Effect of broodstock nutrition on reproductive performance of fish

Aquaculture 197 Ž2001. 25–42 www.elsevier.nlrlocateraqua-online Effect of broodstock nutrition on reproductive performance of fish a M.S. Izquierdo a,) , H. Fernandez-Palacios , A.G.J. Tacon b ´ a GIA, Grupo de InÕestigacion ´ en Acuicultura, P.O. Box 56, 35200 Telde, Las Palmas, Canary Islands, Spain b The Oceanic Institute, Makapuu Point, Waimanalo, HI 96795, USA Received 1 October 2000; accepted 31 December 2000 Abstract In many cultured fish species, particularly in those new for aquaculture, unpredictable and variable reproductive performance is an important limiting factor for the successful mass production of juveniles. An improvement in broodstock nutrition and feeding has been shown to greatly improve not only egg and sperm quality but also seed production. Gonadal development and fecundity are affected by certain essential dietary nutrients, especially in continuous spawners with short vitellogenic periods. Thus, during the last two decades, more attention has been paid to the level of different nutrients in broodstock diets. However, studies on broodstock nutrition are limited and relatively expensive to conduct. Lipid and fatty acid composition of broodstock diet have been identified as major dietary factors that determine successful reproduction and survival of offspring. Some fish species readily incorporate dietary unsaturated fatty acids into eggs, even during the course of the spawning season. Highly unsaturated fatty acids ŽHUFA. with 20 or more carbon atoms affect, directly or through their metabolites, fish maturation and steroidogenesis. In some species, HUFA in broodstock diets increases fecundity, fertilization and egg quality. As in higher vertebrates, vitamin E deficiency affects reproductive performance, causing immature gonads and lower hatching rate and survival of offspring. For example, elevation of dietary a-tocopherol levels has been found to reduce the percentage of abnormal eggs and increase fecundity in the gilthead seabream Ž Sparus aurata.. Ascorbic acid has also been shown to play an important role in salmonid reproduction, where the dietary requirement of broodstock was higher than that of juveniles. Among different feed ingredients, cuttlefish, squid and krill meals are recognized as valuable components of broodstock diets. The protein component of cuttlefish and squid together with their optimal concentration of HUFA appear to be responsible for their positive effect on ) Corresponding author. 0044-8486r01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 8 1 - 6 26 M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 reproductive performance. Both polar and nonpolar lipid fractions of raw krill were found to effectively improve egg quality. q 2001 Published by Elsevier Science B.V. Keywords: Broodstock; Nutrition; Spawn; Egg quality; Fatty acids; Vitamin E 1. Introduction Broodstock nutrition is without doubt one of the most poorly understood and researched areas of finfish nutrition. To a large extent, this has been due to the necessity of suitable indoor or outdoor culture facilities for maintaining large groups of adult fish and the consequent higher cost of running and conducting extended broodstock feeding trials. However, as in human and livestock nutrition ŽLeboulanger, 1977., it is clear that the dietary nutrient requirements of broodstock will be different from those of rapidly growing juvenile animals. Moreover, as in other animals, it is also clear that many of the deficiencies and problems encountered during the early rearing phases of newly hatched finfish larvae are directly related to the feeding regime Žincluding nutrient level and duration. of the broodstock. The aim of this paper is to review the major studies conducted to date on the effects of broodstock nutrition on reproductive performance of farmed fish. 2. Effect of food restriction Food restriction itself can seriously affect spawning success. A reduction in feeding rate has been reported to cause an inhibition of gonadal maturation in several fish species, including goldfish Ž Carassius auratus, Sasayama and Takahashi, 1972., European seabass Ž Dicentrachus labrax, Cerda´ et al., 1994a. and male Atlantic salmon Ž Salmo salar, Berglund, 1995.. In seabass, after 6 months of feeding broodstock with a half food ration, growth rates decreased and spawning time was delayed and eggs as well as newly hatched larvae were smaller than those obtained from fish fed full rations ŽCerda´ et al., 1994a.. In female seabass, the detrimental effects of food restriction were associated with reduced plasma estradiol levels ŽCerda´ et al., 1994a.. However, the expression of the GtH genes was not affected by food restriction in mature female goldfish ŽSohn et al., 1998.. 