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Total lipid and fatty acid composition of brill eggs
Scophthalmus rhombus L. relationship between lipid
composition and egg quality
Ismael Hachero Cruzado1, Marcelino Herrera1, Daniel Quintana1, Ana Rodiles1, Jose¤ I. Navas1,Antonio Lorenzo2 & EduardoAlmansa3
1Instituto de Investigacio¤ n y Formacio¤ n Agraria y Pesquera deAndaluc|¤ a (IFAPA), CentroAgua del Pino, Huelva, Spain2Dept. Biolog|¤ a Animal (U.D.I. Fisiolog|¤ a), Fac. Biolog|¤ a. Univ. La Laguna, deTenerife, Spain3Centro OceanograŁ ¢co de Canarias, Instituto Espan� ol de Oceanograf|¤ a, deTenerife, Spain
Correspondence: I Hachero Cruzado, Instituto de Investigacio¤ n y Formacio¤ n Agraria y Pesquera deAndaluc|¤ a (IFAPA), CentroAgua del
Pino, Carretera Cartaya ^ Punta Umbr|¤ a s/n, E^21450 Cartaya, Huelva, Spain. E-mail: [email protected]
Abstract
The present study examines the total lipid (TL) andfatty acid (FA) composition in eggs of brill Scophthal-mus rhombus L. and the possible relationships withtheir quality parameters. Wild broodstocks werecaught and maintained in captivity until eggs werecollected. A lipid characterization of each egg batchwas conducted in TL, lipid classes (LC) and FA of TL.The TL content was lower than the values reportedfor other £at¢sh species, showing high levels of sterolesters (SE). High viability rates were related to higherlipid reserves. Higher cholesterol (CHO) was linked tohigher egg viability, whereas SE could have an oppo-site e¡ect. Comparison of female quality showed thatlipid composition was more related to egg batchesthan to individual females. However, multivariateanalysis did not show a clear correlation between li-pid composition and brill egg quality, neither as indi-vidual components nor as awhole pro¢le of LC or FA.Our results suggest that some lipid components(phosphatidylcholine, CHO, SE, monounsaturated, ei-cosapentaenoic acid) could be related to di¡erencesin spawning quality, although these were not theonly factors involved in these di¡erences. Thus,these lipid components could be considered to be de-scriptors of the di¡erences found in the rates of brillquality.
Keywords: brill, eggs, lipid class, fattyacid compo-sition, multivariate analysis, egg quality
Introduction
The diversi¢cation of aquaculture, with the introduc-tion of new high-value species, is one of the main ob-jectives that must be achieved for the development ofmarine ¢n¢sh farming. Brill Scophthalmus rhombus L.is a promising species for aquaculture in the south-ern Atlantic^Mediterranean coast, due to its highmarket price and rapid growth rate (Robert & Vianet1988; Arneri, Collella & Gianetti 2001). Brill is apleuronectiform, which inhabits coastal waters from5 to 50m deep along the northeastern Atlantic andthe Mediterranean Sea. It has an external morphol-ogy similar to that of turbot, but it is thinner and nar-rower. Also, brill presents small, smooth scales in theskin, without a bony tubercule. The colour is oftenolive green, with dark and light spots (Bauchot1987).Large-scale production of brill is seriously limited
by high larval mortality rates (Hachero 2006) and bypoor or variable egg quality in captive broodstocks(Hachero-Cruzado, Garc|¤ a-lo¤ pez, Herrera, Vargas-chaco¡, Mart|¤ nez-rodr|¤ guez, Mancera & Navas2007). There are some possible causes of poor eggquality in hand-stripped eggs from captive ¢sh: (1)overripening of ovulated eggs inside the lumen ofthe ovary (turbot Psetta maxima L., McEvoy 1984;Atlantic halibut Hippoglossus hippoglossus L., Nor-berg,Valkner, Huse, Karlsen & Ler˛y Grung1991); (2)e¡ects of water temperature £uctuations (turbot, De-vauchelle, Alexandre, Le Corre & Letty1988; Atlantichalibut, Brown, Shields & Bromage 2006); (3) charac-
Aquaculture Research, 2011, 42, 1011^1025 doi:10.1111/j.1365-2109.2010.02684.x
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teristics of the sperm (turbot, Suquet, Billard, Cosson,Normant & Fauvel 1995; Atlantic halibut,Tvedt, Ben-fey, Martin-robichaud & Power 2001); and (4) the bio-chemical composition of eggs (turbot, McEvoy,Holland & McEvoy 1993). Previous studies of ourgroup have dealt with the ¢rst and the second causebut a biochemical study of the eggs has not beenconducted.In principle, ¢sh eggs contain all the nutrients ne-
cessary for growth and energy provision during em-bryogenesis of most ¢sh species. Moreover, the larvautilizes these nutrients during the lecithotrophicphase, before exogenous feeding, to support bothhomoeostasis and development (Mourente & VaŁ zquez1996).Thus, egg-nutrient composition can be ausefulapproach to the studyof the nutritional requirementsof ¢sh larvae (Ostrowski & Divakaran1991; Rainuzzo1993; Sargent 1995; Mourente & VaŁ zquez 1996; Abi-Ayad, Kestemont & Me¤ lard 2000).Lipids, like other dietary components, play a signif-
icant role in ¢sh nutrition (Sargent, McEvoy, Estevez,Bell, Bell, Henderson & Tocher1999). In eggs, the lipidreserves are used by the developing embryo, both assubstrates for energy metabolism and as structuralcomponents in membrane biogenesis (Heming &Buddington1988; Rainuzzo1993; Finn1994; Sargent1995). Neutral lipids are generally considered to bethe most important energy reserve in marine ¢sheggs and larvae (Vetter, Hodson & Arnold1983; Blax-ter1988). However, it has also been shown that phos-phoglycerides are utilized not only for cell divisionand organogenesis but also for energy by some spe-cies (Tocher, Fraser, Sargent & Gamble1985; Rainuzzo1993; Sargent1995).Within neutral and polar lipids, polyunsaturated
fatty acids (PUFA) are basic components in embryo-nic physiology, as theyare considered to be structuralcomponents during organogenesis (brain, retina,muscle, etc.) (Bell & Tocher 1989; Navarro & Sargent1992; Sargent1995) and precursors of physiologicallyactivemolecules suchas prostaglandins and other ei-cosanoids (Bell, Sargent & Raynard 1992; Sargent1995). Yolk composition should, ideally, suit the em-bryo’s requirements and its constituent lipids, includ-ing PUFA contents.Several studies have showna relationship between
the biochemical composition of the egg and embryoas well as larva quality (Rodriguez, Pe¤ rez, Izquierdo,Mora, Lorenzo & FernaŁ ndez-Palacios 1993; Fiogbe¤1996; Abi-Ayad, Me¤ lard & Kestemont1997; Rodr|¤ guez,Pe¤ rez, D|¤ az, Izquierdo, FernaŁ ndez-Palacios & Lorenzo1997; Kestemont, Cooremans, Abi-Ayad & Me¤ lard
1999). These studies have established that the bio-chemical composition of ¢sh eggs is species speci¢cand varies both qualitatively and quantitatively(Sargent, Henderson & Tocher 1989). Therefore, toevaluate the in£uence of egg biochemical composi-tion on spawning quality, speci¢c studies areneeded within a new species to improve its culturetechniques.In this context, we seek to determine the relation-
ship between the quality of egg batches from di¡erentfemales throughout the spawning season and the li-pid class (LC) as well as the fatty acid (FA) composi-tion of brill eggs.
