Associations between tissue fatty acid composition and physiological traits of performance and metabolism in the seabass (Dicentrarchus labrax)

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1 3429 The Journl of Experimentl Biology 209, Published by The Compny of Biologists 2006 doi: /jeb Associtions between tissue ftty cid composition nd physiologicl trits of performnce nd metbolism in the sebss (Dicentrrchus lbrx) A. Chtelier 1, *, D. J. McKenzie 1, A. Prinet 1, R. Glois 1, J. Robin 2, J. Zmbonino 2 nd G. Clireux 1 1 CNRS-Ifremer UMR 10, Centre de Recherche sur les Écosystèmes Mrins et Aqucoles, Plce du Séminire, BP 5, L Houmeu, Frnce nd 2 Unité mixte Nutrition, Aquculture, Génomique Inr-Ifremer-Bordeux 1, Lbortoire Adpttion Reproduction Nutrition des Poissons, Ifremer, Centre de Brest, BP 70, Plouzné, Frnce *Author for correspondence (e-mil: Aurelien.Chtelier@crhl.ulvl.c) Accepted 24 My 2006 Sebss were fed for 4 months with diets where the lipid ws provided s either cnol oil (CO), plm oil (PO) or fish oil (FO), to generte diversity in their tissue ftty cid (FA) composition nd investigte how this influenced mjor trits of exercise performnce, crdic performnce nd respirtory metbolism. In prticulr, bsed upon previous observtions, we investigted the hypothesis tht enriching the fish tissues with oleic nd linoleic cids (OA, 18:1n-9 nd LA, 18:2n-6, respectively) from the CO nd PO diets would improve mximum exercise nd crdic performnce, nd increse erobic metbolic scope. This proved to be the cse; exercise respirometry on bss fitted with crdic flow probes reveled tht those fed CO nd PO diets hd significntly higher criticl swimming speed (U crit ) thn those fed the FO diet. The improved swimming performnce in the CO nd PO groups ws ccompnied by higher mximum crdic output (Q) nd net crdic scope, nd higher ctive metbolic rte (AMR) nd erobic scope (AS) thn in the FO group. Summry Anlysis of tissue FA composition reveled tht the fish fed the CO nd PO diets hd ccumulted significntly higher levels of OA nd LA in their hert nd muscle thn the fish from the FO group, which hd significntly higher levels of highly unsturted FA of the n-3 series, such s EPA nd DHA (20:5n-3 nd 22:6n-3, respectively). Principl components nlysis reveled significnt positive ssocitions between tissue OA nd LA content nd U crit, mximum Q, the increse in Q during exercise, AMR nd erobic scope. There ws negtive ssocition between these physiologicl trits nd tissue content of EPA. Therefore, diet composition is n environmentl fctor tht cn generte significnt phenotypic diversity in mjor physiologicl trits of performnce nd metbolism in the sebss, with incresed intke of FAs such s OA nd LA leding to improved crdiorespirtory performnce. Key words: sebss, Dicentrrchus lbrx, swimming, metbolism, crdiovsculr performnce, ftty cid, diet. Introduction The study of exercise performnce in fishes hs long history but, to dte, most work hs focused on investigting the mechnisms contributing to propulsion (e.g. Webb, 1993; Webb, 1998; Videler, 1993), the energetics of ctivity metbolism (e.g. Bemish, 1978; Jones nd Rndll, 1978; Webb, 1993), nd the use of locomotor cpcity s guge of fish stress level nd/or wter qulity (e.g. Jin et l., 1998; McKenzie et l., 2003). Logic would dictte tht the bility to swim should fctor into the Drwinin fitness of mny fish species nd it is, therefore, widely ssumed tht swimming performnce will be criticl to the success of individul fish. Intr-specific diversities in performnce, nd the potentil sources of such diversity hve, however, received little ttention (reviewed in Kolok, 1999; Plut, 2001; Nelson et l., 2002) despite the fct tht this is the rw mteril upon which nturl selection might ct. Clerly, individul physiologicl diversity my hve genetic or environmentl origins but, t present, we hve little understnding of the reltive contributions from ech of these sources. One environmentl fctor tht is emerging s significnt source of physiologicl diversity in fish is diet qulity, nd prticulrly the reltive intke nd subsequent tissue ccumultion of certin ftty cids (FAs) (Tocher, 2003; McKenzie, 2005). Fishes ccumulte FAs from their diet, storing them s neutrl lipids (tricylglycerols) nd inserting them into membrnes s polr phosphoglycerides (Srgent et l., 1999; Tocher, 2003). McKenzie et l. (McKenzie et l., 1998) found direct positive reltionship between the sustined erobic exercise performnce of Atlntic slmon (Slmo slr) nd their muscle levels of oleic cid (OA; 18:1n-9) nd linoleic cid (LA; 18:2n-6). On the other hnd, highly unsturted FA of the n-3

2 3430 A. Chtelier nd others series (n-3hufa), nmely EPA (20:5n-3) nd DHA (22:6n-3), hve lso been reported to hve beneficil effects upon the exercise performnce of Atlntic slmon (Wgner et l., 2004). Indeed, the sme n-3hufa hve number of effects upon the crdiorespirtory physiology of fish (McKenzie, 2001; McKenzie, 2005) tht might impinge upon erobic exercise performnce. When compred with individuls fed diet rich in sturted ftty cids (SFA, such s 14:0 nd 16:0), whole-niml metbolic oxygen demnd (stndrd nd routine metbolic rtes) ws significntly lower in Adritic sturgeon (Acipenser nccrii) nd Europen eels (Anguill nguill) fed diet rich in n-3 HUFA (McKenzie, 2001). These reltive effects of SFA nd n- 3HUFA might influence erobic swimming bility if chnges in stndrd nd routine metbolic rtes influence metbolic scope for erobic ctivities (Fry, 1971). Furthermore, in vitro experiments hve