Egg quality determinants in cod (Gadus morhua L.): Egg performance and lipids in eggs from farmed and wild broodstock

Transcription

1 Egg qulity determinnts in cod (Gdus morhu L.): Egg performnce nd lipids in eggs from frmed nd wild roodstock Guillume Slze 1, Dougls R. Tocher 1, Willim J. Roy 1 & Derek A. Roertson 2 1 Institute of Aquculture, University of Stirling, Stirling FK9 4LA, UK 2 Mchrihnish Mrine Frm Ltd, Mchrihnish, Argyll PA28 6PZ, UK Keywords: lipid, ftty cid, phosphtidylinositol, rchidonic cid, eggs, wild, frmed, Atlntic cod Gdus morhu L. Running title: Lipids in frmed nd wild cod eggs Correspondence: DR Tocher, Institute of Aquculture, University of Stirling, Stirling FK9 4LA, United Kingdom. Tel. No: ; Fx No: ; Emil: d.r.tocher@stir.c.uk 1

2 Astrct Lipids nd essentil ftty cids, prticulrly the highly unsturted ftty cids, 20:5n-3 (eicospentenoic cid; EPA), 22:6n-3 (docoshexenoic cid; DHA) nd 20:4n-6 (rchidonic cid, AA) hve een shown to e crucil determinnts of mrine fish reproduction directly ffecting fecundity, egg qulity, htching success, lrvl mlformtion nd pigmenttion. In Atlntic cod (Gdus morhu L.) culture, eggs from frmed roodstock cn hve much lower fertilistion nd htching rtes thn eggs from wild roodstock. The present study imed to test the hypothesis tht potentil qulity nd performnce differences etween eggs from different cod roodstock would e reflected in differences in lipid nd ftty cid composition. Thus eggs were otined from three roodstock, frmed, wild/fed nd wild/unfed, nd lipid content, lipid clss composition, ftty cid composition nd pigment content were determined nd relted to performnce prmeters including fertilistion rte, symmetry of cell division nd survivl to htching. Eggs from frmed roodstock showed significntly lower fertilistion rtes, cell symmetry nd survivl to htching rtes thn eggs from wild roodstock. There were no differences in totl lipid content or the proportions of the mjor lipid clsses etween eggs from the different roodstock. However, eggs from frmed roodstock were chrcterised y hving significntly lower levels of some quntittively minor phospholipid clsses, prticulrly phosphtidylinositol. There were no differences etween eggs from frmed nd wild roodstock in the proportions of sturted, monounsturted nd totl polyunsturted ftty cids. The DHA content ws lso similr. However, eggs from frmed roodstock hd significntly lower levels of AA, nd consequently significntly higher EPA/AA rtios thn eggs from wild roodstock. Totl pigment nd stxnthin levels were significntly higher in eggs from wild roodstock. Therefore, the levels of AA nd phosphtidylinositol, the predominnt AA-contining lipid clss, nd egg pigment content were positively relted to egg qulity or performnce prmeters such s fertilistion nd htching success rtes, nd cell symmetry. Arevitions: AA, rchidonic cid (20:4n-6); DHA, docoshexenoic cid (22:6n-3); EPA, eicospentenoic cid (20:5n-3); HUFA, highly unsturted ftty cids ( 20 crons 3 doule onds); PI, phosphtidylinositol; PUFA, polyunsturted ftty cids. 2

3 Introduction Significnt progress hs een mde recently in the culture of Atlntic cod (Gdus morhu L.) nd the life cycle hs een closed, llowing independence from wild fisheries (Brown & Puvnendrn 2002, Brown, Minkoff & Puvnendrn 2003). However, s with mny other new mrine species, the min issues for the further development of culture is the production of good qulity eggs, s well s lrvl feeding (Srgent, Tocher & Bell 2002; Brown et l. 2003). Kjesu (1989) reported extremely vrile fertilistion rtes (from 0 to 100%) with cod eggs from second-genertion frmed roodstock. Tody, cod htcheries cn still experience significnt prolems in otining sufficient good qulity eggs from frmed roodstock. Eggs from frmed fish cn hve vrile fertilistion rtes rnging from < 10% in poor tches to > 90% for the est tches, nd losses during the incution nd lrvl rering stges re often greter thn with eggs from wild roodstock. Nutrition is generlly ccepted to influence fish performnce, nd Mrshll, Yrgin & Lmert (1999) showed cler correltion etween oil levels nd fecundity in wild popultions of cod. Although there hve een nutritionl trils with cod (Lie, Lied & Lmertsen 1986; Olsen, Henderson & Pedersen 1991; Dos Sntos, Burkow & Joling 1993; Moris, Bell, Roertson, Roy & Morris 2001; Lll & Nnton 2002; Hemre, Krlsen, Mngor-Jensen & Rosenlund 2003: Hemre, Krlsen, Eckhoff, Tviet, Mngor-Jensen & Rosenlund 2004) including lrve (Vn Der Meeren 1993; Bskerville- Bridges & Kling 2000,; Clln, Jordn & Kling 2003), there re few studies on cod roodstock nutrition. In contrst, for other fish species, it is well estlished tht nutritionlly optimised diets re prticulrly crucil in roodstock mngement for the provision of good qulity eggs nd, therefore, lrve (Ashton, Frkvm & Mrch 1993; Czesny & Drowski 1998; Gllgher, Prmore, Alves & Rulifson 1998, Srgent et l. 2002). Thus, low fertilistion rtes, nd poor egg nd lrvl qulity cn often e directly relted to diet composition of the roodstock (Pvlov, Kjorsvik, Refsti & Andersen 2004). Essentil ftty cids (EFA) of the n-3 nd n-6 series re especilly importnt, nd hve een shown to ffect fecundity, egg qulity, htching success, nd mlformtion in mrine fish (Pvlov et l. 2004). In prticulr, mny studies with mrine fish hve demonstrted the importnce of three highly unsturted ftty cids (HUFA), 20:5n-3 (eicospentenoic cid; EPA), 22:6n-3 (docoshexenoic cid; DHA) nd 20:4n-6 (rchidonic cid, AA) (Srgent, Bell, McEvoy, Tocher & Estevez 1999; Srgent, McEvoy, Estevez, Bell, Bell, Henderson & Tocher 1999). Neurl tissues, such s rin nd eyes, contin very high levels of DHA (Srgent, Bell, Bell, Henderson & Tocher 1995), nd therefore DHA is very importnt for pelgic fish eggs nd lrve (including cod) which hve high percentge of 3