Material and methods
Collection and management of broodstocks
Brill broodstock used in this studywere caught in theGulf of CaŁ diz [seawater (SW), Spain] between 2002and 2004. The ¢sh were tagged with an intramuscu-lar passive integrated transponder tag (Trovan ID100A; EID Ibe¤ rica, Madrid, Spain) and stocked intofour groups, in tanks of 4.2m2 (5m3; 1.2m waterdepth) located indoors. The broodstock groups werecomposed of three to four females:two to three males.The tanks were supplied with running SW (200%turnover per day) with 37^39 g L�1 salinity and 5^9 ppm oxygen. Water temperature £uctuated natu-rally (10^23 1C), except during summer, when it wasmaintained below 23 1C (titanium-plate heat exchan-ger; Alfa Laval, Rudeboksv�gen, Sweden). During theexperimental period, ¢sh were fed (at 14:00 hours, ata maximum daily ration of 2% of body mass): withfrozen squid Loligo spp. (twice a week), blue whitingMicromesistius poutassou (Risso) (twice a week), fro-zen musselsMytilus spp. (once aweek), European pil-chard Sardina pilchardus (Walbaum) (once a week)and dry pellets speci¢c for broodstock (Skretting,Spain) (once aweek).
Gamete collection and arti¢cial fertilization
During the spawning season (mid-January to lateMarch), all females were periodically checked for theexpression of hyaline eggs.To collect freshly ovulatedeggs and thus avoid overripening inside the ovary lu-men (McEvoy 1984), the stripping frequency was se-lected individually by daily checking of the femalesduring the beginning of the spawning season, toestablish their approximated ovulatory rhythms
Total lipid and fatty acid composition of brill eggs I H Cruzado et al. Aquaculture Research, 2011, 42, 1011^1025
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according to their ¢rst ovulation date. In themajorityof them, a 3-day stripping frequency was shown to bee¡ective. However, the females that could not bestripped successfully at the third day were checkeddaily during the following days until eggs were re-leased. Tanks were also provided with egg collectorsand when eggs were detected on anunexpected ovu-lation day, the females in the tank were stripped foreggs. Before handling, ¢sh were anaesthetized in 2-phenoxyethanol (500 ppm), which has been testedpreviously by our group (Hachero-Cruzado et al.2007), and no e¡ects on egg quality were observed.Thereafter, eggs were hand-stripped in a dry 2 L plas-tic beaker and kept at room temperature (10^15 1C)until fertilization (ca. 20min). The total number ofeggs was calculated volumetrically under dry condi-tions, counting the eggs in one 500 mL aliquot and ex-trapolating it to the total volume of eggs collected,measured in a calibrated cylinder. The same aliquotwas used to calculate the viability rate and to mea-sure, after adding SW, the diameter of 10 randomlychosen viable eggs. Eggs were estimated to be viablewhen they showed a perfect spherical and translu-cent aspect (McEvoy 1984; Fauvel, Omnes, Suquet &Normant 1992). Each egg batch was fertilized at 18^20 1C with freshly stripped sperm from one male,using a volume ratio eggs:sperm:SW of 100:0.2:100.Sperm density was not measured, but all sperm usedfor fertilization was viscous and had high motility.After gentle mixing, the fertilization mixture was leftfor 3min (Suquet et al. 1995) before adding more SWto a total volume of 1.5 L. Thirty minutes later, the£oating eggs were collected and one sample of 50eggs was incubated in 12-well culture plates ¢lledwith 3mL of ¢ltered SW (2 eggswell�1) at tempera-tures ranging from13.2 to19.8 1C (mean temperature:15.7 1C) in order to estimate the fertilization andhatching rates. Two hours after beginning incuba-tion, the number of eggs with cell division (two- tofour-cell stage) was counted and the fertilization ratewas calculated. Five days after fertilization, the num-ber of larvae in the plate was counted and the hatch-ing rate was calculated. All the operations wereperformed during the morning.
Biochemical analysis
A total of 29 egg batches from 10 females weresampled for analysis of moisture, total lipids (TL), LCand FA in TL extracts throughout the study period(21 January to 17 March). Eggs obtained at tempera-
tures higher than 16.5 1C, in broodstocks or incuba-tion tanks, were not included in the comparativeanalyses, given that high temperatures a¡ect thespawning quality (Hachero-Cruzado et al. 2007). Themoisture content was determined from 500mgsamples using the method of Horwitz (1980). Total li-pid was extracted with chloroform:methanol (2:1v/v)containing 0.01% of butylated hydroxytoluene (BHT)as an antioxidant (Christie1982).The organic solventwas evaporated under a stream of nitrogen and thelipid content was determined gravimetrically. Lipidclasses were separated by one-dimensional double-development high-performance thin-layer chromato-graphy (HPTLC) using methyl acetate/isopropanol/chloroform/methanol/0.25% (w/v) KCl (25:25:25:10:9by volume) as the polar solvent system and hexane/diethyl ether/glacial acetic acid (80:20:2 by volume)as the neutral solvent system. Lipid classes werequanti¢ed by charring with a copper acetate reagent,followed by calibrated scanning densitometry usinga Shimadzu CS-9001PC dual wavelength £ying spotscanner (Shimadzu, Kyoto, Japan) (Olsen & Hender-son1989). Total lipid extracts were subjected to acid-catalysed transmethylation for 16:00 hours at 50 1C,using1mL of toluene and 2mL of1% sulphuric acid(v/v) in methanol. The resulting fatty acid methylesters (FAME) were puri¢ed by thin-layer chromato-graphy (TLC), and visualized with iodine in chloro-form:methanol (2:1 v/v) 98% (v/v) containing 0.01%BHT (Christie 1982). Before transmethylation, henei-cosanoic acid (19:0) was added to theTL as an inter-nal standard. Fatty acid methyl esters were separatedand quanti¢ed using a Shimadzu GC 2010 (Shimad-zu) gas chromatograph equippedwith a £ame-ioniza-tion detector (250 1C) and a fused silica capillarycolumn RTX ^ WAXTM (Restek, Bellefante, PA, USA)(10m � 0.1mm i.d.). Helium was used as a carriergas and the initial oven temperature was 150 1C,followed by an increase at a rate of 90 1Cmin�1 to a¢nal temperature of 250 1C for 3min. IndividualFAME were identi¢ed by reference to authentic stan-dards and to awell-characterized ¢sh oil.Butylated hydroxytoluene, potassium chloride, po-
tassium bicarbonate and 2 0,7 0-dichloro£uoresceinwere supplied by Sigma Chemical (St Louis, MO,USA). Thin-layer chromatography (20 cm � 20 cm �0.25mm) and HPTLC (10 cm � 10 cm � 0.15mm)plates, pre-coated with silica gel (without £uorescentindicator), were purchased from Macheren-Nagel(Dˇren, Germany). All organic solvents for GC usedwere of reagent grade and were purchased fromPanreac (Barcelona, Spain).
