1, respectively)in Atlantic halibut were within the range reported forother flatfish such as Pacific halibut, Hippoglossusstenolepis (Davis and Schreck, 2005) and turbot,Scophthalmus maximus (Mugnier et al., 1998; Person-Le Ruyet et al., 1998, 2002, 2003; Irwin et al., 1999;Pichavant et al., 2002).

Baseline plasma glucose levels (22.0–29.0 mg dL−1)were similar to those observed in other studies withAtlantic (Hemre et al., 1992; Staurnes, 2001) and Pacific(Davis and Schreck, 2005) halibut. In this study, thermalstress leads to maximum glucose levels ranging from29.5 to 37.1 mg dL−1. However, plasma glucose levelsof approximately 90 mg dL−1 were reported in halibutsubjected to a cold temperature shock from 8–9 °C to1 °C (Staurnes, 2001). The further decrease in plasmaglucose concentration observed after 12 h in all treat-ments, was probably due to the over 24 h feed with-drawal. Fish fed diets with oxidized oils of 7.5 meq kg−1

(diets B and C) exhibited similar plasma glucoseconcentration to that of fish fed diet A. However, thegroup fed the highest oxidation diet with vitamin Esupplementation (diet E) presented lower overallglucose levels than control fish, as well as groups feddiets B and C. Although the reason for this is unclearand further studies are required to fully explain thisobservation, it may be possible that the highly oxidizeddiet affected glucose turnover (inhibited synthesis orincreased catabolism), especially upon supplementationwith the anti-oxidant. Studies examining the glycogenlevels in fish fed highly oxidized diets could beimportant to elucidate the mechanisms underlying thisresponse. Ortuño et al. (2003) also observed reducedblood glucose levels after multiple stressors (waterstirring, crowding, 2 min exposure to air) in seabreamfed vitamin E supplemented diets. Independently of thedifferences obtained in our study, fish fed diet E,presented the classical increase in glucose after stress,

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which suggests that their ability to respond to stress wasnot compromised. Furthermore, the plasma glucoselevels observed in all sampling times in fish fed diet Ewere within the physiological range for the species. Theeffects of dietary oil oxidation and vitamin E in stressresponse are still unclear and need to be furtherinvestigated. Taken together, these results demonstratethat like most teleostean fish, juvenile Atlantic halibutcan elicit the neuroendocrine (cortisol) and metabolic(glucose) responses when exposed to heat stress.

Several studies have demonstrated the negative effectsof feeding oxidized oils on fish growth, survival,histopathology and haematology (Cowey et al., 1984;Baker and Davies, 1996a,b, 1997; Guarda et al., 1997;Begg et al., 2000; Huang and Huang, 2004). For theparameters measured, different levels of oxidized oil didnot influence the ability of halibut to respond to asubsequent stressor, independently of vitamin E supple-mentation. The lipid oxidation levels tested were stillwithin acceptable limits, according to feed industry prac-tices, andmay not have been sufficient to strongly affect theresponse of halibut to an acute stress. It is also possible thatthe ability of fish to counteract lipid peroxidation in tissues,induced by the ingestion of oxidized diets, is speciesspecific and size dependent (Mourente et al., 2002; Tocheret al., 2003). Growth and survival were affected byoxidized oil (42 meq kg−1) in 0.31 g halibut fed dietscontaining about 62.8–64.5% PUFA (Tocher et al., 2003).Therefore, larger fish, like the ones used in the presentexperiment (42±11.7 g), are probably more developed andable to cope with the stress imposed by the diets. In largersize halibut (149 g) growth performance and survival werenot negatively affected by feeding oxidized oils of53.3 meq kg−1 peroxidation value (Alves Martins, 2007).

No increase in RBC hsp70 level was observed in fishexposed to a +6 °C heat shock. Erythrocyte cellularmembranes, rich in highly unsaturated fatty acids, areconsidered to be highly sensitive to lipid peroxidation, andred blood cells have been shown to be a sensitive tissue toheat shock, exhibiting increased levels of hsp70 totemperature stress (Smith et al., 1999; Currie et al., 2000;Lund et al., 2003). The lack of hsp response following heatshock was also reported in the Antarctic fish Trematomusbernacchii exposed to an acute heat shock (immediatetransfer) from−1.5 to 10 °C for 2 h (Hofmann et al., 2000),in Atlantic cod (Gadus morhua) subjected to acutetemperature increments (1 °C day−1, from 11 to 16.5 °C;Zakhartsev et al., 2005), and in haddock (Melanogrammusaeglefinus) exposed to an acute heat shock from 10 to15 °C for 1 h (Hosoya et al., 2007). However, the fact thatsimilar levels of hsp70, possibly in their constitutive form(hsc70), were detected in all dietary treatments suggests

that the activity of this protein family is important forhalibut. The present results also indicated that the oxidationof the diets did not pose a challenge to the erythrocytes, inspite of increased peroxidation products and lowerpolyunsaturated fatty acid content in tissues of ∼27 ghalibut, at the end of the feeding trial (Lewis-McCrea andLall, 2007). Further, both the haematocrit and erythrocytefragility did not vary with dietary lipid oxidation and/orpresence of vitamin E at the end of the same period (Lewis-McCrea and Lall, 2007), indicating that the oxidativelevels imposed by the diets were not deleterious enough tothe fish. There are no previous studies in fish investigatingthe effects of oxidized diets on the heat shock proteinresponse. However, in mammals hsps have been seen toconfer cellular protection against oxidant injury in ratintestinal epithelial cells (Urayama et al., 1998) andastrocytes (Russo et al., 2001). Similarly, up-regulation ofactivators of proteasomal proteins were observed in ratsfed oxidized fat (Sülzle et al., 2004). An appropriate intakeof anti-oxidants, such as vitamin E, has been shown toincrease hsp levels in response to a stressor (Peng et al.,2000; Howard et al., 2002).

In conclusion, juvenile Atlantic halibut fed dietscontaining oxidized fish oil up to a peroxide value of15 meq kg−1 were able to cope with temperature stress,regardless of dietary vitamin E content. These diets mayhave not caused an extreme imbalance between anti-oxidants and pro-oxidants in the organism, possibly dueto an efficient endogenous anti-oxidant system in thisfish size, as opposed to smaller animals (Tocher et al.,2002, 2003). Furthermore, plasma cortisol and glucose,but not hsp70, seemed to be adequate indicators of heatshock stress in juvenile halibut.

Acknowledgements

This work was supported by AquaNet-Canada'sResearch Network in Aquaculture, project AP33,“Nutritional Strategies to Improve Lipid Utilization inDiets for Commercially Important Canadian FinfishSpecies”. Dulce AlvesMartins was financially supportedby grant BD/9301/2002 (FCT, Portugal). The authorswould like to thank Ronald Melanson, Carla Walbourne,Audrey Butt and Ryan Gibbs for their technicalassistance. Special thanks to Joyce Milley and SeanTibbetts for their scientific input in this research project.

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