4 neurl tissue in reltively smll ody mss (Srgent et l. 2002). This highlights the importnt roles tht lipids, including specific lipid clsses nd ftty cids, hve in egg nd lrvl development nd thus the importnce of lnced dietry lipids in roodstock diets for optiml emryonic development. Both the solute nd reltive quntities of ech ftty cid pper to e importnt, ut this vries etween species (Srgent et l. 1999,). The primry hypothesis investigted in the present study ws tht differences in qulity nd performnce etween eggs otined from different cod roodstocks would e reflected in differences in lipid nd ftty cid composition of the eggs. Specificlly, this study compred the totl lipid content, lipid clss composition, nd ftty cid composition of totl lipid, s well s lipid-solule pigment (crotenoids) content in eggs otined from wild nd frmed roodstock popultions. These mesurements were relted to some sic performnce prmeters such s fertilistion rte, symmetry of cell division nd survivl to htching. Mterils nd methods Animls nd diet Eggs were otined from roodstock cod t commercil cod htchery on the west cost of Scotlnd. Three groups of roodstock, termed frmed (F), wild/fed (WF) nd wild (W) were used. The frmed roodstock (F) were htched in spring 2002 nd tnk rered, nd therefore were entirely htchery rered frmed fish. Two groups consisting of round 100 nd 200 fish held in two tnks were used for egg collection. The wild/fed roodstock (WF) were cptured in nd round the Clyde estury y seine net etween Septemer 2003 nd Ferury A totl of 355 fish were distriuted etween two tnks. These fish were fed the sme commercil or formulted diet s for the frmed roodstock from the time of their cpture until spwning. At the eginning of April 2004, third group of 27 freshly cught cod were otined from the wild. These fish were kept in single tnk nd spwned nturlly over period of two dys efore spwning cesed. These fish constituted the wild roodstock (W) group nd were not fed prior to the cesstion of spwning. All wild fish were qurntined nd tested for diseses prior to their utilistion y the htchery. The precise sex rtio in ech tnk ws unknown. Tnks were 5 m dimeter nd 2 m deep with volume of 40 m 3. The wter inlet ws tngentil, nd the outlet centrl. For egg collection, secondry outlet ws opened t the surfce, llowing wter to overflow through the collector. The wter flow ws 30 Lmin -1, llowing replcement rte of over 100 % per dy. The density of fish ws round 15 kgm -3 for ll tnks. The frm operted flowthrough system, with wter pumped from the se nd filtered (200 µm elt filter nd 60 nd 10 µm 4

5 drum filters) efore UV disinfection. Oxygen ws mesured through centrl proe nd mintined t 90 % sturtion, slinity ws ocenic nd constnt t 34 gl -1. Wter temperture ws controlled nd mintined t 8 C through n dditionl tnk wter inlet supplying chilled wter. F nd WF fish were fed y hnd twice dy with commercil pellets (Dn-Ex 115mm; 58% crude protein, 17% crude lipids, Dnfeed, Denmrk) until stition, t out 0.33 % iomssdy -1. From one month prior to, nd throughout the spwning seson, cod were fed with the ove pelleted feed in the morning (40 % of the dily rtion), nd in the fternoon they received mturing roodstock diet (Breed M; 62 % crude protein, 16 % crude lipids, INVE Aquculture, Belgium) (60 % of the dily rtion). The ftty cid compositions of the diets re given in Tle 1. Smpling protocols Eggs were collected over two-week period t the end of Mrch nd the eginning of April Eggs were spwned spontneously nd fertilised in the tnk. Ech morning, eggs were collected nd rought into the htchery nd live (floting) eggs smpled. A smple of eggs ws oserved under the inoculr microscope, nd the percentge fertilistion nd the symmetry spect recorded s descried elow. Approximtely 1 g of eggs were plced into 2 ml glss vils contining 1 ml of chloroform/methnol (2:1 y volume) contining 0.01% utylted hydroxytoluene (BHT) s ntioxidnt, nd stored t -20 C prior to nlysis. A further eggs for dry weight determintions were smpled in triplicte, wshed with distilled wter nd lotted to remove wter, nd plced in plstic microcentrifuge tues nd frozen t -20 C. Mesurement of egg performnce or qulity prmeters The percentge of fertilistion ws estimted y counting rndom smples of t lest 300 eggs s 3 tches of round 100 eggs per smple point. As cod usully spwn during the night, egg development hd egun y the time of collection nd, therefore, the symmetry of the first cell divisions ws lso estimted in these smples. A numericl score ws llocted to ech tch of eggs in order to reduce the degree of sujectivity in the estimtion. The score, description nd qulity rting were s follows. 1, represented tch with lmost ll eggs showing symmetry (very poor qulity); 2, most eggs symmetric (poor); 3, out 50 % of eggs showing symmetry (verge); 4, mjority of eggs hve good symmetry (good) to 5, no symmetry oserved (very good). It ws not possile to follow individul tches of eggs through to htching under norml commercil procedures s pooled tches eggs re 5