Aquaculture Research, 2011, 42, 1011^1025 Total lipid and fatty acid composition of brill eggs I H Cruzado et al.
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Statistical analysis
Lipid-composition results are presented as means �SD. The data were checked for normal distributionusing the one-sample Kolmogorov^Smirno¡ test aswell as for homogeneity of the variances usingLevene’s test and, when necessary, arcsin transfor-mation was performed. To all data expressed as per-centage, arcsin transformation (Fowler, Cohen &Jarvis 2002) was applied directly. A Student t-test(Zar1984) was performed to compare the di¡erencesbetween groups with di¡erent spawning qualities.Reference values for spawning quality have been se-lected from the mean values observed in previousstudies of our group (Hachero-Cruzado et al. 2007).Principal component analysis (PCA) of LC and FA ineggswas used to reveal possible groups based on theirmost important quality parameters (viability, fertili-zation and hatching rates). The possible relationshipsbetween LC and the FA pro¢le of eggs with all theparameters related to spawning quality were ana-lysed using Pearson’s correlation; this correlationwas made between the axes plotted from the PCA(based on LC and FAcomposition) in eggs and spawn-ing-quality parameters. In all statistical tests,Po0.05 was considered to be statistically signi¢cant.The statistical analysis was performed using the SPSS
package (version 13.0) from SPSS (IBM Company,Chicago, IL, USA).
Results
The spawning season lasted for 2 months (19 Janu-ary to 21March); a total of 102 egg batches were col-lected with a total production of approximately3654250 eggs. The total eggs collected were ob-tained from13 females, but a continuous supply wasfrom only four females. The egg batches from thesefour females (good females) represented 50% of thetotal egg batches (data not shown) and had signi¢-cantly higher viability and fertilization rates thanthose found for the remaining females (bad females).We studied the lipid composition of 29 randomlyas-
signed egg batches produced by10 females. The qual-ity characteristics of these egg batches are shown inTable 1. Batch fecundity ranged from 8624 to80850 eggs ovulation�1, with a mean of 39944eggs ovulation�1. The diameter of viable eggs rangedfrom1.35 to1.53mm, with a mean of1.42 � 0.04mm.Egg viabilityand fertilization rates were relativelyhighin almost all batches, both parameters exceeding 80%in 20 and 21 of 29 egg batches analysed respectively.
The hatching rate was in general low and had veryhigh variability. The hatching rates were higher than70% in only six egg batches and null in eight. Theother data ranged between 8% and 48% hatch.Twenty-one out of the 29 egg batches analysed werecollected from‘good’ females.TheTL content of the 29 egg batches analysed ran-
ged from 1.93 to 13.70 (%DW), with a mean of7.42 � 4.28 (%DW). The lipid-class composition isshown in Fig. 1. Both parameters showed a highvariability, which could be related to di¡erences inegg quality. Total neutral lipid predominated(73.60%), with sterol ester (SE) as the main LC. Totalpolar lipid represented only 26.40% and phosphati-dylcholine (PC) was the major LC in this group.The relationship between lipid-class contents
(%DW) in eggs and their viability is shown inTable 2.Signi¢cant di¡erences were found between eggswith di¡erent viability rates. It is noteworthy that thehighest viability was found in eggs with higher per-centages inTL, although the di¡erences were not sig-ni¢cant. However, the lipid-class contents showedclear signi¢cant di¡erences in sphingomyelin (SM),PC, phosphatidylglycerol (PG) and cholesterol (CHO),with higher percentages in high-viability eggs.WhenLCwas expressed as a percentage of TL (Fig.2), similartendencies were found, although, in this case, low-viability eggs displayed higher SE levels.However, egg lipid-class content (%DW) did not
show relevant di¡erences when we separated theegg batches based on fertilization rates (Table 2). Onlywhen we compared LC as a percentage of TL did we¢nd higher levels of phosphatidylserine (PS) in eggbatches with high fertilization rates (data notshown).To test the possible relationship between lipidcontents and hatching rates, we compared LC con-tents from egg batches with hatching rates higherthan 70% with respect to egg batches having nullhatching rates. Statistical comparison (Table 3) re-£ected a higher signi¢cant content of SE in null-hatching egg batches. In addition, when LCs were ex-pressed as a percentage of TL (data not shown), CHOwas signi¢cantly higher in eggs with hatching rateshigher than 70%. Egg batches from the females withhigher fecundity (good females) were compared withthe batches from the remaining females (bad fe-males) (Table 3). No signi¢cant di¡erences werefound either in %TL or in %DW of the LC betweenthe two eggs-batch groups.Figure 3a^c shows the distribution of di¡erent egg
batches according to the axes resulting from PCA oftheir LC composition. Figure 3a shows this egg-batch
Total lipid and fatty acid composition of brill eggs I H Cruzado et al. Aquaculture Research, 2011, 42, 1011^1025
1014 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1011^1025
