Development of broodstock diets for the European Sea Bass ( Dicentrarchus labrax) with special emphasis on the importance of n−3 and n−6 highly unsaturated fatty acid to reproductive performance

Development of broodstock diets for the European Sea Bass ( Dicentrarchus labrax) with special emphasis on the importance of n−3 and n−6 highly unsaturated fatty acid to reproductive performance

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  Ž . Aquaculture 177 1999 85–97 Development of broodstock diets for the European ž / Sea Bass  Dicentrarchus labrax  with specialemphasis on the importance of   n y 3 and  n y 6highly unsaturated fatty acid to reproductiveperformance Michael Bruce  a, ) , Ferdinand Oyen  b , Gordon Bell  a ,Juan F. Asturiano  b , Bruce Farndale  a , Manuel Carrillo  b ,Silvia Zanuy  b , Jesus Ramos  b , Niall Bromage  a a  Institute of Aquaculture, Uni Õ ersity of Stirling, Stirling, Scotland, FK9 4LA, UK  b  Instituto de Acuicultura, Torre de la Sal, Consejo Superior de In Õ estigaciones Cientificas, Madrid, Spain Accepted 1 October 1998 Abstract Commercially fabricated diets allow greater control over the composition of biochemicalcomponents and reduce the risks of disease introduction, which are significant concerns whenusing the wet fish diets commonly used for most farmed marine broodstocks. However, satisfyingthe dietary lipid requirements of marine broodstock using artificial diets has proved difficult, Ž . particularly with respect to their highly unsaturated fatty acid HUFA composition. Two groupsof mature sea bass, each divided between three replicated tanks, were fed two dry pelleted dietsover a 2-year period, encompassing two spawning seasons. The first diet contained a good qualityNorthern Hemisphere meal and oil; the second differed only in the source of oil, which was Ž . substituted with tuna orbital oil TOO . The use of TOO in the dry pelleted formulation allowedthe manipulation of   n y 3 and  n y 6 HUFA in the resulting eggs, specifically arachidonic acid Ž . Ž . Ž 20:4  n y 6; AA , eicosapentaenoic acid 20:5  n y 3; EPA and docosahexaenoic acid 22:6 . n y 3; DHA . The results showed that dietary manipulation of these HUFA could improve levelsand ratios of AA, EPA and DHA which were transferred to the resulting eggs with improvementsin early survival and hatching success repeated over successive spawning seasons. The dry diet Ž containing TOO facilitated comparable reproductive performance to the wet fish diet  Boops . boops  which has previously been considered the most effective broodstock diet. The improve- ) Corresponding author. Tel.: q 44-178-646-7929; Fax: q 44-178-647-2133; E-mail: 0044-8486 r 99 r $ - see front matter q  1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0044-8486 99 00071-X  ( ) M. Bruce et al. r  Aquaculture 177 1999 85–97  86 ments in reproductive performance are discussed in relation to the proportion of these HUFA with Ž . respect to each other in total egg lipid and the phospholipid classes phosphatidylcholine PC , Ž . Ž . phosphatidylinositol PI and phosphatidylethanolamine PE and to their potential impact oneicosanoid formation. Finally, this study has shown that a commercially fabricated diet can besuccessfully used as sensitive investigative tool for aquaculture research. q 1999 Elsevier ScienceB.V. All rights reserved. Keywords:  Essential fatty acid; Arachidonic acid; Docosahexaenoic acid;  Dicentrarchus labrax ; Diets;Spawning 1. Introduction During the teleost maturation cycle, females of mature broodstocks require largequantities of macro- and micronutrients to be made available for transfer to thedeveloping oocytes. This involves a considerable mobilisation of stored materials anddiversion of dietary nutrients into gonad development. These processes require the fineco-ordination of many aspects of the maternal system, ranging from the higher, more Ž general control of brain hormones, e.g., GnRH and GtH Bromage and Cumaranatunga, . 1988; Kah et al., 1994 to the more localised actions of prostaglandins and leukotrienes Ž on final maturation, ovulation and spawning Mustafa and Srivastava, 1989; Sorbera et . Ž . al., 1998 . The requirements for essential fatty acids EFA as membrane components tomaintain both structure and function have been well-documented by a number of authors Ž . Castell et al., 1972; Sargent et al., 1995 . In comparison to most mammalian cells Ž . which contain arachidonic acid 20:4  n y 6; AA as their major highly unsaturated fatty Ž . acid HUFA , fish contain high levels of the  n y 3 series HUFA eicosapentaenoic acid Ž . Ž . Ž 20:5  n y 3; EPA and docosahexaenoic acid 22:6  n y 3; DHA Henderson and . Tocher, 1987; Sargent et al., 1995 . However, despite the excess of EPA in fish cells,AA has a vital function as the main precursor for a wide variety of biologically active Ž compounds known collectively as the eicosanoids Tocher and Sargent, 1987; Bell et al., . 