6 used in the norml opertion of the cod htchery. However, two rndomly chosen individul tches of eggs from frmed (F) nd wild/fed (WF) roodstock were followed through to htching in smll reserch incutors. The incutors were 70 L cylindro-conicl tnks ech receiving se wter, s descried ove, through tngentil pipe with njo filter outlet. An ir stone situted under the filter prevented clogging nd provided wter movement. At the ottom of the incutor purge ws instlled. Briefly, pir of incutors nd their support were disinfected with iodine, rinsed thoroughly with htchery wter, nd set up s descried ove efore receiving eggs. One incutor received F eggs nd the other WF eggs (F1 nd WF1 tches). Cod eggs require out 90 degree-dys to htch, so with wter temperture of 7 C, it took round 13 dys for the eggs to htch nd efore second pir of tches (F2 nd WF2) could e incuted. When ll the lrve hd htched, the survivl t htching ws clculted y sutrcting the dily-recorded mortlity from the initil weight of eggs put in the incutor. Eggs from the wild (W) roodstock were not investigted s these fish were still in qurntine nd eggs could not e used in the commercil frm. Lipid nlyses Totl lipid ws extrcted from eggs nd lrve smples y homogenising in 20 volumes of ice-cold chloroform/methnol (2:1, v/v) using rotting Teflon proe/glss homogeniser. Similrly, totl lipid ws extrcted from diet smples y homogenising in 20 volumes of chloroform/methnol (2:1, v/v) with n Ultr-Turrx tissue disrupter (Fisher Scientific, Loughorough, U.K.). Totl lipid ws prepred ccording to the method of Folch, Lees & Slone-Stnley (1957) with non-lipid impurities removed y wshing with 0.88 % (w/v) KCl. The weight of lipid ws determined grvimetriclly fter evportion of solvent under strem of oxygen-free nitrogen nd overnight desicction in vcuo. Seprtion of lipid clsses ws performed y high-performnce thin-lyer chromtogrphy (HPTLC). Approximtely 10 µg of totl lipid ws pplied s 2 mm streks nd the plte developed to two-thirds distnce with methyl cette/isopropnol/chloroform/methnol/0.25 % queous KCl (25:25:25:10:9, y vol.). After desicction, the plte ws fully developed with isohexne/diethyl ether/cetic cid (85:15:1, y vol.). The lipid clsses were visulised y chrring t 160 o C for 15 min fter sprying with 3 % (w/v) queous cupric cette contining 8 % (v/v) phosphoric cid nd quntified y densitometry using Cmg 3 TLC Scnner (Cmg, Muttenz, Switzerlnd) nd wincats softwre (Henderson & Tocher 1992). The identities of individul lipid clsses were confirmed y comprison with reference to the Rf vlues of uthentic stndrds run longside smples on HPTLC pltes nd developed in the ove solvent systems. 6

7 Ftty cid methyl esters (FAME) from egg, lrve nd diet totl lipids were prepred y cidctlysed trnsesterifiction of totl lipid ccording to the method of Christie (1982). Extrction nd purifiction of FAME ws performed s descried y Tocher & Hrvie (1988). FAME were seprted nd quntified y gs-liquid chromtogrphy (Crlo Er Veg 8160, Miln, Itly) using 30 m x 0.32 mm i.d. cpillry column (CP Wx 52CB, Chrompk, London, U.K.) nd on-column injection. Hydrogen ws used s crrier gs nd temperture progrmming ws from 50 o C to 150 o C t 40 o Cmin -1 nd then to 230 o C t 2.0 o Cmin -1. Individul methyl esters were identified y comprison with known stndrds nd y reference to pulished dt (Ackmn 1980; Tocher & Hrvie 1988). Dt were collected nd processed using the Chromcrd for Windows (version 1.19) computer pckge (Thermoquest Itli S.p.A., Miln, Itly). Pigment nlysis Smples (1 ml) of egg totl lipid were tken, solvent evported under oxygen-free nitrogen, nd redissolved in 500 µl isohexne (Bru, Kostic & Olson 1993). Totl crotenoid pigment ws mesured spectrophotometriclly t 470 nm using the E 1% (w/v) of Seprtion nd quntifiction of stxnthin ws performed y HPLC s descried in detil previously (Bell, McEvoy, Tocher & Srgent 2000). Briefly, the chromtogrphic system ws equipped with 5 µm Hypersil ODS column (4.6 mm x 25 cm, Cpitl HPLC, Broxurn, Scotlnd), Wters Model 501 pump, nd stxnthin ws detected t 470 nm nd quntified using n externl stndrd of stxnthin (Roche, Henor, Englnd) using Wters 490E multiwvelength UV/vis detector (Millipore (U.K.) Ltd., Wtford, Englnd). An isocrtic solvent system ws used contining ethyl cette/methnol/wter (20:72:8 v/v/v) t flow rte of 1 mlmin -1. Mterils BHT ws otined from Sigm Chemicl Co. (Poole, U.K.). HPTLC (10 cm x 10 cm x 0.15 mm) nd TLC (20 cm x 20 cm x 0.25 mm) pltes, precoted with silic gel 60 (without fluorescent indictor) were otined from Merck (Drmstdt, Germny). All solvents were HPLC grde nd were otined from Fisher Scientific UK, Loughorough, Englnd. 7

8 Sttisticl nlysis All dt were presented s mens ± SD with n vlues s noted. Differences etween roodstock origins of eggs were nlysed using one-wy ANOVA with Tukey s post-test using GrphPd Prism (v4.00 for Windows, GrphPd Softwre, Sn Diego Cliforni USA, Differences etween men vlues were regrded s significnt when P < 0.05 (Zr 1984). Results Egg performnce or qulity The fertilistion rte ws over three-fold higher for eggs from the wild (W nd WF) roodstocks compred to eggs from the frmed (F) roodstock (Fig.1A). In ddition, significntly higher symmetry scores were oserved with eggs from W nd WF roodstock compred to eggs from F roodstock (Fig.1B). Similrly, in the tches of eggs tht were le to e followed through incution, fertilistion rte nd symmetry were higher in the egg tches from the WF roodstock compred to the tches from the F roodstock (Tle 2). Highest survivl to htching (57.2 %) ws oserved with tch WF2 which lso showed the highest fertilistion rte nd symmetry, wheres the lowest survivl to htching (17.4 %) ws oserved in tch F2 which lso showed the lowest fertilistion rte nd symmetry score (Tle 2). Lipid content nd lipid clss composition of eggs There ws no difference etween eggs from the different roodstock in lipid content, whether presented s percentges of wet or dry weight of the eggs (Fig.2). Around 95 % of the egg totl lipid ws ccounted for y the four min lipid clsses, phosphtidylcholine (~ 40 %), phosphtidylethnolmine (~ 15%), tricylglycerol nd cholesterol (ech ~ 20 %) (Fig.3). There were no differences in the proportions of these mjor lipid clsses in the eggs from the different roodstock other thn the percentge of phosphtidylcholine eing higher in eggs from F roodstock compred to eggs from the W roodstock (Fig.3A). There were lso no significnt differences in most of the minor lipid clsses including sphingomyelin, free ftty cids nd steryl esters (dt not shown). However, the proportion of phosphtidylinositol (PI) showed gret differences etween eggs from the different roodstock with the rnk order eing W > WF > F with ll differences eing highly significnt (Fig.4). A similr pttern 8