distribution divided into two groups according totheir viability rates. Figure 3b separated two groupsbased on their fertilization rates and, ¢nally, Fig. 3cmade the division based on their hatching rates. Inall ¢gures, Axis 1 explains up to 76.15% of systemvariability. PG, PS and phosphatidylethanolaminewere the LCs with the highest weight on this axis.Axis 2 explained only 9% of the variability, with ahigh correlationwith SM and triacylglycerol (TG). Inany case, the three analyses did not discriminate thedi¡erent quality rates of egg batches based on theirLC composition.The fatty acid composition of TL from eggs is
shown in Fig. 4. Polyunsaturated fatty acid was themajor group, duemainly to n-3 HUFA [especiallydoc-osahexaenoic acid (DHA) and eicosapentaenoic acid(EPA)]. Other relevant FA s were16:0 and18:1n-9. Ta-ble 4 compares the FAcontents (mgmg�1DW) of eggswith di¡erent viability rates. The di¡erences are
slight, as only the ratio of monounsaturated fattyacids (MUFA):PUFA displayed higher values in high-viability eggs. This tendency was more evident whenFAwas expressed as a percentage of weight (data notshown) where MUFA and 18:2n-6 showed the mostsigni¢cant di¡erences, with higher levels in high-via-bility eggs with respect to low-viability eggs. This in-crease is re£ected in the ratios of MUFAwith respectto PUFA and saturated FA.Di¡erences in the fertilization rates are not re-
£ected in the FA content from both groups of eggbatches (Table 4). Di¡erences were observed onlywhen we compared FA as a percentage of weight,where EPA is the only relevant FAthat showed higherpercentages in eggs with high fertilization rates withrespect to eggs with low fertilization rates (data notshown). Similarly, the hatching rate did not di¡er sig-ni¢cantly in the FA content (Table 5). Only 18:2n-6was higher in egg batches with a null hatching rate
Table 1 Egg batches quality characteristics sampled for biochemical analysis
Spawningdate
Femalecode
Malecode
Batch fecundity(no. of eggsovulation� 1)
Viability(%)
Fertilization(%)
Hatching(%)
Egg diameter(mm)
21-01-2005 F2D2 D559 33 800 100 84 15 –
25-01-2005 F5C2 F4AF 54 516 99 84 48 –
28-01-2005 D89B E949 35 076 81 94 0 1.42
28-01-2005 F2D2 E949 36 088 92 89 80 1.41
31-01-2005 D89B C439 54 880 96 46 25 1.42
02-02-2005 F2D2 D1F4 30 000 92 55 18 1.43
02-02-2005 F5C2 D1F4 46 560 93 89 88 1.44
03-02-2005 D89B E73C 31 820 96 71 47 1.41
03-02-2005 F64E E73C 77 040 58 82 72 1.42
06-02-2005 D89B C439 54 784 96 55 17 1.42
09-02-2005 D89B C439 50 344 96 93 8 1.43
16-02-2005 F64E D559 44 408 72 91 0 1.37
16-02-2005 D89B D559 67 860 79 100 10 1.43
16-02-2005 F2D2 C439 22 500 93 100 86 1.44
18-02-2005 D59C C439 42 380 80 – – 1.53
21-02-2005 D59C E949 24 660 70 100 0 1.35
21-02-2005 F5C2 E949 13 824 96 62 0 1.50
21-02-2005 ED70 E949 16 640 90 92 48 1.44
22-02-2005 D89B E73C 41 160 82 88 14 1.47
23-02-2005 B4C9 C3A0 8624 61 0 – 1.43
23-02-2005 F2D2 CAF8 17 472 84 0 – 1.45
24-02-2005 A743 F4AF 27 740 68 100 0 1.45
24-02-2005 F5C2 F4AF 55 360 99 100 40 1.42
24-02-2005 ED70 F4AF 80 850 92 100 80 1.37
01-03-2005 F2D2 D1F4 40 600 96 100 80 1.40
03-03-2005 D89B D559 39 008 82 88 0 1.38
10-03-2005 F2D2 B106 18 432 89 100 – 1.42
17-03-2005 E8A4 E949 46 240 56 94 0 1.44
17-03-2005 C5B8 E949 45 696 57 90 0 1.36
Mean 39 944 81 80 31 1.42
SD 18 005 20 27 32 0.04
Aquaculture Research, 2011, 42, 1011^1025 Total lipid and fatty acid composition of brill eggs I H Cruzado et al.
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compared with those with hatching rates higherthan 70% when expressed as a percentage of weight(data not shown).Finally, when we compared the FA content of egg
batches from good females and bad females, only the
ratios of EPA:DHAand arachidonic acid (AA):EPAdif-fered between the two groups (Table 5). However, in acomparison of percentage data, it bears mentioningthe opposite tendencies observed between EPA andDHA, given that EPAwas signi¢cantly higher in egg
Figure1 Lipid-class content (%DW) of brill eggs. Data arepresented as mean � SD (n526). SM, sphingomyeline;PC, phosphatidylcholine; PS, phosphatidylserine; PI,phosphatidylinositol; PG, phosphatidylglycerol; PE, phos-phatidylethanolamine; CHO, cholesterol; FFA, free fattyacids;TG, triacylglycerols; SE, sterol esters.
Table 2 Compositionof total lipids (%DW) and lipid classes (%DW) of eggs, with di¡erent viability rates (lower orhigher than70%) and di¡erent fertilization rates (lower or higher than 70%)
Viability (%) Fertilization (%)
Vo70 (n5 3) V470 (n5 23) Fo70 (n5 5) F470 (n517)
Total lipid 3.65 � 0.98 7.59 � 4.41 7.29 � 5.50 6.83 � 4.10
Sphingomyelin 0.01 � 0.00� 0.04 � 0.03 0.04 � 0.03 0.03 � 0.03
Phosphatidylcholine 0.40 � 0.18� 1.66 � 1.11 1.23 � 1.25 1.48 � 1.06
Phosphatidylserine 0.06 � 0.01 0.21 � 0.13 0.13 � 0.13 0.20 � 0.14
Phosphatidylinositol 0.03 � 0.02 0.08 � 0.06 0.05 � 0.04 0.08 � 0.06
Phosphatidylglycerolw 0.00 � 0.00� 0.03 � 0.03 0.03 � 0.03 0.03 � 0.02
Phosphatidylethanolamine 0.10 � 0.08 0.38 � 0.25 0.27 � 0.29 0.35 � 0.26
Cholesterol 0.47 � 0.13� 1.25 � 0.62 1.02 � 0.80 1.14 � 0.60
Triacylglycerol 0.27 � 0.11 1.59 � 1.30 1.57 � 1.74 1.30 � 1.20
Sterol ester 2.31 � 0.54 2.94 � 1.42 2.96 � 1.30 2.77 � 1.41
Total polar lipid 0.61 � 0.29 2.41 � 1.57 1.74 � 1.76 2.17 � 1.54
Total neutral lipid 3.05 � 0.69 5.78 � 2.90 5.55 � 3.78 5.22 � 2.68
Results represent means � SD.�Signi¢cant di¡erences (Po0.05).wAlso includes phosphatidic acid, and cardiolipin.
Figure 2 Lipid-class composition of low (black bars,n53) and high (grey bars, n523) viability rates in eggs.Data are presented asmean � SD. �Signi¢cant di¡erences(Po0.05). SM, sphingomyeline; PC, phosphatidylcholine;PS, phosphatidylserine; PI, phosphatidylinositol; PG, phos-phatidylglycerol; PE, phosphatidylethanolamine; CHO,cholesterol; FFA, free fatty acids; TG, triacylglycerols; SE,sterol esters.
