Jesús Ramos

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

Commercially fabricated diets allow greater control over the composition of biochemical components and reduce the risks of disease introduction, which are significant concerns when using the wet fish diets commonly used for most farmed marine broodstocks. However, satisfying the dietary lipid requirements of marine broodstock using artificial diets has proved difficult, particularly with respect to their highly unsaturated fatty acid (HUFA) composition. Two groups of mature sea bass, each divided between three replicated tanks, were fed two dry pelleted diets over a 2-year period, encompassing two spawning seasons. The first diet contained a good quality Northern Hemisphere meal and oil; the second differed only in the source of oil, which was substituted with tuna orbital oil (TOO). The use of TOO in the dry pelleted formulation allowed the manipulation of n−3 and n−6 HUFA in the resulting eggs, specifically arachidonic acid (20:4 n−6; AA), eicosapentaenoic acid (20:5 n−3; EPA) and docosahexaenoic acid (22:6 n−3; DHA). The results showed that dietary manipulation of these HUFA could improve levels and ratios of AA, EPA and DHA which were transferred to the resulting eggs with improvements in early survival and hatching success repeated over successive spawning seasons. The dry diet containing TOO facilitated comparable reproductive performance to the wet fish diet (Boops boops) which has previously been considered the most effective broodstock diet. The improvements in reproductive performance are discussed in relation to the proportion of these HUFA with respect to each other in total egg lipid and the phospholipid classes phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) and to their potential impact on eicosanoid formation. Finally, this study has shown that a commercially fabricated diet can be successfully used as sensitive investigative tool for aquaculture research.

Reproductive performance in male European sea bass (Dicentrarchus labrax, L.) fed two PUFA-enriched experimental diets: a comparison with males fed a wet diet

Abstract Reproductive performance of male European sea bass ( Dicentrarchus labrax ) fed a wet diet (WD) was compared to that of fish fed two commercial pelleted diet enriched with polyunsaturated fatty acids (PUFAs) by the use of Northern hemisphere fish oil (ST) or tuna orbital oil (RO). Broodstock growth, spermiation duration, milt production, milt spermatozoa density, sperm motility, milt lipid composition, and fertilization rates were compared during the reproductive season. RO and ST males exhibited longer spermiation periods producing statistically higher milt volumes and milt spermatozoa densities as compared to WD; no differences in quality or motility of sperm were observed between groups. Although the fertilization rates of RO, ST and WD milt at 3 and 24 h after fertilization were similar (88–90%), significantly higher rates of embryonic and larval survival were observed at 48 and 72 h after fertilization from eggs fertilized with ST (13.9% and 15.5%) and RO milt (20.9% and 20.6%) as compared to WD (1.0% and 1.2%). Analysis of milt PUFA profiles revealed several differences between groups. Although total PUFAs were increased in all groups as compared to diet PUFA composition, a greater increase was noted for ST and RO. In January and March, fish fed the WD exhibited more weight gain and attained significantly higher weights, respectively, than RO fish. Results showed that although fish fed the WD displayed increased weight gain, reproductive performance was enhanced in males fed the commercially fabricated diets possibly reflecting benefits of PUFA-enrichment.

Influence of nutritional composition of diet on sea bass, Dicentrarchus labrax L., reproductive performance and egg and larval quality

Abstract Two isocaloric formulated diets varying in protein and carbohydrate content (D1 = 51% (P)rotein, 13% (L)ipid, 10% (C)arbohydrate; D2 = 34% P, 14% L, 32% C) were fed to 2-year-old sea bass broodstock starting 6 months before spawning to determine their effects on reproductive performance and egg and larval survival. Fish were distributed in 8-m3 tanks, supplied with aerated running sea water (salinity 37.8‰, pH 8.3) and maintained under ambient photoperiod and temperature conditions. Before the experiment, fish were acclimated to the artificial diets by feeding D1 for 30 days. After this period, fish were fed D1 and D2 twice a day (1.5% of body weight per day). Fish fed D2, especially females, showed significantly diminished growth (weight, length and CF) and specific growth rates (P ≤ 0.05) over the first months. However, the dietary treatments did not have any effect on the histomorphology of ovarian development or gonad proximate composition. At spawning, D2 fish exhibited different reproductive performances compared to D1 fish. They did not have any peak in the production of eggs and showed a slightly extended spawning period. The mean number of eggs produced per female and the relative fecundity of fish given D2 diet were also appreciably lower than found for the D1 group. Although the egg proximate composition was the same regardless of dietary treatment, the eggs of the D2 fed fish had significantly lower buoyancy and hatchability (P ≤ 0.05) than those from D1 fish. In addition, more than 50% of hatched larvae from the D2 group showed body deformities. Taken together, these results indicate that the reproduction of sea bass and the survival of the progeny are greatly affected by the levels of energy-yielding nutrients in broodstock diets, and that a dietary protein level and a carbohydrate content of 32–33% have adverse consequences on sea bass reproduction. The sea bass dietary requirements to produce viable eggs seem also to be very specific since the hatching rates observed for both treatment groups were lower than those reported in other feeding trials in which natural diets were used.

Genetic, hormonal and environmental approaches for the control of reproduction in the European sea bass (Dicentrarchus labrax L.)