9 ws oserved with the proportions of group of minor lipid clsses including phosphtidic cid, phosphtidlglycerol nd crdiolipid tht cnnot e resolved from ech other in this solvent system (Fig.4). The proportion of PI in incuted eggs just prior to htch ws higher in the tches from WF roodstock compred to F roodstock (verging 5.3 nd 4.1, respectively), with the significntly highest nd lowest levels eing mesured in eggs from WF2 nd F2, respectively (Fig.5). Ftty cid composition of egg totl lipid There ws no difference in the proportions of totl sturted, totl monounsturted, totl n-3 polyunsturted ftty cids (PUFA), nd totl PUFA in totl lipid etween eggs from the different roodstocks (Tle 1). The percentge of egg DHA did not vry etween roodstocks, ut EPA ws lower in eggs from WF roodstock compred to the other groups (Fig.6A) resulting in higher DHA/EPA rtio in eggs from WF roodstock (Fig.6B). However, the most striking difference in ftty cid composition ws the low level of AA oserved in eggs from F roodstock (Fig.6B) tht resulted in considerly higher EPA/AA rtio (Fig.6A). The percentge of AA in incuted eggs just prior to htch ws significntly lower in oth tches from F roodstock compred to the tches from the WF roodstock (verging 1.1 nd 2.7, respectively) (Fig.5). Consequently, the EPA/AA rtio in incuted eggs just prior to htch ws significntly higher in the tches from F roodstock compred to WF roodstock (verging 11.8 nd 5.5, respectively) (Fig.5). Pigment content of eggs Totl pigment levels s mesured spectrophotometriclly were significntly lower in eggs from the F roodstock compred to eggs from the W nd WF roodstocks (Fig.7). The level of the mjor crotenoid pigment, stxnthin, reflected this eing significntly lower in eggs from the F roodstock compred to eggs from the wild roodstocks (Fig.7). Discussion Broodstock nutrition is vitl to producing high qulity eggs nd lrve, nd dietry lipid nd ftty cid contents nd compositions re known to e importnt fctors in determining the success of the developing emryos nd lrve (Tndler, Hrel, Koven & Kolkovoski 1995; Izquierdo, Fernndez- 9

10 Plcios & Tcon 2001). Although, the ftty cid nd lipid clss compositions of eggs re generlly more conserved nd reltively less influenced y diet thn other fish tissues, it is possile to lter the ftty cid composition of fish eggs in reltively smll ut potentilly importnt wys. The primry im of this work ws chieved in tht eggs from the different roodstocks were shown to e chrcterised y different levels of qulity/performnce s determined y fertilistion rte, symmetry nd survivl to htching, nd tht this ws ssocited with differences in egg lipid, ftty cid nd pigment compositions. Specificlly, the eggs tht displyed the est performnce with the highest fertilistion rte, est symmetry nd highest survivl to htching nd hence, could e regrded s of the higher qulity, were chrcterised y higher levels of PI, AA nd crotenoid pigments. Good symmetry is chrcterized y cells of the sme size, with poor symmetry eing chrcterised y cells of gretly different sizes, for instnce t the 4-cell stge there could e one lrge cell nd three very smll cells. Previously, Pickov, Dutt, Lrson & Kiessling (1997) demonstrted tht this prmeter could e n importnt prmeter in terms of egg qulity, influencing htching success in cod. These uthors compred egg lipid ftty cid composition nd some performnce prmeters in two different cod stocks from the Bltic nd Skgerrk. The Bltic stock were cught over period, held in cptivity nd fed for period of up to two yers, wheres the ocenic Skgerrk cod were essentilly wild nd unfed. Symmetry ws higher in the eggs from Skgerrk stock nd this ws correlted with htching rte. A similr reltionship ws oserved in the present study. Improved egg performnce nd thus qulity in the present study ws shown to e relted to the level of AA in the cod eggs. Severl previous studies hve shown tht egg qulity criteri, including htching nd fertilistion rtes, nd survivl in the erly lrvl stges, were positively correlted with incresed levels of AA nd n-3hufa in oth se rem (Sprus urt L.) (Hrel, Tndler, Kissil & Appleum 1992; Fernndez-Plcios, Izquierdo, Roin, Vlenci, Slhi & Vergr 1995; Rodriguez, Cejs, Mrtin, Bdi, Smper & Lorenzo 1998), se ss (Dicentrrchus lrx L.) (Bruce, Oyen, Bell, Asturino, Frndle, Crrillo, Znuy, Rmos & Bromge 1999) nd Atlntic hliut (Hippoglossus hippoglossus L.) (Mzorr, Bruce, Bell, Dvie, Alorend, Jordn, Rees, Ppnikos, Porter & Bromge 2003). Furthermore, in the erlier study on cod descried ove, Pickov et l. (1997) reported tht eggs from the ocenic Skgerrk stock, which hd the superior htching rte, contined twice s much AA in the totl phospholipid frction compred to eggs from the Bltic stock. Unexpectedly, there ws no ssocition in the present study etween egg qulity nd performnce nd the levels of n-3hufa, EPA nd DHA. Incresingly though, ttention is eing focussed not simply on the overll levels of specific ftty cids such s AA, EPA nd DHA, ut on the reltive proportions of 10