Total lipid and fatty acid composition of brill eggs I H Cruzado et al. Aquaculture Research, 2011, 42, 1011^1025
1016 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1011^1025
batches from good females, whereas DHAwas higherin eggs from bad females (Fig. 5). These di¡erenceswere re£ected in the EPA:DHA and AA:EPA ratios inboth groups of females.Figure 6a^c shows the distribution of di¡erent egg
batches according to the axes resulting from the PCAof their FA contents: Fig. 6a divided the batches intotwo groups according to their viability rates, Fig. 6bseparated them based on their fertilization rates and¢nally Fig. 6c divided them based on their hatchingrates. In all ¢gures, Axis 1 explains up to 87.5% ofthe system variability. The majority of the FAwas re-presented on this axis, although it was notable thatPUFA, such as 22:5n-3, AA and DHA, presentedstronger correlations. Axis 2 explained 4.5% of the
variability with a high correlation with minor FAsuch as15:0. As with PCA from LC, it was not possibleto discriminate any quality rates of egg batches basedon their FA composition.Table 6 and 7 shows the possible correlations be-
tween quality parameters and the values of each eggbatch based on the PCA analysis forAxes1and 2. Nostrong correlation was found in any case. Signi¢cantrelationships were observed between Axis 2 from li-pid-class PCA and ovulation temperature (Table 6).Also, it is remarkable that correlation with egg dia-meter showed a P-value of 0.056. On the other hand,correlationwithaxes for the FA PCAanalyses showeda relationship between Axis 2 and fertilization ratesand number of ovulations (Table 7).
Table 3 Composition of total lipids (%DW) and lipid classes (%DW) of eggs, with di¡erent hatching rates (null hatching ratesor470%) and females of di¡erent quality (good-quality females and bad-quality females)
Hatching (%) Quality females
H5 0 (n5 6) H470 (n5 5) Good females (n518) Bad females (n5 8)
Total lipid 6.78 � 2.77 5.15 � 3.18 7.67 � 4.38 6.78 � 4.22
Sphingomyelin 0.03 � 0.02 0.02 � 0.02 0.04 � 0.03 0.04 � 0.02
Phosphatidylcholine 1.05 � 0.70 0.99 � 0.98 1.65 � 1.07 1.40 � 1.19
Phosphatidylserine 0.14 � 0.10 0.12 � 0.10 0.21 � 0.13 0.16 � 0.14
Phosphatidylinositol 0.05 � 0.03 0.07 � 0.06 0.10 � 0.08 0.07 � 0.06
Phosphatidylglycerolw 0.03 � 0.03 0.02 � 0.02 0.04 � 0.03 0.03 � 0.03
Phosphatidylethanolamine 0.23 � 0.16 0.25 � 0.26 0.40 � 0.26 0.29 � 0.27
Cholesterol 0.86 � 0.49 0.92 � 0.59 1.26 � 0.61 1.03 � 0.67
Triacylglycerol 1.04 � 0.89 0.74 � 1.02 1.61 � 1.37 1.32 � 1.07
Sterol ester 3.37 � 0.72� 1.72 � 0.90 3.06 � 1.36 2.45 � 1.05
Total polar lipid 1.52 � 0.99 1.47 � 1.42 2.42 � 1.54 1.98 � 1.67
Total neutral lipid 5.27 � 1.80 3.38 � 2.08 5.93 � 2.86 4.80 � 2.58
Results represent means � SD.�Signi¢cant di¡erences (Po0.05).wAlso includes phosphatidic acid, and cardiolipin.
Figure 3 Distribution of di¡erent eggs batches on the axes resulting from PCAof their LC composition, classi¢ed accord-ing to: (a) viability rates470% (&) ando70% (. ), (b) fertilization rates470% (&) ando70% ( . ) and (c) hatchingrates470% (&) ando70% (. ).
Aquaculture Research, 2011, 42, 1011^1025 Total lipid and fatty acid composition of brill eggs I H Cruzado et al.
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Discussion
The present study found relatively high potential egg-viability and fertilization rates, higher than those re-ported previously for brill (Hachero-Cruzado et al.2007) or for turbot (Devauchelle et al.1988), althougha high variability was observed. The fertilization va-lues were higher than those reported for turbot byOmnes, Normant, Suquet & Fauvel (1991) at a pilotscale and by Chereguini, Garcia De La Banda, Ra-sines and FernaŁ ndez (1999) at an experimental scale.The hatching values were low and similar to thosereported for brill (Hachero-Cruzado et al. 2007) andturbot (Fauvel et al.1992), although signi¢cantly low-er than those found by McEvoy (1984) for the samespecie. For all these parameters, we found high varia-bility among individuals and egg batches, similar tothose reported for brill in a previous study (Hachero-Cruzado et al. 2007).A possible cause for the high variability noted in
quality parameters could be the lipid composition ofegg batches. In this sense, we have analysed the egg
Figure 4 Fatty acid composition (mg mg�1DW), in termsof saturates, monounsaturated and PUFAas well as impor-tant fattyacids. Data (n529) are presented as mean � SD.Sat, total saturated; Mono, total monounsaturated; PUFA,total polyunsaturated; ARA, arachidonic acid; EPA, eico-sapentaenoic acid; DHA, docosahexaenoic acid.
Table 4 Fatty acid composition of total lipid of eggs, with di¡erent viability rates (lower or higher than 70%) and di¡erentfertilization rates (lower or higher than 70%) (mgmg DW)
Fatty acids
Viability (%) Fertilization (%)
Vo70 (n5 3) V470 (n5 23) Fo70 (n5 5) F470 (n5 20)
14:0 0.42 � 0.10 1.29 � 0.88 1.28 � 1.12 1.12 � 0.83
15:0 0.15 � 0.09 0.29 � 0.25 0.31 � 0.25 0.26 � 0.24
16:0 3.01 � 0.63 7.96 � 5.03 7.74 � 6.18 7.02 � 4.78
16:1n-7 0.77 � 0.24 2.38 � 1.65 2.34 � 2.10 2.06 � 1.56
18:0 0.81 � 0.21 1.90 � 1.13 1.85 � 1.40 1.71 � 1.08
18:1 n-9 1.43 � 0.43 4.59 � 3.17 4.53 � 4.03 3.97 � 2.98
18:2 n-6 0.14 � 0.03 0.42 � 0.29 0.41 � 0.36 0.36 � 0.27
18:3n-3 0.07 � 0.03 0.22 � 0.16 0.22 � 0.21 0.19 � 0.15
20:4 n-6 0.44 � 0.02 0.89 � 0.50 0.86 � 0.55 0.81 � 0.49
20:5 n-3 1.31 � 0.54 3.64 � 2.28 3.31 � 2.86 3.28 � � 2.21
22:5 n-3 0.53 � 0.17 1.45 � 0.97 1.35 � 1.14 1.28 � 0.92
22:6 n-3 3.39 � 0.91 8.57 � 5.47 7.97 � 6.30 7.59 � 5.20
Saturated 4.48 � 0.84 11.7 � 7.39 11.43 � 9.12 10.33 � 7.03
Monounsaturated 2.84 � 0.79 8.86 � 6.08 8.80 � 7.85 7.67 � 5.68
PUFA 6.52 � 1.79 17.14 � 10.95 16.09 � 13.16 15.21 � 10.42
n-3 5.53 � 1.69 14.60 � 9.36 13.53 � 11.12 12.98 � 8.93
n-6 0.74 � 0.07 1.79 � 1.09 1.74 � 1.31 1.59 � 1.03
n-9 1.53 � 0.46 4.94 � 3.42 4.86 � 4.33 4.27 � 3.22
n-3 HUFA 5.35 � 1.62 14.01 � 8.95 12.96 � 10.58 12.47 � 8.55
n-3/n-6 7.41 � 1.49 7.85 � 0.89 7.25 � 1.12 7.90 � 0.89
EPA/DHA 0.38 � 0.07 0.44 � 0.06 0.41 � 0.08 0.44 � 0.05
AA/EPA 0.36 � 0.11 0.27 � 0.06 0.33 � 0.10 0.27 � 0.07
Mono/PUFA 0.44 � 0.02� 0.51 � 0.07 0.51 � 0.06 0.49 � 0.08
Mono/saturated 0.63 � 0.06 0.73 � 0.10 0.70 � 0.11 0.72 � 0.10
Results represent means � SD.�Signi¢cant di¡erences (Po0.05).PUFA,polyunsaturated fatty acids; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; AA, arachidonic acid.