demonstrted tht herts from the sturgeon fed the diet enriched in SFA were unble to mintin performnce when oxygen supply ws reduced, unlike herts from sturgeon fed the n-3hufa (Agnisol et l., 1996). The myocrdium is predominntly erobic muscle, nd crdic performnce is believed to be one of the mjor fctors underlying erobic exercise cpcity in ctive teleosts (Frrell, 1997; Frrell, 2002; Clireux et l., 2005; Clrk et l., 2005). The mechnisms for these effects of tissue FA re not known, lthough they presumbly derive from the mnner in which different neutrl tricylglycerols re used s oxidtive fuels, nd/or from chnges in cell function nd metbolism consequent to chnges in the phosphoglyceride composition of membrnes (McKenzie, 2001; McKenzie, 2005). The objective of the current study ws, therefore, to investigte how the reltive tissue levels of OA, LA, SFA nd n-3hufa influenced trits of exercise performnce, respirtory metbolism nd in vivo crdic performnce in the Europen sebss (Dicentrrchus lbrx). Sebss re ctive predtory teleosts tht feed primrily upon smller fish nd crustcens, which they cpture by pursuit (Pickett nd Pwson, 1994). Young stges re subject to predtion by pelgic fishes nd severl species of bird (Pickett nd Pwson, 1994). The species performs substntil migrtions nd is profoundly euryhline (Chtelier et l., 2005), colonising environments rnging from offshore ocenic wters to inshore, brckish nd freshwters in esturies nd costl lgoons (Pickett nd Pwson, 1994). Thus, exercise bility should be correlte, nd predictor, of the fitness of n individul sebss in its nturl environment. The vilbility of prticulr FAs (e.g. n-3hufa nd LA) cn disply lrge sptil nd sesonl fluctutions between esturine nd costl mrine foodwebs (Glois et l., 1996), so the different environments colonised by the sebss cn be expected to provide different diets. Three groups of fish were fed for 4 months with one of three diets in which lipids were provided s either cnol oil (CO), plm oil (PO), or cod liver oil, fish oil (FO), to supply different mounts of dietry OA, LA, SFA nd n-3hufa. Exercise respirometry nd crdic flow probes were then employed to mesure suite of physiologicl trits of metbolism nd performnce. The dietry oils provided complex mixture of FA longside specific ones of interest. Therefore, tissue levels of FA nd the suite of trits were ll mesured upon the sme fish, such tht principl components nlysis (PCA) could be performed to highlight ssocitions between physiologicl trits nd specific FA in the tissues. We investigted the hypothesis tht fish with tissues rich in OA nd LA (from CO nd PO) would exhibit improved exercise performnce by comprison with those with tissues rich in n-3hufa (from FO). We lso investigted the hypothesis tht n improved exercise performnce in sebss with tissues rich in OA nd LA would be linked to greter erobic metbolic scope nd in vivo crdic performnce. Furthermore, we investigted the hypothesis tht fish with tissues rich in SFA (from PO) would hve higher metbolic rte thn the fish with tissues rich in n-3hufa (from FO). Tissue polr nd neutrl lipid frctions were nlysed seprtely, to provide some insight into whether ny ssocitions between physiologicl trits nd tissue FA levels might derive from chnges to membrne polr lipid composition, or from the qulity of neutrl lipids s oxidtive fuels (McKenzie, 2001; McKenzie, 2005). Mterils nd methods All experiments were performed t the CNRS/Ifremer Centre de Recherche sur les Écosystèmes Mrins et Aqucoles (CREMA, l Houmeu, Frnce). Experimentl diets The experimentl diets were prepred t the Ifremer Centre de Nutrition des Poissons (Brest, Frnce) s 4.5 mm pellets. The bsl composition of these diets ws identicl. The ddition of CO s dietry lipid provided FA mixture dominted by OA, LA nd -linolenic cid (18:3n-3). Plm oil (PO) provided dietry FA mixture dominted by OA nd LA but lso by plmitic cid (16:0), SFA. The fish oil (FO) provided mixture of mny FA but, in prticulr, reltively high quntities of EPA nd DHA, n-3hufa tht were only present t low levels in the two vegetble oils. The FA composition of the three diets is shown in Tble 1. Experimentl nimls nd feeding Europen sebss (Dicentrrchus lbrx L.) with men mss (±s.d.) 200±10 g nd length 26.56±0.28 cm were obtined from commercil supplier on Ile de Ré (Chrente Mritime, Frnce). They were mintined t CREMA in 1 m 3 fibreglss tnks (wter volume pproximtely 400 liters) under nturl photoperiod. Tnks were provided with biofiltered sewter (SW) t temperture of 20 C nd slinity of 30. A totl of 72 fish were slightly nesthetised, fitted with subcutneous pssive integrted trnsponder (PIT) tg for individul identifiction, then llocted rndomly to one of 6 experimentl groups (2 replictes per diet, 12 individuls per replicte). The fish were then llowed 1 month of cclimtion to the previling holding conditions, during which they were fed commercil diet (Br D Perform Ntur 4.5; Sic du Gouessnt, Lmblle, Frnce). They were then fsted for 2