3. Effects of nutrition on fecundity of broodstock fish Several methods have been developed to assess the egg quality of fish ŽKjorsvik et al., 1990; Fernandez-Palacios et al., 1995.. One of the parameters, fecundity, has been ´ used to determine egg quality, which is also affected by a nutritional deficiency in broodstock diets. Fecundity is the total number of eggs produced by each fish expressed either in terms of eggsrspawn or eggsrbody weight. Reduced fecundity, reported in several marine fish species, could be caused either by the influence of a nutrient M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 27 imbalance on the brain–pituitary–gonad endocrine system or by the restriction in the availability of a biochemical component for egg formation. The elevation of dietary lipid levels from 12% to 18% in broodstock diets for rabbitfish Ž Siganus guttatus . resulted in an increase in fecundity and hatching ŽDuray et al., 1994., although this effect could also be related to a gradual increase in the dietary essential fatty acid content. Indeed, one of the major nutritional factors that has been found to significantly affect reproductive performance in fish is the dietary essential fatty acid content ŽWatanabe et al., 1984a,b.. Fecundity in gilthead seabream Ž Sparus aurata. was found to significantly increase with an increase in dietary n y 3 HUFA Žpolyunsaturated fatty acids with 20 or more carbon atoms, essential for marine fish. levels up to 1.6% ŽFernandez-Palacios et al., 1995., and similar results have been ´ reported in other sparids ŽWatanabe et al., 1984a,b,c, 1985a,b.. However, studies on the reproductive performance of Nile tilapia Ž Oreochromis niloticus ., as indicated by the number of females that spawn, spawning frequency, number of fry per spawning and total fry production over a 24-week period, show that the performance was much higher in fish fed a basal diet supplemented with soybean oil Žhigh in n y 6 fatty acids, essential for this fish species; Watanabe, 1982. and relatively low in fish fed a 5% cod liver oil supplemented diet Žhigh in n y 3 fatty acids. ŽSantiago and Reyes, 1993.. Fish fed the diet containing cod liver oil showed the highest weight gain ŽSantiago and Reyes, 1993.. With the exception of salmonids and turbot Ž Scophthalmus maximus ., muscle lipid reserves are utilised during the maturation of the ovaries ŽLie et al., 1993.. In sparids, the fatty acid composition of the female gonad is greatly affected by the dietary fatty acid content, which in turn significantly influences egg quality in a short period of time ŽHarel et al., 1992.. Thus, in gilthead seabream, the fatty acid composition of eggs is directly affected by the n y 3 HUFA content of the broodstock diet. Both the n y 3 fatty acid and n y 3 HUFA content of gilthead seabream eggs increased with an increase in n y 3 HUFA dietary levels, mainly due to the increase of 18:3n y 3, 18:4n y 3 and 20:5n y 3 ŽEPA, eicosapentaenoic acid. contents in the eggs ŽFernandez-Palacios et al., ´ 1995.. A positive correlation was observed between the levels of n y 3 HUFA in the diet and the eggs with the EPA concentration being more readily affected by dietary n y 3 HUFA than DHA Ždocosahexaenoic acid.. Rainbow trout Ž Oncorhynchus mykiss . fed an n y 3 deficient diet during the last 3 months of vitellogenesis produced a moderate effect on the incorporation of DHA into egg lipid whereas EPA concentration decreased by 50% ŽFremont et al., 1984.. However, the levels of other fatty acids in the ´ eggs were not affected by the fatty acid composition of the diet. Selective retention of DHA has also been found during embryogenesis ŽIzquierdo, 1996. and during starvation ŽTandler et al., 1989. denoting the importance of this fatty acid for the developing embryo and larvae. Polyunsaturated fatty acids can also regulate eicosanoid production, particularly prostaglandins, which are involved in several reproductive processes ŽMoore, 1995., including the production of steroid hormones and gonadal development such as ovulation. Fish ovaries have a high capacity to generate eicosanoids, among them prostaglandin E ŽPGE. derived from cycloxygenase action and leukotrienes LTB 4 and LTB 5 derived from lipoxygenase action ŽKnight et al., 1995.. Inhibitors of the latter enzyme reduced the gonadotropin-induced maturation of European seabass oocytes 28 M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 ŽAsturiano, 1999., suggesting that products derived from lipoxygenase action could also be involved in oocyte maturation. This fact has been demonstrated in mammals, where some leukotrienes ŽLTB 4 . enhance the steroidogenic action of LH ŽSullivan and Cooke, 1985.. In other fish species such as cod Ž Gadus morhua. a clear effect of essential fatty acid on fecundity was not observed in fish fed commercial diets coated with different types of oils ŽLie et al., 1993.. In a long-term feeding trial with cod, broodstock were fed diets coated with soybean, capelin or sardine oils. It showed a relatively small effect on the fatty acid