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1018 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1011^1025
batches to test this hypothesis. Our results showed thatthe TL contents of brill eggs (7.42 � 4.28%DW) werelower than the values found in turbot eggs (14.2^17.0%DW;McEvoy et al.1993) and those found in Sene-gal sole eggs (11.7%DW;VaŁ zquez, GonzaŁ lez, Rodriguez&Mourente1994) and Diplodus sargus (16.54%DW; Ce-jas, Almansa, Je¤ rez, Bolan� os, Felipe & Lorenzo 2004).The higher percentages of NL in brill eggs (73.6%TL),which reach an NL:PL ratio of 2.78, were noteworthy.This is clearly higher than the values found in turboteggs, which showed a ratio between1.1 and1.5 (McE-voy et al.1993), but more similar to the values for Sene-gal sole with1.98 (VaŁ zquez et al.1994).The high NL content was due mainly to SE
(44%TL), together with lower levels of TG and CHO.Senegal sole eggs also showed a predominance of TGand SE, although in this case, theTG percentage wasclearly higher than the SE percentage.This pro¢le re-vealed an important presence of LC related to energyreserves (suchas SE andTG) (Vetter et al.1983; Blaxter1988), which is usually consumed after hatching
Table 5 Fattyacid compositionof total lipid of eggs, with di¡erent hatching rates (null hatching rates or470%) and di¡erentquality females (good-quality females and bad-quality females) (mgmg�1DW)
Fatty acids
Hatching (%) Quality females
H5 0 (n5 6) H470 (n5 6) Good females (n5 21) Bad females (n5 8)
14:0 1.05 � 0.66 0.81 � 0.66 1.32 � 0.92 1.08 � 0.75
15:0 0.26 � 0.16 0.20 � 0.24 0.30 � 0.25 0.24 � 0.14
16:0 6.77 � 3.41 4.94 � 3.53 8.02 � 5.12 7.17 � 4.71
16:1 n-7 1.92 � 1.21 1.54 � 1.35 2.41 � 1.69 2.14 � 1.60
18:0 1.68 � 0.90 1.24 � 0.76 1.95 � 1.15 1.61 � 1.00
18:1 n-9 3.56 � 2.23 3.10 � 2.69 4.74 � 3.23 3.67 � 2.73
18:2 n-6 0.35 � 0.20 0.24 � 0.19 0.42 � 0.29 0.37 � 0.28
18:3 n-3 0.19 � 0.12 0.13 � 0.11 0.23 � 0.16 0.17 � 0.14
20:4 n-6 0.83 � 0.39 0.56 � 0.31 0.90 � 0.51 0.81 � 0.46
20:5 n-3 3.15 � 1.85 2.42 � 1.75 3.72 � 2.28 2.93 � 2.16
22:5 n-3 1.29 � 0.75 0.88 � 0.69 1.46 � 0.98 1.31 � 0.98
22:6 n-3 7.61 � 3.80 5.28 � 3.86 8.52 � 5.44 8.07 � 5.72
Saturated 9.97 � 5.12 7.34 � 5.29 11.84 � 7.55 10.30 � 6.72
Monounsaturated 7.01 � 4.25 5.84 � 5.08 9.04 � 6.21 7.50 � 5.49
PUFA 15.04 � 7.95 10.72 � 7.89 17.26 � 10.99 15.27 � 10.93
n-3 12.84 � 6.83 9.14 � 6.77 14.67 � 9.36 13.08 � 9.48
n-6 1.57 � 0.78 1.09 � 0.69 1.80 � 1.11 1.60 � 1.03
n-9 3.82 � 2.40 3.30 � 2.88 5.09 � 3.49 3.98 � 2.99
n-3 HUFA 12.35 � 6.52 8.79 � 6.46 14.07 � 8.94 12.61 � 9.10
n-3/n-6 8.02 � 0.56 7.98 � 1.21 7.90 � 0.89 7.75 � 1.07
EPA/DHA 0.40 � 0.09 0.47 � 0.04 0.45 � 0.04� 0.36 � 0.04
AA/EPA 0.29 � 0.07 0.25 � 0.07 0.26 � 0.06� 0.33 � 0.08
Mono/PUFA 0.45 � 0.05 0.52 � 0.09 0.51 � 0.08 0.48 � 0.05
Mono/saturated 0.68 � 0.09 0.74 � 0.13 0.73 � 0.10 0.69 � 0.08
Results represent means � SD.�Signi¢cant di¡erences (Po0.05).PUFA,polyunsaturated fatty acids; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; AA, arachidonic acid.
Figure 5 Fatty acid composition [% total fatty acids(TFA)] of females of di¡erent quality (good-quality femalesn521and bad-quality females n58), in terms of impor-tant fatty acids (EPA and DHA). EPA, eicosapentaenoicacid; DHA, docosahexaenoic acid. �Signi¢cant di¡erences(Po0.05).
Aquaculture Research, 2011, 42, 1011^1025 Total lipid and fatty acid composition of brill eggs I H Cruzado et al.
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(Cunha & Planas 1997; Cejas et al. 2004). Brill eggscontain a single oil globule, a feature that has beenassociated with the predominance of TG and SE in¢sh eggs (Russel1976; Cejas et al.2004). However, thismorphological feature is usually linked to highamounts of TL (415%DW; Cejas et al. 2004), whichhas not been reported in brill.
Whenwe compared the lipid composition from eggbatches with di¡erent viability rates, signi¢cant dif-ferences were apparent. In this way, the TL contentwas higher in high-viability eggs (470%). This in-crease was due to a signi¢cant increase in severalLCs such as SM, PC and CHO.These data suggest thatviability could be related to high egg-lipid reserves.
Figure 6 Distribution of di¡erent eggs batches in the axes obtained from PCA of their FA composition, classi¢ed accord-ing to: (a) viability rates470% (&) ando70% (. ), (b) fertilization rates470% (&) ando70% (. ) and (c) hatchingrates470% (&) ando70%(. ).