3 Ftty cids nd exercise in se bss 3431 Tble 1. Ftty cid composition of lipids in the diets s percentge of totl ftty cids Ftty cid CO PO FO 14: : : : :1n :1n :1n :1n :1n :1n :2n :4n :3n :4n :4n :5n :5n :6n SFA MUFA PUFA n3/n FA, ftty cids; FO, fish oil diet; CO, cnol oil diet; PO, plm oil diet; SFA, sturted ftty cids; MUFA, monounsturted ftty cids; PUFA, polyunsturted ftty cids. For the individul FA, only those tht occurred t more thn 0.5% of lipids re reported, with the exception of 20:4n-6. weeks prior to the beginning of the feeding protocol, t which point they ccepted the novel feeds egerly. Fish were then fed by hnd dily to stition, with their designted experimentl diet. After 4 months the fish hd pproximtely doubled in mss. Dily growth nd condition fctor were clculted s follows: Dily growth = {[(M f M i )/M i ] 100} / n, Condition fctor = (M f /BL 3 ) 100, where M f is finl mss in kg, M i is initil mss in kg, n is the number of feeding dys nd BL is body length in cm. Surgicl preprtion At this stge of the experiment, bss with men mss nd fork length FL of 395±10 g nd 30.58±0.28 cm, respectively, were nesthetised with tricine methne sulphonte (MS-222) t concentrtion of 0.1 g l 1, nd trnsferred to n operting tble where their gills were irrigted with erted wter contining 0.05 g l 1 MS-222. A 2S-type Trnsonic (EMKA, Pris, Frnce) ultrsound flow probe (resolution 0.1 ml min 1 ; bsolute ccurcy ±15%) ws plced round the ventrl ort, s described (Axelsson et l., 2002). Following surgery, the nimls were llowed 48 h recovery in opque PVC chmbers provided with flow of wter. Exercise nd crdic performnce Swimming respirometry ws performed using n utomted Brett-type swim-tunnel respirometer designed to exercise fish in non-turbulent wter flow with uniform velocity profile (McKenzie et l., 2001). Fish were trnsferred individully to the respirometer nd llowed to recover for t lest 12 h (overnight) t current speed of 0.5 BL s 1. At this low current speed bss rested on the bottom nd mintined position by gentle sculling of their pectorl fins nd occsionl til flicks. The following dy, fish were exposed to progressive increments in swimming speed t 1, 2, 3, 3.5 nd 4 BL s 1, every 30 min, until ftigue. Fish were considered to be ftigued when they were unble to remove themselves from the posterior screen of the swimming chmber despite gentle encourgement by sudden increses in current velocity. Mesurements of O 2 uptke (M O 2, in mg kg 1 h 1 ) were collected utomticlly t ech swimming speed with the custom-designed dt-cquisition system described elsewhere (McKenzie et l., 2001) nd custom-mde softwre (G. Guillou, CREMA). Wter O 2 sturtion in the seled respirometer ws mesured with n Orbisphere clrke-type polrogrphic oxygen electrode nd ssocited meter (Orbisphere Lbortory, Genev, Switzerlnd). The mesurements of M O 2 during exercise were used to derive the notionl metbolic rte of the immobile fish (IMR) (McKenzie et l., 2003b). The mximum metbolic rte of ctivity (AMR) ws identified during swimming (this occurred t speeds pproching criticl swimming speed U crit ) nd used to clculte net erobic scope (AS) reltive to IMR (McKenzie et l., 2003b). U crit ws clculted in BL s 1 (Brett, 1964). At ech swimming speed, crdic output (Q) ws mesured in ml min 1 kg 1 with the signl from the flowprobe displyed on the Trnsonic mplifier nd cquired by PC with the customdesigned lbview softwre described elsewhere (Axelsson et l., 2002). The signl ws used to clculte hert rte f H in bets min 1 nd, together with the dt for Q, used to clculte crdic stroke volume V SH, in ml bet 1 kg 1. Crdic scope during exercise ws clculted s Q mx Q routine. Q mx lwys occurred t swimming speeds pproching U crit, Q routine ws tken s the lowest Q mesured when the fish ws swimming very gently t speed of 5 cm s 1, prior to the exercise protocol. f H,routine nd V SH,routine were derived from the mesures of Q routine. Increse in f H nd V SH during exercise ws clculted, respectively, s f H,mx f H,routine nd V SH,mx V SH,routine. After experiments, nimls were rpidly removed from the respirometer nd killed with blow to the hed. The ventricle nd piece of white muscle were tken nd stored t 80 C until ftty cid nlysis. Tissue ftty cid nlysis Totl lipid extrction nd mesurement Lipids were extrcted from tissues following procedure derived from tht of Folch et l. (Folch et l., 1957). A double step extrction ws crried out on rehydrted smples by grinding them in chloroform:methnol mixtures (1:2 then 2:1, v/v) with n ll-glss Potter homogeniser. For ech smple, the

4 3432 A. Chtelier nd others two homogentes were filtered on GF/F pre-combusted filters (Whtmn, Brentford, UK) nd pooled in conicl glss centrifuge tube. Following the ddition of 1% NCl solution, the crude extrct seprted into two phses. After decnttion, the lower phse contining lipids ws recovered by suction nd stored in Teflon-cpped glss tubes t 20 C until nlysed. Totl lipids were mesured with Chromrod SIII Itroscn TH-10 system (TLC-FID, Itron Lbortories, Tokyo, Jpn) connected to CR3A integrtor (Shimdzu, Kyoto, Jpn). For ech smple, four successive volumes (1 l) of concentrted extrct liquot were spotted on Chromrod with 2 l microsyringe. After drying in dessictor, the rod ws red directly by the Itroscn without ny development. A clibrtion curve bsed on mixture of pure stndrds (Sigm-Aldrich, St Louis, MO, USA) ws used to clculte the totl lipid concentrtion of the lipid extrcts (Prrish nd Ackmn, 1985). Seprtion of polr nd neutrl lipids Totl lipids were seprted into polr nd neutrl frctions by high performnce liquid chromtogrphy (HPLC) (Kontron Anlyticl, Zürich, Switzerlnd) coupled with n ELSD-500 detector (Alltech, Deerfield, IL, USA). The seprtion ws crried out on preprtive 50 mm 10 mm i.d. stinless steel column filled with n SPE Si-diol phse (IST, Mid-Glmorgn, UK). The polrity grdient ws obtined with incresing proportions of methnol in chloroform. The column effluent ws by-pssed in such wy tht 90% of the seprted lipid frctions were