11 these essentil ftty cids (Pvlov et l. 2004). Thus the DHA/EPA rtio in cod eggs ws shown previously to e correlted positively with egg qulity criteri (Pickov et l. 1997). However, in the present study, the DHA/EPA rtio ws not ssocited with performnce of the eggs, wheres there ws cler reltionship etween the EPA/AA rtio nd egg qulity with lower rtios in eggs displying etter performnce. Thus, in future studies it will e importnt to estlish the optimum rtio of DHA/EPA/AA in cod eggs (Bell, Frndle, Bruce, Nvs & Crrillo 1997; Bruce et l. 1999). The present study lso investigted the reltionship etween egg lipid content nd lipid clss composition nd egg qulity, nd showed tht PI ws lso positively correlted with performnce prmeters. This ws interesting s PI in fish hs unique ftty cid composition eing rich in 18:0, ut reltively low in PUFA which is predominntly C 20, prticulrly AA. This pttern hs een commonly reported in vrious neurl tissues from vriety of fish species (Tocher 1995) including cod rins nd retins (Tocher & Hrvie 1988). However, the high AA content of PI hs lso een reported in whole turot (Psett mxim L.), dogfish (Scyliorhinus cnicul) rectl glnds nd rinow trout Oncorhynchus mykiss (Wlum) spleen (Tocher 1995) s well s vrious cod tissues (Bell 1989; Bell & Dick 1990) nd mrine fish eggs, including cod (Tocher & Srgent 1984). Vrious long estlished cultured fish cell lines lso showed this composition, nd it ws noteworthy tht neither 35 yers in culture, nor direct supplementtion of PUFA to the cell lines olished the pttern (Tocher 1995). Severl nutritionl studies on Atlntic slmon (Slmo slr L.) lso showed tht dietry effects did not olish these ptterns (Tocher 1995). The composition of PI in fish, so similr to tht in mmmls, showing selective retention of AA, hs suggested potentil role for this lipid clss in eicosnoid metolism lthough this hs never een directly demonstrted (Tocher 2003). AA is known to e the primry precursor of eicosnoids, highly ctive ftty cid derivtives including prostglndins nd leukotrienes, in fish, despite the high levels of EPA which cts more s modultor of eicosnoid metolism (Srgent et l. 2002; Tocher 2003). Consequently, the EPA/AA rtio is n importnt determinnt of eicosnoid lnce, nd ws significntly higher in eggs from the F roodstock nd negtively correlted with the egg performnce. The results of the present study my suggest tht it is not simply the overll level of AA nd the EPA/AA rtio tht is importnt, ut very possily the level of AA in PI tht is n importnt determinnt of egg qulity in cod. Eggs from oth the W nd W/F roodstock were lso chrcterised y hving higher totl pigment nd stxnthin levels correlting with etter performnce. Interestingly, the vrition in eggs from wild fish ws high showing tht different tches hd quite different pigment levels. The reson for this vrition in wild eggs ws uncler. Astxnthin is effectively trnsferred from the roodstock to 11

12 the eggs in slmonids nd cod (Grung, Svendsen & Lien-Jensen 1993), nd positive effects were demonstrted in se rem nd yellowtil (Seriol llndi L.) (Wtne & Miki 1993; Verkunpiriy, Mushike, Kwno & Wtne 1997), yet the precise requirements (qulittive nd quntittive) for high egg qulity hve not een clerly defined. Therefore, there is emerging evidence tht egg pigment content is fctor tht is t lest consistent with differences in egg performnce. This finding is prticulrly interesting, since pelgic eggs re usully colourless. However, unripe ovries of cod re ctully right ornge, nd eggs only turn trnsprent s they mture. Two lterntive explntions hve een dvnced for this phenomenon (Pvlov et l. 2004). One suggestion is tht pelgic eggs re in the upper lyer of the se, where phytoplnkton produce oxygen nd, therefore, the oxygen supply is good nd there is no requirement for supplementry pigment to improve oxygen trnsport. The lterntive explntion is tht coloured eggs re esier prey for predtors. Therefore, it ws perhps surprising to find tht eggs from the W nd WF roodstock were more pigmented thn eggs from F roodstock. However, neither of these explntions ccount for the potentil nti-oxidnt properties of crotenoid pigments, which possily my e significnt fctor in determining egg qulity nd susequent egg performnce (Cowey, Bell, Knox, Frser & Youngson 1985). The present study ws not nutritionl tril ut rther sought to ssocite some sic prmeters of performnce with specific chemicl nlyses of the eggs under stndrd operting procedures in the htchery. However, the results suggest tht dietry level of 0.3 % AA (s in diet A) my not e sufficient to enle roodstock to mintin n dequte level of AA in the eggs when fed over n extended period. The reltively short term nutritionl effects, rought out y feeding wild roodstock for period of months prior to spwning, hd some effect ut not sufficient to gretly ffect egg qulity s eggs from WF fish were more similr to eggs from W fish thn F fish oth in composition nd performnce despite eing fed exctly the sme regime s F fish. This regime included specific mturing roodstock diet with n incresed level of AA. However, mny studies hve found tht egg ftty cid compositions cn e ffected y roodstock diets in vrious species, including se rem (Mourente & Odriozol 1990; Fernndez-Plcios et l. 1995; Almnso, Perez, Cejs, Bdi, Villmndos & Lorenzo 1999), se ss (Bell et l. 1997), striped jck (Pseudocrnx dentex L.) (Vssllo-Agius, Wtne, Mushike, Kwno & Stoh 1998), yellowtil (Verkunpiriy, Wtne, Mushike, Kiron, Stoh & Tkeuchi 1996) nd hliut (Mzorr et l. 2003). In cod, it ws shown tht the ftty cid composition of eggs ws very similr to the vitellogenin composition, nd tht the ftty cid composition of vitellogenin ws highly conserved nd only ffected y diets of extreme composition (Silversnd, Norerg, Holm, Lie & Hux 1995). The lterntive sitution to extreme diets is feeding diets with lesser differences ut over longer time. The differences in lipid, ftty cid nd 12

13 pigment composition etween the eggs from WF nd F fish will undoutedly e relted to diet ut clerly over much longer time period mesured in yers nd genertions rther thn few months. Broodstock diets, like most quculture diets, re hevily sed on mrine fish products nd hve sufficient AA for on-growing t lest. However, in the light of severl studies (Bell et l. 1997; Pickov et l. 1997; Mzorr et l. 2003), including the present one, showing the importnt ssocition etween egg AA content nd egg qulity nd performnce in mrine fish, considertion of AA supplementtion to roodstock diets my e required. The inclusion of phospholipid-rich products my lso e prudent (Bell et l. 1997). Acknowledgements We cknowledge the support of the Se Fish Industry Authority (Sefish) for prtil funding of this project. We grtefully cknowledge the stff of the Mrine Environmentl Reserch Lortory (MERL), Mchrihnish, Argyll nd Mchrihnish Mrine Frm Ltd, for their invlule technicl ssistnce nd logisticl support. References Ackmn R.G. (1980) Fish lipids, Prt 1. In: Advnces in Fish Science nd Technology (ed. y J.J. Connell), pp Fishing News Books, Frnhm, UK. Almnso E., Perez M.J., Cejs J.R., Bdi P., Villmndos J.E. & Lorenzo A. (1999) Influence of roodstock gilthed serem (Sprus urt L.) dietry ftty cids on egg qulity nd egg ftty cid composition throughout the spwning seson. Aquculture 170, Ashton H.J., Frkvm D.O. & Mrch B.E. (1993) Ftty cid composition of lipids in the eggs nd levins from wild nd cultured chinook slmon (Oncorhynchus tshwytsch). Cn. J. Fish. Aqut. Sci. 50, Bru A.B., Kostic D. & Olson J.A. (1993) New simplified procedures for the extrction nd simultneous high-performnce liquid chromtogrphy nlysis of retinol, tocopherols nd crotenoids in humn serum. J. Chromtogr. 617, Bskerville-Bridges B. & Kling L.J. (2000) Development nd evlution of microprticulte diets for erly wening of Atlntic cod (Gdus morhu) lrve. Aqucult. Nutr. 6,