composition of eggs from fish fed the fish oils, however, the egg n y 3 HUFA concentration was significantly reduced in fish fed soybean oil ŽLie et al., 1993.. These results may be due to a low essential fatty acid ŽEFA. requirement of cod broodstock, compared with sparids, which possibly allowed them to derive EFA from the residual lipid present in the fish meal component of the experimental diet in order to satisfy their physiological needs. Apart from dietary EFA deficiencies causing detrimental effects in fish, their excess has been also reported to have a negative effect on reproductive performance of fish. For example, high levels of dietary n y 3 HUFA reduced the total amount of eggs produced by gilthead seabream broodstock despite an increase in egg n y 3 HUFA concentration ŽFernandez-Palacios et al., 1995.. Since reduced fecundity was associated with high ´ n y 3 HUFA egg content, increased EFA content alone should not be used as a criterion to assess the egg quality of gilthead seabream broodstock. High dietary n y 3 HUFA levels could affect the brain–pituitary–gonad endocrine axis since both EPA and DHA have been found to reduce in vitro the steroidogenic action of gonadotropin in the ovary of teleost fish ŽMercure and Van Der Kraak, 1995.. This is similar to mammals where an increased dietary level of n y 3 fatty acids delays the onset of puberty ŽZhang et al., 1992.. Other nutrients which have been shown to affect fecundity include vitamin E ŽIzquierdo and Fernandez-Palacios, 1997; Fernandez-Palacios et al., 1998. and ascorbic ´ ´ acid ŽBlom and Dabrowski, 1995.. An increase in dietary a-tocopherol levels up to 125 Fig. 1. Reproductive performance in gilthead seabream fed diets containing different levels of vitamin E. M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 29 Table 1 Biochemical composition of gilthead seabream eggs on broodstock fed several vitamin E and ny3 HUFA levels Žmgrkg and % dry weight Žd.w... Vit ErHUFA Total lipids Ž% d.w.. ny3 HUFA Žarea %. EPA Žarea %. DHA Žarea %. Vit E Žmgrkg. 22r1.6 55r1.7 125r1.6 2010r1.4 190r2.2 28.2 a 27.4 a 24.9 ab 24.8 b 23.0 b 27.1a 27.1a 25.9 a 25.3 a 27.5a 3.72 a 3.6 a 4.58 ab 4.04 a 5.32 b 21.36 a 21.23 a 19.18 a 19.03 a 19.62 a 101.3 a 106.7 a 106.7 a 207.1b 115.5a mgrkg resulted in an improvement in fecundity of gilthead seabream as expressed by the total number of eggs producedrfemale and egg viability ŽFig. 1; authors’ unpublished data.. However, the reduced fecundity observed in broodstock fed a diet deficient in a-tocopherol was not associated with reduced vitamin E content of eggs, and only very high dietary vitamin E levels Ž2020 mgrkg. were found to increase egg atocopherol content ŽTable 1; authors’ unpublished data.. In other species such as turbot ŽHemre et al., 1994. or Atlantic salmon ŽLie et al., 1993., vitamin E was mobilized from peripheral tissues during vitellogenesis although the plasma vitellogenin content was not affected, suggesting that lipoproteins may be involved in the transport of vitamin E during this period ŽLie et al., 1993.. Vitamin C content of rainbow trout eggs reflected the content of this nurient in the diet and was associated with improved egg quality ŽSandnes et al., 1984.. Changes in the vitamin C content of cod ovaries did not significantly affect hatching rates ŽMangor-Jensen et al., 1993.. Again these results suggest that the biochemical composition of eggs should not be used as the sole criteria to determine egg quality, despite the fact that several authors ŽSandnes et al., 1984; Craik, 1985; Harel et al., 1994. have suggested that the chemical composition of fish eggs is related to spawning success since nutrients stored in the egg must satisfy nutritional demands for embryonic development and growth. Dietary antioxidant requirements increase during reproduction ŽIzquierdo and Fernandez-Palacios, 1997; ´ Fernandez-Palacios et al., 1998.. This may be related to the formation of oxygen radicals ´ during steroid hormone biosynthesis as observed in higher vertebrates. For example, levels of antioxidant compounds were correlated with progesterone levels in bovine corpus luteum suggesting the activation of antioxidative mechanisms to cope with steroidogenesis dependent oxyradical formation ŽRapoport et al., 1998.. Dietary tryptophan, a precursor of the neurotransmitter serotonin, may positively affect gonad maturation in both males and females. Supplementation of 0.1% tryptophan in the diets of ayu Ž Plecoglossus altiÕelis . resulted in a significant increase in the serum testosterone levels thus advancing time of spermiation in males and induced maturation of females ŽAkiyama et al., 1996.. 