recovered for ftty cid methyl ester (FAME) nlyses, while the detector mesured 10%. FAME preprtion, purifiction nd mesurement FAME from the seprted lipid frctions were obtined fter bse-ctlysed trnsesterifiction with sodium methylte (0.5 mol l 1, 1 h, 80 C) (Christie, 1984). FAME were purified by HPLC, using preprtive 100 mm 10 mm i.d. stinless steel column filled with n SPE Si-NH 2 phse (IST). The polrity grdient ws obtined by incresing the proportions of chloroform nd methnol in heptne. As bove, the column effluent ws by-pssed so tht 90% of the purified FAME ws recovered for gs chromtogrphy, while the detector mesured 10%. Purified FAME were then nlysed by gs chromtogrphy (Pckrd 439) on BPX-70 highly polr cpillry column 30 m 0.25 mm i.d m film (SGE, Courtboeuf, Frnce), with hydrogen s crrier gs t 1.1 ml min 1. The temperture grdient rn from 90 to 210 C t rte of 1 C min 1. Identifiction of FAME ws completed by comprison with pure individul stndrds (Sigm-Aldrich), stndrd mixtures (Supelco, Bellemonte, PA, USA) nd hydrogented smples. Sttisticl nlysis Differences mongst the dietry groups for ny given vrible were ssessed by one-wy nlysis of vrince (ANOVA) with Bonferroni post-hoc tests to identify where significnt differences ly. In those cses where the groups did not exhibit homogeneity of vrince, Kruskl Wllis nonprmetric ANOVA nd Mnn Whitney post-hoc tests were used. Links between hert polr lipids nd physiologicl trits were explored by Principl Component Anlysis (PCA) followed by Person correltion tests, using SttBox6 softwre. Only FA tht represented t lest 1% of tissue FA in t lest one lipid frction (polr or neutrl) were used for the PCA nd Person correltion tests. A probbility of less thn 5% (P<0.05) ws tken s the fiducil level for sttisticl significnce. Results The FO group exhibited significntly higher dily percentge increse in mss thn the other two dietry groups; men specific growth rtes (± s.e.m.) were 0.51±0.10, 0.31±0.03 nd 0.36±0.03% dy 1 in the FO, CO nd PO groups, respectively. The FO group lso exhibited significntly higher men condition fctor, nmely 1.46±0.04, 1.36±0.02 nd 1.33±0.03 in the FO, CO nd PO groups, respectively. Exercise performnce, crdic performnce nd respirtory metbolism Sebss fed the CO nd PO diets chieved men (± s.e.m.) U crit of 3.2±0.04 nd 3.24±0.05 BL s 1, respectively, which were not significntly different (Fig. 1). In contrst, fish fed FO only chieved U crit of 2.93±0.12 BL s 1, which ws significntly lower thn the other two groups (Fig. 1). There ws no significnt difference in Q routine between diets (Fig. 2), nor in routine V SH nd f H (Tble 2). There were, however, differences in the crdic response to exercise. As shown in Fig. 2, Q mx ws significntly higher in CO nd PO fish thn in those from the FO group. As consequence, the increse in Q during exercise ws lso significntly higher for CO nd PO fish (Fig. 2). In ll fishes, the incresed Q during Ucrit (BL s 1 ) CO PO FO Diet Fig. 1. Effects of diet on criticl swimming speed (U crit ) in the three groups of sebss fed diets where lipids were provided s either cnol oil (CO, green column), plm oil (PO, red column) or fish oil (FO, blue column). Vlues re mens ± s.e.m.; N=6 in ll cses. A dissimilr letter indictes significnt difference (P<0.05) between the dietry tretments. b

5 Ftty cids nd exercise in se bss 3433 exercise ws primrily consequence of incresed f H, with smller contribution from increses in V SH (Tble 2), but there were no differences in the mxim for these ltter two vribles mongst the three diets (Tble 2). IMR ws not significntly different between diets (Fig. 3) but there were significnt differences in respirtory metbolism during exercise. Exercise elicited n exponentil increse in M O 2 in ll fish (dt not shown) until plteu ws reched just prior to ftigue (U crit ). As shown in Fig. 3, AMR ws significntly higher in the CO nd PO groups reltive to the FO group. As direct consequence, net erobic scope ws lso significntly higher in the CO nd PO groups, reltive to the FO group (Fig. 3). Tissue FA composition As nticipted, there ws cler influence of dietry FA supply upon the FA composition of neutrl nd polr lipids in the hert (Tble 3) nd muscle (Tble 4). In polr lipids of the hert (Tble 3), the PO fish hd higher levels of steric cid thn found in the other two groups. Both the CO nd PO fish hd higher levels of OA nd LA in their polr lipids thn the FO fish did (Tble 3). On the other hnd, the FO fish hd higher Increse in Q (ml min 1 kg 1 ) Qmx (ml min 1 kg 1 ) Qroutine (ml min 1 kg 1 ) b b CO PO FO Diet Aerobic scope (mg O2 kg 1 h 1 ) Active MR (mg O2 kg 1 h 1 ) Immobile MR (mg O2 kg 1 h 1 ) b b CO PO FO Diet Fig. 2. Effects of diet on routine crdic output Q, mximum crdic output Q mx, nd the resultnt increse in crdic output during exercise Q routine, in the three groups of sebss fed diets where lipids were provided s either cnol oil (CO, green column), plm oil (PO, red column) or fish oil (FO, blue column). Vlues re mens ± s.e.m.; N=6 in ll cses. A dissimilr letter indictes significnt difference (P<0.05) between the dietry tretments. Fig. 3. Effects of diet on immobile metbolic rte, ctive metbolic rte, nd erobic scope, in the three groups of sebss fed diets where lipids were provided s either cnol oil (CO, green column), plm oil (PO, red column) or fish oil (FO, blue column). Vlues re mens ± s.e.m.; N=6 in ll cses. A dissimilr letter indictes significnt difference (P<0.05) between the dietry tretments. MR, metbolic rte.