14 Bskerville-Bridges B. & Kling L.J. (2000) Erly wening of Atlntic cod (Gdus morhu) lrve onto microprticulte diet. Aquculture 189, Bell J.G., Frndle B.M., Bruce M.P., Nvs J.M. & Crillo M. (1997) Effects of roodstock dietry lipid on ftty cid compositions of eggs from se ss (Dicentrrchus lrx). Aquculture 149, Bell J.G., McEvoy J., Tocher D.R. & Srgent J.R. (2000) Depletion of α-tocopherol nd stxnthin in Atlntic slmon (Slmo slr) ffects utoxidtive defence nd ftty cid metolism. J. Nutr. 130, Bell M.V. (1989) Moleculr species nlysis of phosphoglycerides from the ripe roes of cod (Gdus morhu). Lipids 24, Bell M.V. & Dick J.R. (1990) Moleculr species composition of phosphtidylinositol from the rin, retin, liver nd muscle of cod (Gdus morhu). Lipids 25, Brown J.A. & Puvnendrn V. (2002) Development of Atlntic cod lrviculture in Newfoundlnd. Bull. Aqucult. Assoc. Cn. 102 (1), 5-7. Brown J.A., Minkoff G. & Puvnendrn V. (2003) Lrviculture of Atlntic cod (Gdus morhu): progress, protocols nd prolems. Aquculture 227, Bruce M., Oyen F., Bell J., Asturino J.F., Frndle B., Crrillo M., Znuy S., Rmos J. & Bromge N. (1999) Development of roodstock diets for the Europen Se Bss (Dicentrrchus lrx) with specil emphsis on the importnce of n-3 nd n-6 highly unsturted ftty cid to reproductive performnce. Aquculture 177, Clln C., Jordn A. & Kling L.J. (2003) Reducing Artemi use in the culture of Atlntic cod (Gdus morhu). Aquculture 219, Christie W.W. (1982) Lipid Anlysis, 2nd edn. pp Pergmon, Oxford, UK. Cowey C.B., Bell J.G., Knox D., Frser A., & Youngson A. (1985) Lipids nd ntioxidnt systems in developing eggs of slmon (Slmo slr). Lipids 20, Czesny S. & Drowski K. (1998) The effect of egg ftty cid concentrtions on emryo viility in wild nd domesticted wlleye (Stizostedion vitreum). Aqut. Living Resour. 11, Dos Sntos J., Burkow I.C. & Joling M. (1993) Ptterns of growth nd lipid deposition in cod (Gdus morhu L.) fed nturl prey nd fish-sed feeds. Aquculture 110, Fernndez-Plcios H., Izquierdo M.S., Roin L., Vlenci A., Slhi M. & Vergr J.M. (1995) Effect of n-3hufa level in roodstock diets on egg qulity of gilthed se rem (Sprus urt L.). Aquculture 132,

15 Folch J., Lees M. & Slone-Stnley G.H. (1957) A simple method for the isoltion nd purifiction of totl lipids from niml tissues. J. Biol. Chem. 226, Gllgher M.L., Prmore L., Alves D. & Rulifson R.A. (1998) Comprison of phospholipid nd ftty cid composition of wild nd cultured striped ss eggs. J. Fish Biol. 52, Grung M., Svendsen Y.S. & Lien-Jensen S. (1993) The crotenoids of eggs of wild nd frmed cod (Gdus morhu). Comp. Biochem. Physiol. 106B, Hrel M., Tndler A., Kissil G.W. & Appleum S. (1992) The kinetics of nutrient incorportion into ody tissues of gilthed serem S. urt femles nd the susequent effects on egg composition nd egg qulity. Isr. J. Aqucult. 44, 127. Hemre G., Krlsen O., Eckhoff K., Tviet K., Mngor-Jensen A. & Rosenlund G. (2004) Effect of seson, light regime nd diet on muscle composition nd selected prmeters in frmed Atlntic cod, Gdus morhu L., Aqucult. Res. 35, Hemre G.-I., Krlsen O., Mngor-Jensen A. & Rosenlund G. (2003) Digestiility, of dry mtter, protein, strch nd lipid y cod, Gdus morhu: comprison of smpling methods. Aquculture 225, Henderson R.J. & Tocher D.R. (1992) Thin-lyer chromtogrphy. In: Lipid Anlysis: A Prcticl Approch (ed. y R.J. Hmilton & S. Hmilton), pp IRL Press, Oxford, UK. Izquierdo M.S., Fernndez-Plcios H. & Tcon A.G.J. (2001) Effect of roodstock nutrition on reproductive performnce of fish. Aquculture 197, Kjesu O.S. (1989) The spwning ctivity of cod. J. Fish Biol., 34, Lll S.P. & Nnton D. (2002) Nutrition of Atlntic cod. Bull. Aqucult. Assoc. Cn. 102, Lie O., Lied E & Lmertsen G. (1986) Liver retention of ft nd of ftty cids in cod (Gdus morhu) fed different oils. Aquculture 59, Mrshll C.T., Yrgin N.A. & Lmert Y. (1999) Totl lipid energy s proxy for totl egg production y fish stocks. Nture 402, Mzorr C., Bruce M., Bell J.G., Dvie A., Alorend E., Jordn N., Rees J., Ppnikos N., Porter M., & Bromge N. (2003) Dietry lipid enhncement of roodstock reproductive performnce nd egg nd lrvl qulity in Atlntic hliut (Hippoglossus hippoglossus). Aquculture 227, Mourente G. & Odriozol J.M. (1990) Effect of roodstock diets on lipid clsses nd their ftty cid composition in eggs of gilthed se rem (Sprus urt L.). Fish Phsiol. Biochem. 8, Moris S., Bell J.G., Roertson D.A., Roy W.J. & Morris P.C. (2001) Protein/lipid rtios in extruded diets for Atlntic cod (Gdus morhu L.): effects on growth, feed utiliztion, muscle composition nd liver histology. Aquculture 203,