4. Effect of broodstock nutrition on fertilization Certain dietary nutrients also exert a marked effect on fertilization. Dietary eicosapentaenoic ŽEPA. and arachidonic acid ŽAA. levels show a correlation with fertilization 30 M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 rates in gilthead seabream broodstock ŽFernandez-Palacios et al., 1995, 1997.. Since ´ sperm fatty acid composition depends upon the essential fatty acid content of broodstock diet in species such as rainbow trout ŽWatanabe et al., 1984d, Labbe et al., 1993. and European seabass ŽAsturiano, 1999., it is possible that sperm motility and in turn fertilization would be affected. Particularly in salmonids, where cryopreservation of sperm is currently utilized, sperm fatty acid composition could be a factor that determines the membrane integrity after freeze-thawing. However, Labbe et al. Ž1993. did not find any effect of dietary fatty acids Ž n y 3 and n y 6 polyunsaturated fatty acids. on sperm freeze–thaw fertilizing ability, whereas low membrane cholesterol– phospholipid ratios were correlated with a better sperm freezing resistance ŽLabbe and Maisse, 1996.. Another hypothesis to explain the beneficial effect of EPA and AA on fertilization rates has been proposed by several investigators. Both EPA and AA are involved in cell-mediated functions and are precursors of eicosanoids. EPA is known to be a precursor of prostaglandins ŽPG. from series III, whereas AA is a precursor of PG from series II ŽStacey and Goetz, 1982.. In vitro AA, but not EPA or DHA, stimulates testicular testosterone in goldfish testis through its conversion to prostaglandin PGE 2 ŽWade et al., 1994.. On the contrary, EPA or DHA blocked the steroidogenic action of both arachidonic acid and PGE 2 . Both AA and EPA modulate steroidogenesis in the goldfish testis ŽWade et al., 1994.. Thus, the timing of spermiation may be delayed and subsequently fertilization rates reduced by depressed steroidogenesis caused by a broodstock EFA deficiency or imbalance. Moreover, prostaglandins are also recognized as important pheromones in some teleost fish. Some PGs produced by female goldfish, such as PGFs, have been shown to stimulate male sexual behaviour and synchronize male and female spawnings, thus directly affecting the success in fertilization ŽSorensen et al., 1988.. Other nutrients known to be important for fertilization are vitamin E ŽIzquierdo and Fernandez-Palacios, 1997; Fernandez-Palacios et al., 1998., carotenoids ŽHarris, 1984; ´ ´ Craik, 1985. and vitamin C. Ascorbic acid has been shown to play an important role in salmonid reproduction ŽEskelinen, 1989; Blom and Dabrowski, 1995., and its role in steroidogenesis and vitellogenesis has been reported ŽSandnes, 1991.. The antioxidant function of vitamins C and E can provide an important protective role for the sperm cells during spermatogenesis and until fertilization by reducing the risk of lipid peroxidation, which is detrimental for sperm motility. The ascorbic acid concentration in the seminal fluid reflected the concentration of this vitamin in the broodstock diet and it did not affect semen quality at the beginning of the spawning season ŽCiereszco and Dabrowski, 1995.. However, a deficiency of ascorbic acid reduced sperm concentration and motility during the later part of the spawning period. 5. Effect of broodstock nutrition on embryo development Several nutrients are essential for the normal development of the embryo, and their optimum level in broodstock diets improves egg morphology and hatching rates. The M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 31 percentage of morphologically normal eggs Žas a parameter to determine egg viability. has been found to increase with an increase in the n y 3 HUFA levels in broodstock diets and an incorporation of these fatty acids into the eggs ŽFernandez-Palacios et al., ´ 1995., thus indicating the importance of EFAs for normal development of gilthead seabream eggs and embryo. Gilthead seabream fed EFA deficient diets also showed an increased number of lipid droplets in egg ŽFernandez-Palacios et al., 1997. as also ´ reported in red seabream ŽWatanabe et al., 1984a.. Improved egg quality has been associated with higher total n y 3 fatty acids content in European seabass fed a pelleted diet enriched with high quality fish oil ŽNavas et al., 1996., whereas the comparison between brackish water and seawater cod eggs showed that AA and DHArEPA contents in the PL fraction of eggs are positively correlated with egg symmetry and viability ŽPickova et al., 1997.. These fatty acids play an important structural role as components of phospholipids in fish biomembranes and are associated with the membrane fluidity and correct physiological functions for bound membrane enzymes and cell functions in marine fish ŽBell et al., 1986.. In some species, such as halibut Ž Hippoglossus hippoglossus., the n y 3 PUFA Žpolyunsaturated fatty acids. are also regarded as major energy sources during early embryonic development ŽFalk-Petersen et al., 1989.. Nevertheless, fatty acid composition of fish egg lipids is not only determined by the broodstock diet but is also related to species and stock differences