6 3434 A. Chtelier nd others Tble 2. Routine nd mximum hert rte nd ventriculr stroke volume, nd the resulting increse in ech vrible during exercise, in sebss fed one of three diets where lipids were provided s cnol oil, plm oil or fish oil Vrible CO PO FO f H,routine, 44.0± ± ±5.2 f H,mx 95.1± ± ±2.3 Increse in f H 51.1± ± ±5.5 V SH,routine 0.96± ± ±0.18 V SH,mx 1.28± ± ±0.18 Increse in V SH 0.32± ± ±0.05 f H, hert rte (bets min 1 ); V SH, ventriculr stroke volume (ml); CO, cnol oil; PO, plm oil; FO, fish oil. Vlues re mens ± s.e.m. (N=6 in ll cses). There were no significnt differences between the groups. levels of n-3hufa such s EPA nd its elongtion product 22:5n-3. DHA comprised bout 28% of the hert polr lipids in ll groups, with no differences between them. The groups did not differ in their overll levels of SFA, MUFA nd PUFA but the FO group hd much higher rtio of n-3 to n-6 PUFA in its polr lipids by comprison with the other two groups. In the neutrl lipids, steric cid, OA, LA nd n-3hufa exhibited the sme overll pttern of distribution s seen for the polr lipids (Tble 3). However, the dominnt FA in the neutrl lipids of ll groups ws not DHA but, rther, OA. Furthermore, for their neutrl lipids, the PO group hd higher totl content of SFA thn the other two groups, nd it lso hd higher totl content of MUFA thn the FO group (hert neutrl MUFA levels vried gretly in the CO fishes, Tble 3). Conversely, the PO group hd significntly lower level of totl PUFA thn the CO nd FO groups. Finlly, the FO group, s expected, hd much higher rtio of totl n-3 to n-6 PUFA thn in the other groups, lthough the differences were less pronounced thn in the polr lipids (Tble 3). As cn be seen in Tble 4, skeletl white muscle lso exhibited this generl pttern of diversity in FA distribution between the three dietry groups. Indeed, the differences observed in the white muscle were, if nything, more pronounced thn in the ventriculr muscle (Tble 3 cf. Tble 4). This demonstrtes tht the diets hd generted systemic differences in tissue FA profile mongst the three groups. Principl component nlysis The PCA reveled some cler ssocitions between the mesured physiologicl trits nd levels of prticulr FA in the polr nd neutrl lipids of the hert (Fig. 4). The nlysis lso seprted the groups long the primry xis, with the CO nd Tble 3. Ftty cid composition of polr nd neutrl lipids in ventriculr muscle of sebss fed one of three diets where lipids were provided s cnol oil, plm oil or fish oil Polr FA Neutrl FA CO PO FO CO PO FO 14:0 <0.5 <0.5 < ± ± ±0.4 16:0 16.5± ± ± ± ±0.5 b 11.2±0.6 18:0 5.5± ± ± ± ± ±0.5 16:1n-7 0.3± ± ± ± ± ±0.4 b 18:1n-7 2.7± ±0.1 b 2.8± ± ± ±0.1 18:1n ± ±0.8 b 13.2±0.3 c 27.6±1.4,b 31.7± ±0.9 b 20:1n-9 1.6± ± ±0.1 b 0.8± ± ±0.0 22:1n-11 <0.5 <0.5 < ± ± ±0.4 18:2n-6 9.4± ± ±0.1 b 13.9± ± ±0.6 b 20:2n-6 0.8± ± ± ± ± ±0.2 20:4n-6 3.5± ± ± ± ± ±0.2 18:3n-3 1.9± ±0.1 b 1.0±0.0 b 3.9± ±0.1 b 2.7±0.1 b 18:4n-3 <0.5 <0.5 < ± ±0.1 b 1.0±0.1 20:4n-3 <0.5 <0.5 < ± ± ±0.0 b 20:5n-3 6.7± ±0.6 b 9.8±0.3 c 4.8± ±0.3 b 6.3±0.2 c 22:5n-3 1.6± ± ±0.1 b 1.1±0.0,b 0.7± ±0.3 b 22:6n ± ± ± ±1.6,b 9.7± ±1.0 b SFA 23.0± ± ± ± ±0.7 b 17.0±1.9,b MUFA 22.7± ± ± ±2.4,b 39.0± ±1.0 b PUFA 54.2± ± ± ± ±1.4 b 37.0±1.8,b n-3/n-6 3.0± ± ±0.2 b 1.5± ± ±0.1 b Vlues re mens ± s.e.m., N=7 in ll cses. CO, cnol oil; PO, plm oil; FO, fish oil; FA, ftty cids; SFA, sturted ftty cids; MUFA, monounsturted ftty cids; PUFA, polyunsturted ftty cids, n-3/n-6, rtio of totl n-3 to totl n-6 PUFA. Dissimilr letters indicte significnt differences (P<0.05) between dietry tretments for tht ftty cid frction (polr or neutrl).