Table 6 Correlation coe⁄cient and signi¢cance (in brackets) for date, number of ovulation (no. ovulation), temperature ategg collection (temperature), temperature during the day before egg collection (temperature previous day), egg diameter(mm), viability (%), fertilization (%), hatching (%), quality females and batch fecundity in relation to the axes resulting fromPCAof their lipid classes composition
DateNo.ovulation Temperature
Temperaturepreviousday
Eggdiameter
Viability(%)
Fertilization(%)
Hatching(%)
Qualityfemales
Batchfecundity
Axis 1 � 0.383 0.030 � 0.102 0.080 � 0.191 0.232 0.075 � 0.195 � 0.343 0.137
(0.071) (0.893) (0.643) (0.718) (0.407) (0.287) (0.740) (0.410) (0.110) (0.534)
Axis 2 0.243 0.173 0.288 0.446 0.423 0.176 � 0.024 � 0.042 0.049 0.045
(0.265) (0.430) (0.183) (0.033) (0.056) (0.422) (0.917) (0.859) (0.825) (0.840)
PCA, principal component analysis.
Table 7 Correlation coe⁄cient and signi¢cance (in brackets) for date, number of ovulation (no. ovulation), temperature ategg collection (temperature), temperature during the day before egg collection (temperature previous day), egg diameter(mm), viability (%), fertilization (%), hatching (%), quality females and batch fecundity in relation to the axes obtained fromPCAof their fatty acid composition
DateNo.ovulation Temperature
Temperaturepreviousday
Eggdiameter
Viability(%)
Fertilization(%)
Hatching(%)
Qualityfemales
Batchfecundity
Axis
1
� 0.260 � 0.181 � 0.019 0.156 0.191 0.133 0.142 � 0.287 � 0.121 0.106
(0.199) (0.375) (0.925) (0.446) (0.372) (0.517) (0.499) (0.195) (0.556) (0.607)
Axis
2
0.328 0.541 0.250 0.348 0.009 � 0.026 � 0.455 0.027 � 0.114 0.016
(0.102) (0.004) (0.219) (0.081) (0.968) (0.899) (0.022) (0.905) (0.580) (0.936)
PCA, principal component analysis.
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Phosphatidylcholine, SM and CHO are LCs that dis-play a strong relationship with membrane formation,which plays a vital role during organogenesis (Sar-gent et al.1999). Gime¤ nez, Este¤ vez, Lahnsteiner, Zece-vic, Bell, Henderson, Pin� era and Sanchez-prado(2006) also reported a higher content of NL relatedto high egg quality in Dentex dentex.These changes in the total content of several LC
were similar to the changes observed when we ex-pressed the data as lipid-class proportions (expressedas %TL instead of %DW). In both cases, PC increasedin high-viability egg batches. This implies that the LCpro¢le varied not only by the total amount but also inthe proportions among eggs with di¡erent viabilityrates. Phosphatidylcholine is the most important LC,within PL, and has been described as a structuralmembrane component as well as an energy substrate.Several studies have found that PC is one of the mostimportant lipid components to be used during embry-ogenesis and the larval period to obtain energy, butcould also provide both phosphate and choline for in-termediary metabolism and for macromolecule andneurotransmitter synthesis in addition to the synth-esis of other phospholipids (Tocher et al.1985; Sargent1995). Thus, higher contents of PC in eggs could sug-gest that these eggs have more resources to face a cri-tical phase such as embryogenesis.One of the main factors a¡ecting viability could be
that the stripping of the eggs was not well synchro-nized with the ovulatory patterns in all the females,so that eggs were retained in the ovary lumenand be-came overripe. There are external factors, such astemperature or stress, which might a¡ect the ovula-tion period. In this sense, it has been reported thatwater temperature is able to act on the synchroniza-tion of gamete maturation of last stages, ovulationand spawning (Bromage, Porter & Randal 2001).Moreover, it is widely accepted that exposure of ¢shto stressors induces a physiological response with de-leterious e¡ects on reproductive processes (Barton2002). McEvoy (1984) found that ovulated turboteggs retained in the ovary lumen 1 day before strip-ping yielded 0% hatch, whereas freshlyovulated eggsshowed 490% fertilization and up to 97% hatched.Similar results were observed in Atlantic halibut(Norberg et al.1991). The over-ripening process coulda¡ect the lipid composition of the eggs (Corcobado-On� ate, Coo, Arnaiz, Rua & Olmedo 1993), a¡ectingthe viability rate of eggs and supporting the di¡er-ences found in the lipid-class composition betweenthese egg batches. However, further studies are ne-cessary to test this hypothesis.
The relationship between lipid-class compositionand other quality parameters such as fertilization,hatching or female quality is weaker than the viabilityrelationship. In thisway, the fertilization rate showedasigni¢cantly higher proportion of PS (%TL) in eggswith higher fertilization rates. This increase in PSwas similar to that found when comparing eggs ac-cording to their viability rate. Phosphatidylserine is aminor LC, although it is important in many biologicalprocesses associated with membrane physiology, suchas the apoptotic process (Balasubramanian,Mirnikjoo& Shroit 2007); in addition, PS contained large propor-tions of DHA,which is a FAwith important roles in cellphysiology.Egg batches compared according to their hatching
rates showed higher CHO contents, as with the viabi-lity rate, although the di¡erences in hatching wereobserved in CHO proportions (%TL), whereas di¡er-ences in viability were related to the CHO content(%DW). Furthermore, a signi¢cant positive correla-tion (r50.711, P50.000) was found between CHO(%TL) and hatching rate, although the r-value wasnot high. Taken together, all these data suggest thatCHO could be a factor limiting egg quality, whichcould be related to the CHO presence in the cell mem-brane.An opposite tendency was noted in SE levels,
which were lower in eggs with high viability andhatching rates; in addition, we found a signi¢cant ne-gative correlation (r5 �0.659, P50.002) betweenthe hatching rates and the SE content (%DW). There-fore, high SE levels would be related to low-qualityeggs. In previous studies, SE have generally been de-scribed as potential reserves in teleost eggs andwould probably play a dual role: ¢rst, by storing largeamounts of saturated and MUFA for energy purposesand, second, as a temporary reservoir of importantphysiological PUFA (Tocher et al. 1985; Napolitano,Ratmayake & Ackman 1988). These roles have beenobserved in some marine ¢sh species such as Eur-opean sea bass (Bell, Farndale, Bruce, Navas & Carril-lo 1997), mullet (Spener & Sand 1970), whiteseabream (Cejas et al. 2004) and turbot (Silversand,Norberg & Haux 1996). However, our date suggestthat high levels of SE in brill eggs could re£ect a poorlipid pro¢le, implying a loss in egg quality.Finally, a comparisonwasmade between eggs from
females separated according to their spawning qual-ity, but no signi¢cant di¡erences were detected usingStudent’s t-test. This suggests that egg-lipid composi-tion and quality parameters could be more related todi¡erent egg batches than to the females. Di¡erences
Aquaculture Research, 2011, 42, 1011^1025 Total lipid and fatty acid composition of brill eggs I H Cruzado et al.