1994 ; these are responsible for a wide range of physiological roles including osmoregu-lation, cardiovascular functions, neural control and the functioning of reproductive Ž . systems Mustafa and Srivastava, 1989 .Generally, freshwater teleosts possess the ability to synthesise their own essential Ž long-chain HUFA from the 18 carbon unit precursors linoleic acid; 18:2  n y 6 and . a -linolenic acid; 18:3  n y 3 . Marine teleosts lack this ability due to an apparentdeficiency of the  D 5-desaturase required for the elongation and desaturation of these C 18 Ž . precursors to AA and EPA Sargent, 1995 . Also, the ‘Sprecher shunt’, which convertsEPA to DHA, operates at a level insufficient to supply the quantity of DHA required for Ž . normal growth and development Voss et al., 1991; Buzzi et al., 1997 . Therefore, it isimperative that diets contain sufficient amounts of these long-chain EFA.To meet their essential dietary needs, juvenile marine fish require between 0.5 and Ž . 1.7% of the dry weight of their diet as long-chain  n y 3 fatty acid Sargent et al., 1995 .However, the developing egg and larval stages of fish probably have greater require-ments for  n y 3 HUFA, because of the preponderance of   n y 3 HUFA in their neuraland visual tissue which predominates in the early stages of development. Consequently,  ( ) M. Bruce et al. r  Aquaculture 177 1999 85–97   87 any deficiency in these particular fatty acids can cause developmental abnormalities inthe neural system and may affect their success as visual predators at the onset of  Ž . first-feeding Bell et al., 1995a,b .To date, the most effective way to satisfy the EFA requirements of marine broodstock has been through the use of ‘wet fish’, confirmed by the industry preference for thismethod of broodstock nutrition. The aim of the present experiment was to produce anartificially fabricated diet for sea bass broodstock, optimising the EFA composition toachieve the excellent spawning performance so far only reliably accomplished when Ž . using a ‘wet fish’ diet  B. boops  . 2. Materials and methods 2.1. Broodstock husbandry and egg sampling Ž . Sea bass broodstock 2 years of age of farmed origin were divided into twoexperimental diet groups and maintained on these diets for the duration of two spawningseasons. Each group consisted of three replicate 8000 l tanks containing 6 to 10 femalesin each tank and sufficient males to maintain a 2:1 male-to-female sex ratio. All tanks Ž  y 1 . were supplied with flow-through ambient sea water 21 min and photoperiod, andfish were fed to satiation once daily. Each diet consisted of a 9-mm commercially Ž . fabricated expanded pellet EWOS Technology Centre, Livingston, UK using a low- Ž . temperature Northern hemisphere fish meal LT94 and containing 20% lipid differing Ž . only in the source of fish oil Table 1 . Both diets contained the EWOS standard vitaminand mineral premix. The oil component was sprayed on after the pellets were extruded. Ž . The first of the two diets Control contained a high-quality Northern Hemisphere fish Ž oil, intended to emulate the most successful diet differing only in the srcinal source of  . the fish oil, i.e., a different manufacturer from previous work, in terms of subsequent Ž . Ž . egg and larval performance Navas et al., 1997 . The second diet Ropufa diet used a Ž by-product of the Japanese tuna fishing industry, tuna orbital oil TOO; Ropufa 30, . Ž . Roche Products, Heanor, UK , particularly high in DHA 27.3% , relatively low in EPA Ž . Ž . Ž . 5.4% , and containing significantly more AA 1.8% than standard fish oils 0.5% Ž . Table 2 . The eggs spawned were collected daily from conical mesh traps placed across Table 1Proximate composition of experimental diets Ž . Percentage % Control RopufaMean SEM Mean SEM U Moisture 4.3 0.01 3.4 0.04Protein 50.8 0.77 51.4 0.21Lipid 20.9 0.67 20.3 0.12 U CHO 23.8 0.32 27.8 0.12Ash 10.0 0.08 10.2 0.10Fibre 1.1 0.18 1.2 0.13 U Ž . Denotes a significant difference  p - 0.05 .  ( ) M. Bruce et al. r  Aquaculture 177 1999 85–97  88Table 2Percent fatty acid composition of experimental dietsFatty acid Control Ropufa ‘Wet fish’14:0 5.8 3.8 1.916:0 12.2 18.7 20.116:1s 4.3 4.9 4.118:0 1.6 4.5 8.318:1s 12.7 14.8 10.318:2  n y 6 4.6 4.9 2.618:3  n y 6 0.1 0.2 –18:3  n y 3 1.4 1.2 0.618:4  n y 3 2.4 1.2 0.520:1s 13.6 3.9 1.820:2  n y 6 0.2 0.2 – U 20:4  n y 6 0.4 1.4 4.620:3  n y 3 0.1 0.1 –20:4  n y 3 0.5 0.4 0.2 U 20:5  n y 3 5.8 5.6 6.722:1s 19.4 5.4 0.621:5 0.2 0.2 –22:5  n y 6 0.1 1.2 1.222:5  n y 3 0.6 0.8 2.4 U 22:6  n y 3 7.8 19.5 22.124:1  n y 9 1.0 0.7 – U Ý  Saturates 20.2 28.3 32.5 U Ý  Monoenes 51.0 30.0 18.1 Ý  Dienes 5.1 5.4 3.4 U Ý  PUFA 25.0 37.4 44.6 U Ý  n y 3 18.7 28.4 34.6 U Ý  n y 6 5.4 7.9 10.0 U Ý  n y 9 25.7 17.7 – n y 3: n y 6 ratio 3.4 3.6 3.5 U AA:EPA ratio 0.06 0.25 0.7 U DHA:EPA ratio 1.3 3.5 3.3 U Ž . Denotes a significant difference between Control and Ropufa  p - 0.05 of values tested, i.e., highlightedtext and fatty acid group totals. the out-flow of each tank. Eggs collected thus were always fertilised and derived fromone female as seen by the homogeneity of development. Egg viability was assessed bymeasuring the proportion of floating viable eggs to sinking non-viable eggs, previously Ž . described by Carrillo et al. 1989 . Duplicate samples of 100 eggs from each spawningwere taken for lipid and dry weight measurement. The comparisons to ‘wet fish’ made Ž . with respect to the current experiment refer to the bogue  B. boops  . 2.2. Egg incubation In the first spawning season, a total of six and eight batches, from the Control andRopufa diets, respectively, were incubated at 16 8 C. Samples of eggs from each batch Ž . were incubated using the technique described by Thrush et al. 1993 . The technique  ( ) M. Bruce et al. r  Aquaculture 177 1999 85–97   89 was extended by a further assessment at 48 h post-fertilisation. In addition, incubationswere carried out in parallel using 96-well microtitre plates incubated in triplicate at 16 8 C Ž . utilising a method first described by Shields et al. 1997 for Atlantic halibut. Thefollowing year, due to the effectiveness of the technique and limitations on equipmentand space, all incubations were carried out again in triplicate using the microtitre plate.During the second spawning season, 17 batches were incubated from the Control groupand 13 from the broodstock fed the Ropufa diet. 2.3. Lipid analysis Material destined for lipid analysis was stored in at least 10 volumes of  Ž . Ž . Ž . chloroform r methanol 2:1 v r v with 0.01% w r v butylated hydroxytoluene BHT at y 70 8 C prior to extraction. The amount of total lipid was determined gravimetricallyusing an Ohaus JA200B analytical balance after homogenisation in chloroform r methanol Ž . Ž . 2:1 v r v and extraction using the method of Folch et al. 1957 . Lipid classes were Ž . analysed by double development high-performance thin layer chromatography HPTLC Ž . following the method described by Olsen and Henderson 1989 using 10 = 10 cm silica Ž . gel plates E. Merck, Darmstadt, Germany . The solvent systems used to develop polar Ž . Ž . lipid PL and neutral lipid NL classes were methyl acetate:propan-2- Ž . ol:chloroform:methanol:0.25% aqueous KCl 25:25:25:10:9 by volume and hexane:di- Ž . ethyl ether:acetic acid 80:20:2 v r v r v , respectively. The plates were then sprayed with Ž . copper acetate r phosphoric acid reagent Fewster et al., 1964 and charred at 160 8 C for20 min to visualise the lipid classes and analysed by quantitative scanning densitometryusing a Shimadzu CS9000 densitometer.Fatty acid methyl esters of total lipid were prepared by transesterification of 250  m g Ž . of total lipid with 1% sulphuric acid in methanol and toluene 2:1 v r v at 50 8 C for 16 h Ž . Christie, 1982 . Fatty acid methylesters were purified by thin layer chromatography Ž . Ž . TLC prior to analysis as detailed by Tocher et al. 1985 . Individual phospholipidclasses were isolated by loading 1–2 mg of total lipid onto a 2-cm srcin of a 20 = 20cm 2 TLC plate and separated using the polar lipid solvent system mentioned above Ž . Vitiello and Zanetta, 1978 . Individual phospholipids were identified under UV lightafter lightly spraying the plate with 2,7-dichlorofluorescein in 97% methanol containing0.05% BHT. Each class was then scraped from the silica plate and transmethylated as Ž . described by Christie 1982 . 2.4. Data analysis Replicates were pooled after variation between replicates was tested using the  F  max test followed by a Student’s  t  -test. Difference between dietary treatments was deter-mined by a further Student’s  t  -test. 3. Results 3.1. Diet composition; proximate and fatty acid  Ž . Both experimental diets had essentially the same proximate composition Table 1 , Ž . with only moisture and carbohydrate significantly different  p - 0.05 .
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