7 Ftty cids nd exercise in se bss 3435 Tble 4. Ftty cid composition of polr nd neutrl lipids in white skeletl muscle of sebss fed one of three diets where lipids were provided s cnol oil, plm oil or fish oil Polr FA Neutrl FA CO PO FO CO PO FO 14:0 <0.5 <0.5 < ± ± ±0.3 b 16:0 16.7± ±1.2 b 19.0± ± ±2.5 b 16.3±0.8 18:0 6.1± ± ± ± ±0.6 b 3.1±0.1 b 16:1n-7 0.4± ± ± ± ± ±0.3 b 18:1n-7 1.5± ±0.1 b 1.6± ± ±0.4 b 3.2±0.1 18:1n ± ± ±0.4 b 29.3± ± ±4.0 b 20:1n-9 1.0±0.0,b 0.9±0.1 b 1.1± ± ± ±0.1 20:1n-11 <0.5 <0.5 < ± ± ±0.7 18:2n ± ± ±0.1 b 12.9± ± ±0.4 b 20:2n-6 <0.5 <0.5 < ± ± ±0.0 20:4n-6 1.7± ± ±0.0 b 0.3± ± ±0.0 22:4n-6 0.5± ± ±0.0 <0.5 <0.5 <0.5 18:3n-3 2.6± ±0.2 b 1.0±0.0 c 4.7± ±0.2 b 3.2±0.2 b 18:4n-3 <0.5 <0.5 < ± ± ±0.1 b 20:4n-3 <0.5 <0.5 < ± ± ±0.0 b 20:5n-3 6.4± ± ±0.2 b 3.8± ± ±0.5 b 22:5n-3 1.1± ± ±0.2 b 0.9± ± ±0.3 22:6n ± ± ±1.2 b 7.3± ± ±0.9 SFA 23.8± ±1.6 b 26.5± ± ±3.5,b 23.6±1.1 b MUFA 24.4± ± ±0.4 b 37.3± ± ±3.3 b PUFA 51.8±1.1,b 47.4±2.8 b 54.6± ± ± ±1.9 n-3/n-6 2.8± ± ±0.2 b 1.3± ± ±0.1 b Vlues re mens ± s.e.m., N=7 in ll cses. CO, cnol oil; PO, plm oil; FO, fish oil; FA, ftty cids; SFA, sturted ftty cids; MUFA, monounsturted ftty cids; PUFA, polyunsturted ftty cids, n-3/n-6, rtio of totl n-3 to totl n-6 PUFA. Dissimilr letters indicte significnt differences (P<0.05) between dietry tretments for tht ftty cid frction (polr or neutrl). PO fish loclised close together nd prt from the FO fish (Fig. 4). In prticulr, on xis 1, fishes from the CO nd PO groups with elevted levels of OA, LA nd totl MUFA in their lipids hd high U crit, Q mx, scope to increse Q during exercise, AMR, nd AS (Fig. 4). On the other hnd, fishes from the FO group with high levels of n-3pufa nd high rtio of n-3 to n-6 PUFA hd low vlues for these trits of exercise nd crdic performnce but high vlues for growth nd condition fctor (Fig. 4). The xes 2 nd 3 showed some further ssocitions between tissue FA nd physiologicl trits. These ssocitions were not strictly linked to dietry group but were function of the intrinsic individul diversity mongst ll of the nimls studied (Fig. 4). Axis 3 shows n effect of polr lipids, whereby fishes with high levels of steric cid nd totl SFA tended to hve high IMR, wheres IMR ws low in fishes with high levels of DHA nd totl PUFA in their polr lipids (Fig. 4). Axis 2 shows n effect predominntly of neutrl lipids, whereby fishes with high levels of steric cid, OA, totl SFA nd totl MUFA in neutrl lipids tended to hve low routine V SH, wheres routine V SH ws high in fish with high levels of rchidonic cid (AA) nd DHA in neutrl lipids, or of AA in polr lipids (Fig. 4). These ssocitions between FA nd physiologicl trits were borne out by the Person correltions derived from the PCA. Tble 5 shows the correltions for polr lipids. Specific growth rte showed wek negtive correltion with LA levels nd positive correltion with n-3 to n-6 PUFA rtio. Immobile metbolic rte showed strong positive correltion with steric cid nd totl SFA in polr lipids, nd negtive correltion with DHA nd totl PUFA, this ltter probbly becuse totl PUFA were dominted by DHA (Tble 5). The performnce trits (U crit, Q mx, scope to increse Q during exercise, AMR nd AS) were ll correlted positively with LA nd OA in polr lipids, but negtively with EPA, 22:5n-3 nd rtio of n-3 to n-6 PUFA (Tble 5). Tble 6 shows the correltions for neutrl lipids. Once gin, growth ws correlted negtively with steric cid nd totl SFA levels in neutrl lipids, while both growth nd condition fctor were relted positively with EPA levels. Routine V SH ws correlted positively with AA, DHA nd totl PUFA in neutrl lipids (Tble 6). Performnce trits were relted positively to LA levels in neutrl lipids (but not neutrl OA levels, which did not emerge from the PCA), nd relted negtively to EPA levels nd rtio of n-3 to n-6 PUFA (Tble 6). Discussion The results demonstrte tht diet-relted chnges in the FA composition of the tissues cn hve significnt effects upon

8 3436 A. Chtelier nd others 4 Neutrl FA 2 16:0 18:1n-9 0 SFA 2 MUFA Polr FA 16:0 SFA Trits IMR A B Axis 1 (36%) Axis 1 (36%) Neutrl FA 20:4n-3 20:5n-3 n-3/n-6 Neutrl FA 18:2n-6 18:3n-3 Polr FA 20:5n-3 22:5n-3 n-3/n-6 Polr FA 18:1n-9 18:2n-6 MUFA Trits SGR CF Axis 2 (20%) Axis 3 (15%) Trits U crit Q mx Q inc AMR AS Neutrl FA 20:4n-6 22:6n-3 Polr FA 20:4n-6 Trits r-v SH Polr FA 22:6n-3 PUFA Fig. 4. Projections of individuls onto the plne of three min functions of principl components nlysis of individul dt for ventriculr muscle neutrl ftty cids (FA), polr FA, nd the mesured trits of growth, metbolism nd performnce, in the three groups of sebss fed diets where lipids were provided s either cnol oil (green symbols, N=5), plm oil (red symbols, N=5) or fish oil (blue symbols, N=6). (A) Projections for the first nd second xes, which describe 36% nd 20% of the vrition, respectively. (B) The sme first xis plotted ginst the third, which describes 15% of the vrition. mjor physiologicl trits of performnce nd metbolism in sebss. This overll result is consistent with previous studies showing n impct of tissue FA composition on the crdiorespirtory physiology of fish (Rndll et l., 1992; McKenzie et l., 1995; McKenzie et l., 1997; McKenzie et l., 1998; McKenzie et l., 2000; Agnisol et l., 1996; Wgner et l., 2004). As expected, the best physiologicl performnce ws observed in the nimls fed the CO nd PO diets, rich in OA nd LA. The PCA reveled number of ssocitions between the percentge content of specific FAs in the polr nd/or neutrl pools of ventriculr muscle nd prticulr trits of growth, metbolism nd performnce. The significntly higher U crit mesured in the CO nd PO groups, s compred to the FO group, ws linked to the high levels of OA nd LA in their tissues. These results re consistent with previous study on Atlntic slmon (McKenzie et l., 1998), which found tht incrementl substitutions of fish oil by cnol oil in the dietry lipids led to incrementl increses in swimming performnce, with direct positive reltion between tissue OA nd LA (derived from the dietry cnol oil) nd U crit. The current dt extend these observtions to demonstrte tht improved exercise performnce ws linked to improved