ŽPickova et al., 1997.. Essential fatty acid requirements for sparids broodstock range between 1.5% and 2% n y 3 HUFA in diet ŽWatanabe et al., 1984a,b,c, 1985a,b; Fernandez-Palacios et al., ´ 1995., being higher than those determined for juveniles which range between 0.5% and 0.8% n y 3 HUFA in diet ŽIzquierdo, 1996.. These values are higher than the optimum essential fatty acid levels determined for salmonids which are approximately around 1% n y 3 HUFA ŽWatanabe, 1990.. Free radicals are able to deteriorate egg membranes and membrane integrity. Vitamin E, vitamin C and carotenoids Že.g. astaxanthin., are strong scavengers of active oxygen species and have been shown to have a protective role against the action of free radicals. Although the negative effects of vitamin E deficiency on the reproductive performance of higher vertebrates have been demonstrated since the early 1920s, dietary vitamin E was only shown to be an important nutrient for fish reproduction in 1990, with its deficiency resulting in immature gonads in carp and ayu, and reducing hatching rates and fry survival in ayu ŽWatanabe, 1990.. Increased levels of dietary vitamin E Žup to 2000 mgrkg. in red seabream diets improved percentages of buoyant eggs, hatching rates and percentage of normal larvae ŽWatanabe et al., 1991a.. An increase in the level of dietary a-tocopherol from 22 to 125 mgrkg also significantly reduced the percentage of abnormal gilthead seabream eggs ŽFernandez-Palacios et al., 1997. and resulted in an ´ improvement in the percentage of normal eggs ŽFig. 1; authors’ unpublished data.. The lowest fertility and larval survival rate was reported in eggs from broodstock fed the lowest dietary levels of a-tocopherol. The function of vitamin E as an inter- and intra-cellular antioxidant to maintain homeostasis of labile metabolites in the cell and tissue plasma is well known. In diabetic rats, supplementation of vitamin E in maternal diets also reduces congenital malformations, increasing tocopherol concentrations in maternal, embryonic and fetal tissues ŽSiman and Erikkson, 1997.. In gilthead seabream, a vitamin E level of 250 mg a-tocopherolrkg diet is sufficient to meet the requirements 32 M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 for successful reproduction, however, Hemre et al. Ž1994. have suggested this level is suboptimal for turbot broodstock. The carotenoid content of broodstock diets has also been reported to be important for normal development of fish embryo and larvae. However, for over 50 years there has been a great deal of controversy concerning the relationship between egg carotenoid content and egg quality in salmonids. For a review of carotenoid nutrition and functions Žincluding their effect on egg quality. see Tacon Ž1981., Craik Ž1985., Choubert Ž1986., Torrissen Ž1990. and Torrissen and Christiansen Ž1995.. Reports on the effect of carotenoid egg concentration on egg quality in salmonids have been contradictory. Some authors have reported a positive relationship between egg pigmentation and fertilization as well as survival rates of rainbow trout eggs ŽHarris, 1984; Craik, 1985., whereas others have not observed this response ŽTorrissen, 1984; Craik and Harvey, 1986; Torrissen and Christiansen, 1995.. Differences in the methodology employed by the different authors include age of broodstock, differences in egg carotenoid contents, differences in carotenoids Žastaxanthin, canthaxanthin, etc.. included in the diet or determined in the egg, sample size and even differences in criteria used to determine egg quality. Very few studies have been conducted controlling the level of dietary carotenoid supplied in broodstock diets ŽHarris, 1984; Choubert and Blanc, 1993; Watanabe and Kiron, 1995.. The addition of purified astaxanthin to broodstock diets for red seabream was found to clearly improve the percentage of buoyant and hatched eggs, as well as the percentage of normal larvae ŽWatanabe and Kiron, 1995.. By contrast, the inclusion of b-carotene had no effect on these parameters. Miki et al. Ž1984. have demonstrated the incorporation of dietary canthaxanthin or astaxanthin into red seabream eggs and the absence of the conversion of these carotenoids into b-carotene. It is possible that a lower intestinal absorption of b-carotene compared with that of canthaxanthin or astaxanthin may have affected these results. A preferential absorption and deposition of hydroxy and keto carotenoids was reported in fish by Torrissen and Christiansen Ž1995.. Carotenoids constitute one of the most important pigment classes in fish, with a wide variety of functions including protection from adverse lighting conditions, a provitamin A source, chemotaxis of spermatozoa and antioxidant functions including singlet oxygen quenching. The survival of embryo has also been shown to be affected by the vitamin C content of broodstock diets. This vitamin is necessary for the synthesis