16 Olsen R.E., Henderson R.J. & Pedersen T. (1992) The influence of dietry lipid clsses on the ftty cid composition of smll cod Gdus morhu L. juveniles rered in n enclosure in northern Norwy. J. Exp. Mr. Biol. Ecol. 148, Pvlov D., Kjorsvik E., Refsti T. & Andersen O. (2004) Broodstock nd egg production. In: Culture of cold wter mrine fish (ed. y E. Moksness, E. Kjorsvik & Y. Olsen), 544 pp. Blckwell Pulishing, Oxford, UK. Pickov J., Dutt P.C., Lrson P.O. & Kiessling A. (1997) Erly emryonic clevge pttern, htching success nd egg-lipid ftty cids composition: comprison etween two cod stocks. Cn. J. Fish. Aqut. Sci. 54, Rodriguez C., Cejs J.R., Mrtin M.V., Bdi P., Smper M. & Lorenzo A. (1998) Influence of n-3 highly unsturted ftty cid deficiency on the lipid composition of roodstock gilthed serem (Sprus urt L.) nd on egg qulity. Fish Physiol. Biochem. 18, Srgent J.R., Bell J.G., McEvoy L., Tocher D.R. & Estevez A. (1999) Recent developments in the essentil ftty cid nutrition of fish. Aquculture 177, Srgent J.R., Bell M.V., Bell J.G., Henderson R.J. & Tocher D.R. (1995) Origins nd functions of (n-3) polyunsturted ftty cids in mrine orgnisms. In: Phospholipids: Chrcteriztion, Metolism nd Novel Biologicl Applictions (ed. y G. Cevc & F. Pltuf), pp AOCS Press, Chmpign, Illinois, USA. Srgent J.R., McEvoy L., Estevez A., Bell J.G., Bell M.V., Henderson R.J. & Tocher D.R. (1999) Lipid nutrition of mrine fish during erly development: Current sttus nd future directions. Aquculture 179, Srgent J.R., Tocher D.R. & Bell J.G. (2002) The lipids. In: Fish Nutrition, 3 rd Edition (ed. y J.E. Hlver & R.W. Hrdy), pp Acdemic, Sn Diego, USA. Silversnd C., Norerg B., Holm J.C., Lie O. & Hux C. (1995) Dietry influence on the ftty cid composition of vitellogenin nd the susequent effect on the egg composition in cod (Gdus morhu). Proc. 5 th Internt. Symp. Reprod. Physiol. Fish, The University of Texs t Austin, 2-8 July p Tndler A., Hrel M., Koven W.M. & Kolkovski S. (1995) Broodstock nd lrve nutrition in gilthed serem Sprus urt - new findings on its mode of involvement in improving growth, survivl nd swimldder infltion. Isr. J. Aqucult. 47, Tocher D.R. (1995) Glycerophospholipid metolism. In: Biochemistry nd Moleculr Biology of Fishes, Vol. 4. Metolic nd Adpttionl Biochemistry (ed. y P.W. Hochchk & T.P. Mommsen), pp Elsevier, Amsterdm, Netherlnds. 16

17 Tocher D.R. (2003) Metolism nd functions of lipids nd ftty cids in teleost fish. Rev. Fish. Sci. 11, Tocher D.R. & Hrvie D.G. (1988) Ftty cid compositions of the mjor phosphoglycerides from fish neurl tissues : (n-3) nd (n-6) polyunsturted ftty cids in rinow trout (Slmo girdneri, L.) nd cod (Gdus morhu) rins nd retins. Fish Physiol. Biochem. 5, Tocher D.R. & Srgent J.R. (1984) Anlyses of lipids nd ftty cids in ripe roes of some northwest Europen mrine fish. Lipids 19, Vn Der Meeren T. (1993) Ftty cid composition of unfed cod lrve Gdus morhu L. nd cod lrve feeding on nturl plnkton in lrge enclosures. J. World Aqucult. Soc. 24, Vssllo-Agius R., Wtne T., Mushike K., Kwno K. & Stoh S. (1998) Chemicl components of eggs nd yolksc lrve otined from striped jck roodstock fed on rw fish mix or dry pellets. Fish. Sci. 64, Verkunpiriy V., Mushike K., Kwno K. & Wtne T. (1997) Supplementl effect of stxnthin in roodstock diets on the qulity of yellowtil eggs. Fish. Sci. 63, Verkunpiriy V., Wtne T., Mushike K., Kiron V., Stoh S. & Tkeuchi T. (1996) Effect of roodstock diets on the chemicl components of milt nd eggs produced y yellowtil. Fish. Sci. 62, Wtne T. & Miki W. (1993) Astxnthin: n effective dietry component for red serem roodstock. In: Fish Nutrition in Prctice (ed. y S.J. Kushik & P. Luquet), pp INRA, Pris, Frnce. Zr J.H. (1984) Biosttisticl Anlysis, 2 nd Edition. Prentice-Hll, Englewood Cliffs, USA. 17