mximum crdic performnce, higher ctive metbolic rtes nd greter erobic scope in fish with tissues rich in OA nd LA. It is possible tht the greter erobic scope nd higher swimming performnce of the CO nd PO fish ws consequence of the improved crdic performnce. It hs been rgued tht erobic myocrdil performnce my be primry fctor limiting AMR nd U crit in ctive teleosts (Frrell, 2002; Clireux et l., 2005; Clrk et l., 2005). A recent study hs demonstrted tht intrinsic individul diversities in Q mx re directly linked to prllel diversities in AMR, AS nd U crit in rinbow trout (Clireux et l., 2005). This linkge hs yet to be demonstrted for sebss, but there is some evidence for role of crdic performnce in defining erobic scope nd U crit. In mny ctive teleosts, including rinbow trout nd sebss, both Q nd M O 2 plteu s fish pproch U crit in swim tests (Kiceniuk nd Jones, 1977; Kolok nd Frrell, 1994; Thorresen et l., 1996; Gllugher et l., 2001; Chtelier et l., 2005) nd this hs been tken s circumstntil evidence tht it is limittions to crdic work tht re constrining erobic scope (Frrell, 2002). In the sebss, the simultneous plteu of both Q nd M O 2 during exercise corresponds with the initition of n intermittent burst nd cost swimming pttern (Chtelier et l., 2005) tht indictes recruitment of nerobic white muscle (Dy nd Butler, 1996) nd which precedes ftigue. Visul observtion of the ventrl ortic probe trce t this time of the experiment lso reveled significnt crdic rrhythmis, further circumstntil evidence tht impired crdic performnce ws linked to the onset of ftigue. This rrhythmi lwys occurred t higher swimming speeds in the CO nd PO fish, when compred to the FO nimls. At present, it is only possible to speculte bout the mechnism by which the tissue OA nd LA might exert their effects upon crdiorespirtory performnce. They my be relted to the fct tht erobic metbolism, nd erobic work, is fuelled primrily by -oxidtion of neutrl FA in fish (Hochchk nd Somero, 1984; Richrds et l., 2002). There is in vitro evidence to suggest tht OA nd LA re preferred over other FA, especilly HUFA, s substrtes for -oxidtion (Sidell nd Driedzic, 1985; Henderson nd Srgent, 1985; Egginton, 1996). It is conceivble, therefore, tht higher levels of these preferred substrtes in the tissues might llow the nimls to chieve higher rtes of erobic work (McKenzie, 2001). It ws unexpected, therefore, tht only levels of neutrl LA in tissue lipids emerged s being relted to high

9 Ftty cids nd exercise in se bss 3437 Tble 5. Significnt Person correltions (i.e. coefficient greter thn 0.5 derived from the principl components nlysis) between trits of growth, performnce or metbolism nd the percentge content of polr ftty cids in ventriculr muscle SGR CF U crit Q mx Q inc IMR AMR AS 16: :1n :2n :5n :5n :6n SFA 0.78 MUFA PUFA 0.73 n-3/n SGR, specific growth rte; CF, condition fctor; U crit, criticl swimming speed; Q mx, mximum crdic output; Q inc, increse in Q during exercise; IMR, immobile metbolic rte; AMR, ctive metbolic rte; AS, erobic scope; SFA, sturted FA; MUFA, monounsturted FA; PUFA, polyunsturted FA; n-3/n-6, rtio of totl n-3 to n-6 PUFA. performnce in the PCA, nd tht levels of neutrl OA nd totl neutrl MUFA showed no ssocition. This might rgue ginst such substrte mechnism. The linkge between levels of OA nd LA in polr lipids nd incresed crdic (nd exercise) performnce my lso be result of membrne processes. Studies on isolted mmmlin crdiomyocytes reveled tht extrcellulr ppliction of EPA nd other PUFA produced prompt nd reversible concentrtion-dependent inhibition of the L-type clcium current, thereby limiting clcium entry into the cells (Xio et l., 1997; Lef et l., 1999). It ws proposed tht such effects should protect ginst clcium overlod nd rrhythmi (Xio et l., 1997; Lef et l., 1999). Any such beneficil effect of EPA on crdic rrhythmi ws not reveled in our study. There is, however, preliminry evidence to indicte tht OA hs n inhibitory effect on L-type clcium chnnels nd could, therefore, protect herts ginst rrhythmi t high worklods (A. Chtelier, N. Imbert, J. L. Zmbonino Infnte, D. J. McKenzie nd P. Bois, mnuscript submitted for publiction). Such n effect might hve contributed to the improved crdic performnce in the CO nd PO fish. The reltively poorer exercise performnce of the sebss with tissues rich in EPA nd with high n-3 to n-6 PUFA rtio is lso consistent with previous results (McKenzie et l., 1998) on Atlntic slmon. These results re, however, in direct contrst to those reported by Wgner et l., who found improved exercise performnce in slmon fed diet rich in n-3hufa (Wgner et l., 2004). The explntion for these opposing results presumbly lies in the enormous complexity of fctors within such diet studies. In prticulr, the oils used in dietry studies ll provide complex mixture of FA. Mny of these FA hve specific biologicl roles tht interct with ech other (Srgent et l., 1999), such tht ech study is effectively unique unless gret cre is tken to mtch the ingredients. There my lso be minimum threshold of ction for some FA. In the current study, the reltively poor crdiorespirtory performnce of fish with tissues rich in EPA should not be tken s n indictor of overll reduced fitness, since tissue levels of this FA were correlted positively with high fish growth rtes. It is interesting tht the PCA reveled tht these effects were correlted with EPA (nd its elongtion product 22:5 n-3) rther thn DHA, the other essentil n-3hufa provided in the FO diet. Tble 6. Significnt Person correltions between trits of growth, performnce or metbolism nd the percentge content of neutrl ftty cids in ventriculr muscle SGR CF U crit V SH,routine Q mx Q inc AMR AS 16: :2n :4n :4n :6n SFA 0.56 PUFA 0.54 n-3/n Significnt Person correltions re coefficients greter thn 0.5 derived from the principl components nlysis. SGR, specific growth rte; CF, condition fctor; U crit, criticl swimming speed; r-v SH,routine, routine ventriculr stroke volume; Q mx, mximum crdic output; Q inc, increse in Q during exercise; AMR, ctive metbolic rte; AS, erobic scope; SFA, sturted FA; PUFA, polyunsturted FA; n-3/n-6, rtio of totl n-3 to n-6 PUFA.