of collagen during embryo development. In rainbow trout Ž O. mykiss . broodstock, the requirement for vitamin C was up to eight times higher than that of juveniles ŽBlom and Dabrowski, 1995., although a much lower requirement for ascorbic acid has been reported in broodstock diets for cod ŽMangor-Jensen et al., 1993.. Other investigations with red seabream have shown that dietary phospholipids also improve egg quality ŽWatanabe et al., 1991a,b.. Although the beneficial effects of phospholipids have been attributed to their quencher activity and ability in stabilizing free radicals ŽWatanabe and Kiron, 1995., in some fish species they are important during larval development being preferentially catabolized after hatching and prior to first feeding ŽRainuzzo et al., 1997.. Despite the fact that little is known about the vitamin A requirement during gonadal maturation and spawning, it is considered important for embryo and larval development M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 33 due to its important role in bone development, retina formation and differentiation of immune cells. An increased retinol concentration in liver of turbot has been observed during the gonad maturation as the length of the day increased, whereas retinol content in gonads was reduced during maturation ŽHemre et al., 1994.. Other dietary nutrients which have been found to affect the reproductive performance of marine fish include dietary protein intake. For example, a low protein–high calorie diet caused a reduction in red seabream reproductive performance ŽWatanabe et al., 1984d.. In another sparid, gilthead seabream, a broodstock diet well balanced in essential amino acids, improved vitellogenin synthesis ŽTandler et al., 1995.. Moreover, reduction of dietary protein levels from 51% to 34% together with an increase in dietary carbohydrate levels from 10% to 32% reportedly reduced egg viability in seabass ŽCerdá et al., 1994b.. These diets have been shown to cause alterations in GnRH release in seabass broodstock during spawning ŽKah et al., 1994. and plasma hormonal levels of the gonadotropin GtH II, the latter known to play an important role in oocyte maturation and ovulation ŽNavas et al., 1996.. Further research into the broodstock requirements for thiamin Žvitamin B1 . is needed since there is evidence of its importance for normal embryo and larval development at least in salmonids. For instance, thiamin injection into gravid Atlantic salmon female reduces mortality of progeny ŽKetola et al., 1998.. Also egg or sac fry thiamin concentration is related to reduction of early mortality syndrome in feral lake trout ŽBrown et al., 1998. and Pacific ŽHornung et al., 1998. and Atlantic salmon ŽWooster and Bowser, 2000.. Research should also be directed to establishing the requirement of pyridoxine Žvitamin B 6 . in broodstock diets. Vitamin B 6 is known to be important in the synthesis of steroid hormones and folic acid since its deficiency may result in reduced cell division due to impaired synthesis of DNA and RNA and it has a role in hatchability of eggs ŽHalver, 1989.. Unfortunately, there is no information available on the effect of other B vitamins on fish reproduction. 6. Effects of broodstock nutrition on larval quality Few studies have been able to show the improvement of seed quality through implementation of broodstock nutrition. Increasing lipid levels from 12% to 18% in broodstock rabbitfish produced large newly hatched larvae and an increase in survival 14 days after hatching ŽDuray et al., 1994.. Increased n y 3 HUFA Žparticularly docosahexaenoic acid. levels in broodstock diets were reported to significantly enhance the weight of fish larvae and their resistance to osmotic shock ŽAby-ayad et al., 1997.. In a similar way, increasing n y 3 HUFA levels in broodstock diets for gilthead seabream significantly improved the percentage of live larvae after yolk reabsorption. Moreover, growth, survival and swimbladder inflation in gilthead seabream larvae were improved when fish oil was used instead of soybean oil in broodstock diets ŽTandler et al., 1995.. However, excessive levels of n y 3 HUFA in broodstock diets Žover 2%. caused yolk sac hypertrophy in gilthead seabream larvae and a decrease in larval survival rates ŽFig. 2; Fernandez-Palacios et al., 1995.. This is probably associated with ´ 34 M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 Fig. 2. Effect of increased dietary ny3 HUFA levels at constant vitamin E levels Ž125 mgrkg dry diet. on egg quality and larval survival of gilthead seabream. an increase in antioxidant nutrient requirement since an increase in dietary a-tocopherol levels from 125 to 190 mgrkg prevented the appearance of yolk sac hypertrophy and larval mortality ŽFig. 3; Fernandez-Palacios et al., 1998.. ´ 7. Timing of broodstock nutrition In some fish species such as gilthead seabream or red seabream, egg composition is readily affected