18 Figure Legends Figure 1 Fertilistion rte (percentge; pnel A) nd cell symmetry score (ritrry units; pnel B) of eggs from wild (W), wild/fed (WF) nd frmed (F) roodstock of Atlntic cod, Gdus morhu. Results re presented s mens ± S.D (n = 3, 11, nd 7 for W, WF nd F, respectively). Different letters indicte sttisticlly significnt (P < 0.05) differences etween mens s determined y ANOVA followed y Tukeys multiple comprison test. Figure 2 Totl lipid content of eggs, s percentges of the egg wet (pnel A) nd dry (pnel B) weight, from wild (W), wild/fed (WF) nd frmed (F) roodstock of Atlntic cod, Gdus morhu. Results re presented s mens ± S.D (n = 3, 11, nd 7 for W, WF nd F, respectively). There were no sttisticlly significnt (P > 0.05) differences etween mens s determined y ANOVA. Figure 3 Proportions (percentge of totl lipid) of the quntittively mjor polr (pnel A, phosphtidylcholine nd phosphtidylethnolmine) nd neutrl (pnel B, tricylglycerol nd cholesterol) lipid clsses of eggs from wild (W), wild/fed (WF) nd frmed (F) roodstock of Atlntic cod, Gdus morhu. Results re presented s mens ± S.D (n = 3, 11, nd 7 for W, WF nd F, respectively). Different letters indicte sttisticlly significnt (P < 0.05) differences etween mens s determined y ANOVA followed y Tukeys multiple comprison test. Figure 4 Proportions (percentge of totl lipid) of the quntittively minor phospholipid clsses of eggs from wild (W), wild/fed (WF) nd frmed (F) roodstock of Atlntic cod, Gdus morhu. Results re presented s mens ± S.D (n = 3, 11, nd 7 for W, WF nd F, respectively). Different letters indicte sttisticlly significnt (P < 0.05) differences etween mens s determined y ANOVA followed y Tukeys multiple comprison test. PA/PG/CL, phosphtidic cid/phosphtidylglycerol /crdiolipin which could not e resolved in this solvent system. Figure 5 Levels of phosphtidylinositol (percentge of totl lipid), rchidonic cid (percentge of totl ftty cids; AA) nd the eicospentenoic cid/rchidonic cid rtio (EPA/AA) s determined just prior to htching in two tches of eggs from wild/fed (WF1 nd WF2) nd frmed (F1 nd F2) roodstock of Atlntic cod, Gdus morhu. Results re presented s mens ± S.D (triplicte smples 18

19 from ech tch). Different letters indicte sttisticlly significnt (P < 0.05) differences etween mens s determined y ANOVA followed y Tukeys multiple comprison test. Figure 6 Proportions of docoshexenoic cid (DHA), eicospentenoic cid (EPA), rchidonic cid (AA) (ll s percentge of totl ftty cids), nd the EPA/AA nd DHA/EPA rtios of eggs from wild (W), wild/fed (WF) nd frmed (F) roodstock of Atlntic cod, Gdus morhu. Results re presented s mens ± S.D (n = 3, 11, nd 7 for W, WF nd F, respectively). Different letters indicte sttisticlly significnt (P < 0.05) differences etween mens s determined y ANOVA followed y Tukeys multiple comprison test. Figure 7 Levels of totl pigment nd stxnthin (oth s ng/mg totl lipid) of eggs from wild (W), wild/fed (WF) nd frmed (F) roodstock of Atlntic cod, Gdus morhu. Results re presented s mens ± S.D (n = 3). Different letters indicte sttisticlly significnt (P < 0.05) differences etween mens s determined y ANOVA followed y Tukeys multiple comprison test. 19

20 Tle 1 Ftty cid composition (percentge of weight) of totl lipid from eggs from different roodstocks of Atlntic cod (Gdus morhu ) nd roodstock diets. Wild (W) Wild/Fed (WF) Frmed (F) Diet A Diet B 14:0 1.5 ± ± ± ± ± : ± ± ± ± ± :0 2.4 ± ± ± ± ± 0.0 Totl sturted ± ± ± ± ± : ± ± ± ± ± :1n ± ± ± ± ± :1n ± ± ± ± ± : ± ± ± ± ± : ± ± ± ± ± :1n ± ± ± ± ± 0.1 Totl monoenes 23.6 ± ± ± ± ± :2n ± ± ± ± ± :4n ± ± ± ± ± 0.0 Totl n-6 PUFA ± ± ± ± ± :3n ± ± ± ± ± :4n ± ± ± ± ± :4n ± ± ± ± ± :5n ± ± ± ± ± :5n ± ± ± ± ± :6n ± ± ± ± ± 0.6 Totl n-3 PUFA 46.0 ± ± ± ± ± 1.0 Totl PUFA ± ± ± ± ± 1.0 Vlues re mens ± SD (n = 3, 11 nd 7 for W, WF nd F, respectively, nd 3 for oth diets). Significnce of differences etween mens for eggs were determined y one-wy ANOVA followed, where pproprite, y Tukey's multiple comprison test s descried in the Mterils nd Methods. Vlues within row (eggs only) with different superscript letter re significntly different (P < 0.05). 1, contins 15:0 nd 20:0, present in some smples t up to 0.5%; 2, predominntly n-7 isomer; 3, predominntly n-9 isomer; 4, predominntly n-11 isomer; 5, totls include 20:2n-6, 22:4n-6 nd 22:5n-6 present in some smples t up to 0.5%. Diet A = Dn-Ex, Dnfeed, Denmrk; Diet B = Breed M, INVE Aquculture, Belgium 20

21 Tle 2 Performnce indictors of two tches of incuted eggs from frmed (F1 nd F2) nd wild/fed (WF1 nd WF2) roodstocks of Atlntic cod (Gdus morhu ) Btch Fertilistion rte Symmetry Survivl/htching rte (percentge) (score) (percentge) F F WF WF

22 Fig.1 Fertilistion rte (percentge) A W WF F B W WF F Symmetry (score units) Broodstock 22

23 Fig.2 Egg lipid content (percentge of wet weight) A W WF F Egg lipid content (percentge of dry weight) B W WF F Broodstock 23

24 Fig.3 Content (percentge of totl lipid) A W WF F 0 Phosphtidylcholine Phosphtidylethnolmine Content (percentge of totl lipid) B W WF F 0 Tricylglycerol Lipid clss Cholesterol 24

25 Fig.4 Egg content (percentge of totl lipid) c Phosphtidylinositol Lipid clss PA/PG/CL W WF F 25

26 Fig WF1 WF2 F1 F2 Percentge or rtio d c 2.0 c c 0.0 Phosphtidylinositol AA EPA/AA 26

27 Fig.6 Content (percentge of totl ftty cids) A W WF F 0 DHA EPA EPA/AA Content (percentge of totl ftty cids) B AA Ftty cid/ftty cid rtio DHA/EPA W WF F 27

28 Fig.7 Egg content (ng/mg totl lipid) W WF F 0 Totl pigment Astxnthin 28