10 3438 A. Chtelier nd others The high IMR observed in fish with high tissue totl SFA, reveled in the PCA nd Person correltions, re consistent with previous studies (McKenzie et l., 1997; McKenzie et l., 2000) where sturgeon nd eels fed SFA hd significntly higher rtes of metbolism thn those fed n-3hufa. The results of the PCA indicte tht this effect of SFA on IMR in sebss my hve been result of membrne-relted mechnism, s the ssocitions were only observed for polr lipids. It is lso interesting tht it ws high tissue levels of DHA in polr lipids tht were correlted with low IMR in the PCA, with no other HUFA implicted. Once gin, it is only possible to speculte bout the mechnism(s) for the effects on metbolic rte of SFA versus DHA (McKenzie, 2001). It hs been suggested tht effects of n-3hufa versus SFA on metbolic rte re due to membrnerelted processes, nd prticulrly to differences in energy consumption by membrne-bound ATPses, consequent to chnges in membrne FA composition nd physico-chemicl properties (Hulbert nd Else, 1999; Hulbert et l., 2005). These uthors, however, report tht n-3hufa rise metbolic rte, while SFA lower it, in number of terrestril endotherms (birds nd mmmls) (Hulbert nd Else, 1999; Hulbert et l., 2005). This is exctly opposite to the effects on metbolic rte reported here nd previously (McKenzie et l., 1997; McKenzie et l., 2000). On the other hnd, herts isolted from rts fed with n- 3HUFA (from fish oil) hd significntly lower rtes of myocrdil oxygen consumption thn herts from rts fed SFA (from coconut oil) (Pepe nd McLennn, 2002), which ws ttributed to differences in mitochondril membrne composition tht influenced proton lek (Pepe nd McLennn, 2002). The bsis for such contrsting results is not cler nd, while there cn be no doubt tht dietry nd tissue FA exert profound effects upon the metbolic nd crdiorespirtory physiology of vertebrtes, further work is needed to explore the mechnisms for these effects nd thereby, hopefully, to explin contrsting results. The positive correltion between routine V SH nd tissue totl PUFA levels, nd in prticulr HUFA such s AA nd DHA, cnnot be explined t present. Agnisol et l. found, when working spontneously in vitro, tht herts isolted from sturgeon fed diet rich in n-3hufa hd greter routine V SH, thn herts isolted from sturgeon fed diet rich in SFA (Agnisol et l., 1996). In the present study t 20 C, sebss exhibited much greter AMR, erobic scope, Q mx nd U crit thn the sebss t 15 C studied by Chtelier et l. (Chtelier et l., 2005). This demonstrtes tht temperture exerts profound effect upon the crdiorespirtory performnce of sebss. In both the present study nd tht of Chtelier et l. (Chtelier et l., 2005), the increse in Q during exercise ws ttributble to n increse in f H rther thn V SH. Rpid chnges in wter slinity hd no effect on crdic or swimming performnce (Chtelier et l., 2005). This indictes tht diet my be t lest s importnt n environmentl vrible s slinity for defining the performnce of sebss in their nturl environment. Migrtions of sebss my expose the nimls to entirely different FA vilbilities, prticulrly between mrine foodwebs tht re rich in n-3hufa nd esturine foodwebs where FA such s LA nd OA my be more common (Glois et l., 1996). Exploring the ssocitions between tissue FA composition nd trits of growth, performnce nd respirtory metbolism in wild sebss is, therefore, n interesting venue for future reserch. AMR AS BL CF CO DHA EPA FA FAME f H FL FO HPLC HUFA IMR LA M M O 2 MUFA n-3hufa OA PCA PIT PO PUFA Q SFA SGR SW U crit V SH List of symbols nd bbrevitions ctive metbolic rte erobic scope body length condition fctor cnol oil docoshexenoic cid eicospentenoic cid ftty cid ftty cid methyl ester hert rte fork length fish oil high performnce liquid chromtogrphy highly unsturted ftty cids metbolic rte of the immobile fish linoleic cid mss rte of oxygen uptke monounsturted ftty cids highly unsturted ftty cids of the n-3 series oleic cid principl component nlysis pssive integrted trnsponder plm oil polyunsturted ftty cids crdic output sturted ftty cids specific growth rte sewter criticl swimming speed crdic stroke volume A.C. ws funded by doctorl fellowship from IFREMER nd the Conseil Régionl Poitou-Chrentes. D.J.M. contributed to this study while employed upon the EU project Ethofish. The uthors re grteful to G. Guillou, P. Pineu nd M. Prineu for their ssistnce. References Agnisol, C., McKenzie, D. J., Tylor, E. W., Bolis, C. L. nd Tot, B. (1996). Crdic performnce in reltion to oxygen supply vries with dietry lipid composition in sturgeon. Am. J. Physiol. Regul. Integr. Comp. Physiol. 271, Axelsson, M., Altimirs, J. nd Clireux, G. (2002). Post-prndil blood flow to the gstrointestinl trct is not compromised during hypoxi in the sebss Dicentrrchus lbrx. J. Exp. Biol. 205, Bemish, F. W. H. (1978). Swimming cpcity. In Fish Physiology. Vol. VII (ed. W. S. Hor nd D. J. Rndll), pp New York: Acdemic Press.

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