by the diet within a few weeks of feeding ŽWatanabe et al., 1985b; Fig. 3. Combined effect of dietary ny3 HUFA and vitamin E levels on egg viability, fertilization and larval survival of gilthead seabream. M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 35 Fig. 4. Effects of low a-tocopherol diet on reproductive performance Žfecundity: no. eggsrkg femalerspawn; viable eggs: % total eggs produced. of gilthead seabream. Fernandez-Palacios et al., 1995; Tandler et al., 1995; Fig. 4 Žauthors’ unpublished data... ´ In these species, which are continuous spawners with short vitellogenetic periods, it is possible to improve spawning quality by modification of the nutritional quality of broodstock diets even during the spawning season ŽFernandez-Palacios et al., 1995, ´ 1997, 1998; Tandler et al., 1995.. Similarly, it is possible to improve egg quality and hatching rates of seabass by feeding broodstock with appropriate amounts of HUFA during the vitellogenetic period which is slightly longer than that of sparids ŽNavas et al., 1997.. In batch spawners with up to 6 months of vitellogenesis ŽFremont et al., ´ 1984., such as in salmonids, broodstock must be fed a good quality diet for several months before the spawning season to improve their reproductive performance ŽWatanabe et al., 1984d; Corraze et al., 1993.. Although fatty acid profiles of fish muscle and developing eggs of coho salmon ŽHardy et al., 1990. reflect dietary fatty acid profiles only after 2 months of feeding, Harel et al. Ž1992. have shown that the tissue lipid composition of gilthead seabream broodstock reaches an equilibrium with the dietary lipids after only 15 days of feeding. Turbot could be an exception to this observation because it is important to feed broodstock with high nutritional quality diets during vitellogenesis and spawning periods. The composition of turbot ovaries is more readily affected by the diet during the early stages of gonadal development ŽLie et al., 1993.. 8. Valuable ingredients for broodstock diets Several feedstuffs have been recognized as highly valuable for broodstock nutrition. In gilthead seabream, when broodstock were fed a minced cuttlefish diet or a commer- 36 M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 cial diet with additional minced cuttlefish, a close relationship between the lipids and fatty acid composition of broodstock diets and eggs was found ŽMourente and Odriozola, 1990.. Some authors have suggested that cuttlefish ŽMourente et al., 1989. and squid meal ŽZohar et al., 1995. contain nutritional components which are essential for successful spawning in gilthead seabream. Mourente et al. Ž1989. related this beneficial effect to the high content of EFAs in cuttlefish. However, Watanabe et al. Ž1984a. suggested that the high dietary value of cuttlefish meal was mainly due to the fat-insoluble fraction of the meal. Fernandez-Palacios et al. Ž1997. conducted an ´ experiment to identify the components of squid meal that improve egg quality ŽFernandez-Palacios et al., 1997.. Gilthead seabream broodstock were fed diets based on ´ fish meal, squid meal, defatted fish meal with squid oil or defatted squid meal with fish oil. These authors showed an improvement in egg quality when broodstock were fed the fat-insoluble fraction of squid meal in terms of total number of eggs produced daily Žper kilogram of female. and percentages of viable and fertilized eggs. Squid meal protein, a major component of the fat-insoluble fraction, was reported as having a beneficial effect on egg quality ŽFernandez-Palacios et al., 1997.. Since the amino acid profiles were very ´ similar among the diets used during the study, the superior nutritional value of the squid protein-based diets may have been related to the higher protein digestibility by gilthead seabream ŽFernandez-Palacios et al., 1997.. In fact, slightly higher protein levels were ´ found in the eggs from broodstock fed squid protein-based diets and they also produced about 40% more eggsrkgrfemale than fed fish meal-based diets. Watanabe et al. Ž1991a. reported that high calcium content of fishmeal does not cause a poorer spawning when compared with squid meal. They found that the addition of calcium to a cuttlefish meal-based diet did not affect egg quality of red seabeam. Increased egg production and viability was also observed by Watanabe et al. Ž1984a,b. when red seabream were fed with a cuttlefish meal-based diet. Moreover, the replacement of 50% of the fish meal with cuttlefish meal ŽWatanabe et al., 1984b. resulted in improved egg viability, although the number of eggs produced per female was not affected. The replacement of protein or lipid extracted from squid meal with protein or lipid extracted from soybean meal in diets for gilthead seabream broodstock caused a reduction in hatching and 3-day-old larval survival rates ŽZohar et al., 1995.. This could have been due to the beneficial effect of squid