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in female quality could be due to the non-optimalfeeding condition (individual di¡erences in uptakewithin the same tank). This factor could explain dif-ferences in spawn quality, although it seemed not toa¡ect the lipid composition. In any case, speci¢c ex-periments would be necessary to test the in£uenceof this factor. Exploration of the data with PCAshowed that the lipid-class composition did notclearly distribute the eggs according to their qualityparameters (viability, fertilization or hatching rate).This result suggests that there is no individual LC orgroup that makes eggs batches di¡erent according totheir viability, fertilization or hatching. The lack ofdi¡erences in PCA together with the di¡erences ob-served in Student’s t-test suggest that LC, such as PC,CHO or SE, could be used as a descriptor of the viabi-lity rates but were not enough to explain di¡erencesin quality and presumably there are other factors toexplain this.Finally, we tested a possible correlation among
quality parameters and the LC pro¢le. Only Axis 2,which was correlated with SM and TG, showed lowcorrelation values with egg diameter and tempera-ture from previous days to egg collection. These datasuggest that the content of several LCs, such as TG,could in£uence egg diameter, although more datawould be necessary to reinforce this hypothesis. An-other factor that could in£uence egg diameter is thespawning season. In several species, it has beenshown that egg diameters can decrease during thespawning season (Atlantic cod Gadus morhua L.,Kjesbu, Kryvi & Norberg1996;Senegal sole Solea sene-galensis K., Dinis, Ribeiro, Soares & Sarasquete1999;turbot, McEvoy &McEvoy1991; Mugnier, Guen-noc, Lebegue, Fostier & Breton 2000;Atlantic halibut,Brown et al. 2006), but previous results of our groupshowed that brill egg diameter does not decrease dur-ing the course of the spawning season (Hachero-Cruzado et al. 2007).The FA composition of TL was similar to that re-
ported by Silversand et al. (1996) in turbot eggs. It isremarkable that our FA values were more similar towild eggs than cultured eggs described by theseauthors. The ratio EPA:DHA found in brill (0.43) washigher than the ratio displayed by Senegal sole (0.26)(VaŁ zquez et al.1994), although it was similar to turboteggs (0.25^0.44) (Corcobado-On� ate et al. 1993). TheAA:EPA ratio was more similar to turbot (0.38) (Sil-versand et al. 1996) than to Senegal sole (0.80) (VaŁ z-quez et al.1994).Higher FAcontents were observed in high-viability
eggs, although without signi¢cant di¡erences. Simi-
lar results were observed by Corcobado-On� ate et al.(1993), who reported a reduction (non-signi¢cant) inmost of FA from eggs with lower viability. However,these authors emphasized that PUFA displayed thehighest losses, whereas our data re£ect similar lossesin all FA. These tendencies in the FA contents werenot shared when we expressed data as weight per-centages, indicating that di¡erences are due to the li-pid content, more than the lipid pro¢le. The mostrelevant di¡erence, observed between eggs with dif-ferent viability rates, was the higher MUFA/PUFA ra-tio related to high-viability eggs, which was due tohigh monounsatured contents. These di¡erencescould be related to the loss in egg FA proposed andobserved by Corcobado-On� ate et al. (1993) due toover-ripening.Another relevant ¢nding was the higher percen-
tage of EPA in eggs with high fertilization rates aswell as in eggs from good females. Also, in the lastgroup, DHA showed an opposite trend, with higherpercentages in bad females. Both EPA and DHA be-long to highly unsaturated FA s of the n-3 series (n-3HUFA) and are the most relevant FA in marine ¢sh(Tocher & Sargent 1984). The composition of n-3HUFA eggs has been related to blastomere morphol-ogy and hatching rate in ¢sh (Pickova, Dutta, Lars-sonp.- & Kiessling 1997). Thus, the egg shouldcontain enough of these FA for correct development.In this context, it is worth noting the lower content ofDHA in eggs from good females, but further studiesare necessary to explain this.In this study, no signi¢cant di¡erences were found
in the AA content although we should mention therelevant contributionof AA in the ¢rst principal com-ponents of PCAanalysis, which explained 87% of sys-tem variability. Arachidonic acid is the majoreicosanoid precursor in ¢sh cells (Bell,Tocher & Sar-gent1994) and is involved in the regulation of numer-ous physiological processes (Mustafa & Srivastava1989; Sorbera, Zanuy & Carrielo1998). Our data sug-gest that despite AAvariability, this was not enoughto explain the di¡erences in quality parameters.As with the LC, we examined the data with PCA
but without ¢nding a clear distribution of the eggsaccording to their quality parameters. These data re-inforce the hypothesis that there was no lipid compo-nent, nor a group of them, whichwas able to separateegg batches according to their quality parameters.Therefore, other factors could be involved in the qual-ity di¡erences. Similar to PC, high MUFA:PUFA ratioscould be a descriptor of high-viability rates, and theEPA percentage could be considered to be a good de-
Total lipid and fatty acid composition of brill eggs I H Cruzado et al. Aquaculture Research, 2011, 42, 1011^1025
1022 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1011^1025
scriptor of high fertilization eggs, as well as eggs fromgood females, although their levels did not appear tobe enough to induce egg-quality di¡erences.The multivariate correlation among egg values
and quality parameters did not reveal relevant rela-tionships. Only Axis 2, which was related to 15:0,showed a signi¢cant but weak correlation with thefertilization rates and ovulation number (de¢ned asthe number of an individual egg batch from a singlefemale during the spawning season).These dataweresimilar to those found in the lipid-class analysis. Ingeneral terms, these data are in agreement withthose observed by Corcobado-On� ate et al. (1993),who did not identify a clear relationship between FAcomposition and egg viability in turbot eggs.In conclusion, lipid composition did not show a
clear correlation with the quality of brill eggs, eitheras individual components or as the whole pro¢le ofLC or FA. Our results suggest that some lipid compo-nents (PC, CHO, EE, monounsaturated, EPA) could berelated to di¡erences in spawning quality, althoughthey were not the only factors involved in these dif-ferences. Thus, these lipid components could be con-sidered to be descriptors of di¡erences in quality.
Acknowledgments
This research has been funded by the project INIA‘Establecimiento de una metodolog|¤ a de fecundacio¤ narti¢cial para la obtencio¤ n de puestas de calidad en elparracho’ (TRT2006-00011-C02-01). The authorsthank J. F. Ferrer and M. Rosano at IFAPA and J. M.MaŁ rquez at DesarrolloAgrario y Pesquero S.A. (JuntadeAndaluc|¤ a) for their essential help on ¢sh husban-dry. Dr Antonio Lorenzo HernaŁ ndez is a member ofthe ITB of La Laguna University (Spain).
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