meal or to the detrimental effect of soybean meal. Although it has been shown ŽRobaina et al., 1995. that soybean protein is a potential protein source for partial substitution of fishmeal in gilthead seabream diets, it contains several antinutritional factors that limit the use of this protein supplement. Moreover, an imbalanced fatty acid composition, in terms of high n y 6 polyunsaturated fatty acids and low n y 3 fatty acids together with a lower phosphorous availability ŽRobaina et al., 1995. in broodstock diets based on soybean meal, could also directly reduce spawning quality as both nutrients are essential for reproduction in sparids ŽWatanabe et al., 1984a; Watanabe and Kiron, 1995.. Another feed ingredient, often included in the practical diets for sparids, is raw krill that has a distinct quality of having an enhancing effect on feed intake compared with fishmeal. For example, viable offspring production in red seabream, in terms of the percentage of buoyant eggs, total hatch and normal larvae, was more than doubled when krill was included in broodstock diets ŽWatanabe and Kiron, 1995.. Studies by Watan- M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 37 abe et al. Ž1991a,b. investigating the spawning quality enhancement effect of raw krill has shown that both polar and nonpolar lipid fractions contain important nutritional components for red seabream broodstock. They attributed this positive effect to the presence of phosphatidyl choline and astaxanthin from the polar and nonpolar fractions, respectively. Despite the importance of krill as a spawn quality-enhancing factor for red seabream, there is little published information concerning the nutritional value of raw krill, or its components, for broodstock of other sparids. Recently, yellowtail broodstock fed soft dry pellet diets without krill meal did not show a reduced spawning quality compared with fish fed diets supplemented with 10% krill meal ŽVerakunpiriya et al., 1997.. In addition, a further increase in the krill meal content up to 20% and 30% caused a clear reduction in egg quality which was associated with high levels of astaxantin ŽVerakunpiriya et al., 1997.. 9. Broodstock feeding practices At present, for most cultured fish species, the commercially available so-called Abroodstock dietsB are just larger sized Aon-growingB diets. In practice, many marine fish hatcheries improve the nutrition of their broodstock by feeding them solely on fresh marine by-products or in combination with commercial diets. The most common fresh marine organisms used to feed broodstock fish include squid, cuttlefish, mussels, krill and small crustaceans. The use of these unprocessed fish products often does not provide adequate levels of nutrients needed by broodstock fish and it increases the risk of disease transmission to the parents and offspring including endo and ecto parasites, and bacterial and viral pathogens, etc. The nutritional quality of formulated feeds can be effectively improved. For example, an increase in the levels of dietary n y 3 HUFA up to 2%, with an a-tocopherol content up to 250 mgrkg, and the inclusion of squid meal rather than fishmeal, was found to increase larval production three-fold compared with fish fed commercial diets. Larval quality in terms of growth, survival and swim bladder inflation was also improved ŽTandler et al., 1995.. These changes would increase feed production costs, which would be even higher if diets are developed for each species. It remains to be seen if the manufacture of broodstock diets, specifically for individual fish species of commercial aquaculture potential, would be worth pursuing. However, the benefits of improving the survival and thus increasing the production of marine fish larvae will have far reaching economic return than the initial cost of feeding the broodstock feed. 10. Concluding remarks In summary, information on the nutrient requirements of broodstock fish is limited to a few species. Certain nutrients such as essential fatty acids and antioxidant nutrients have been shown to be particularly important in broodstock nutrition. Their requirements 38 M.S. Izquierdo et al.r Aquaculture 197 (2001) 25–42 during reproduction is higher than those of juveniles, but excess amounts of nutrients or an imbalance can be detrimental for reproduction. Some minerals, such as phosphorous, and other nutritional aspects, such as protein quality, are also known to be important for fish reproduction. The importance of many other nutrients such as vitamin A, vitamin B 6 and folic acid has not yet been established within broodstock feeds and deserve future research. Future Ain vitroB studies may provide clues to function and certain unexplained biochemical mechanisms of certain micronutrients in reproduction of fish; however, these studies should complement Ain vivoB research rather than substitute it. 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