The European sea bass Dicentrarchus labrax is a very important commercial fish in Europe, the production of which by aquaculture has increased several fold during the last decades. Studies on biology, control of reproduction, broodstock management, offspring quality and rearing techniques, as well as large investments in the aquaculture sector, have made possible this rapid increase in production. However, under culture conditions, there are still important problems that negatively influence its industrial production. These are: (1) high percentage of males (70–90%), some of which are precocious at the first year of life; (2) in males, puberty typically appears in the second year of life (at commercialization), 1 year before females; and (3) males grow less than females, being 40% lower in body weight at harvest time. Consequently, reduction of the number of males and/or production of all-female stocks and sterile fish and prevention of puberty are highly desirable goals. The present work reviews the latest scientific advances on the genetic and physiological control of the sex and the process of puberty in the sea bass to provide tools to alleviate some shortcuts in its industrial production.

Vitellogenin, steroid plasma levels and spawning performance of cultured female Senegalese sole (Solea senegalensis).

The Senegalese sole (Solea senegalensis) is a high value market flatfish, which aquaculture is compromised by severe reproductive problems; these are mostly found in soles hatched and raised in captivity (F1 generation). To gain knowledge on the reproductive dysfunctions observed in cultured (F1) Senegalese sole, this work aimed at developing a specific vitellogenin (VTG) ELISA, for the measurement of plasma VTG levels in this species. Profiles of VTG were correlated with those of sexual steroids and spawning performance of an F1 broodstock, during three consecutive years. The Senegalese sole VTG (ssVTG) was purified by precipitation with MgCl(2)-EDTA and anion-exchange chromatography and showed a molecular mass of 172 kDa, by SDS-PAGE. Specific antibodies were obtained and used to develop a competitive ELISA, which had a sensitivity of 3.6 ng ml(-1), and inter- and intra-assay coefficients of variation of 9.5% (n=29) and 6.7% (n=12), respectively. Annual profiles of plasma VTG showed a major peak at pre-spawning, and a second minor rise around autumn, which mirrored plasma profiles of both estradiol (E(2)) and testosterone (T) levels. Spontaneous spawning occurred every year in the spring season, but no fertilized eggs were obtained. In conclusion, this study described, for the first time, the purification and development of a sensitive and specific ELISA for Senegalese sole VTG. The endocrine and spawning data suggested that F1 female broodstock showed normal VTG and steroid releasing profiles in captivity with occurrence of spontaneous spawning, but no fertilization of the eggs was recorded.

Effect of dietary lipid composition on vitellogenin, 17β-estradiol and gonadotropin plasma levels and spawning performance in captive sea bass (Dicentrarchus labrax L.)

Abstract The influence of dietary lipid composition on the reproductive performance of the sea bass ( Dicentrarchus labrax ) and on vitellogenin (VTG), 17 β -estradiol (E2) and gonadotropin II (GtH II) plasma levels has been investigated. The control group was fed with a natural diet consisting of trash fish ( Boops boops ). Two experimental groups were fed with pelleted diets containing different amounts of lipids: Group A was fed with a commercially available diet with a 10% lipid content and Group B fed the base diet enriched to a 22% lipid content using refined fish oil enriched with n −3 fatty acids. The control group exhibited higher egg viability and hatching rate than the experimental groups. The better spawning performance of the control group, with respect to the experimental groups, was associated with differences in endocrine profiles during the reproductive cycle. In the experimental groups, E2 levels were higher than in controls during the period of vitellogenesis. In the profiles of VTG levels, groups A and B exhibited a greater decrease of plasma VTG during the mid spawning time as compared to the control group. Profiles of plasma GtH II levels were determined for the first time in the sea bass and showed a single annual peak during the spawning period. At that time, GtH II levels from groups A and B were higher than in the controls. The present data suggest that dietary lipid composition significantly affects the reproductive performance of the sea bass.

Optimal conditions for the induction of triploidy in the sea bass (Dicentrarchus labrax L.)

Abstract The optimal conditions for the induction of triploidy in the European sea bass ( Dicentrarchus labrax L.) using cold shocks were investigated. Three treatment variables were considered: the time after fertilization when the shock was applied, the temperature of the shock and the duration of the shock. Ploidy was determined by counting the nuclear organizer regions, karyotype analysis and measures of erythrocyte size. A first series of experiments performed at what was subsequently shown to be a sub-optimal shock duration (5 min), demonstrated that the best time after fertilization for cold shock application was 5 min, regardless of temperature of shock (0 or 2 °C). The maximum percent of triploids obtained was 87% at survival rates approximately 90% of that of the untreated controls. In a second series of experiments, this timing (5 min after fertilization) was used to determine the best combination of temperature (0, 2 or 4 °C) and duration of shock (5, 10, 15 or 20 min) for optimal triploid induction. In these experiments, triploidy reached 100%, with survival approximately 80% of controls. However, when the shock temperature was above 0 °C and the duration shorter than 10 min, triploidy induction became highly variable. The optimum treatment parameters for the induction of triploidy in the sea bass were established to be as follows: time after fertilization, 5 min; duration of shock, 10 min; temperature of shock, 0 °C. Pre-shock incubation temperature was 12–13 °C in all cases. These conditions were used to mass-produce triploid sea bass. Results showed that under these conditions triploidy induction could reach 100%, although it was essential to reproduce optimum shock conditions, especially temperature, when dealing with large volumes of eggs. At 8 months of age, triploid sea bass had similar growth than their diploid counterparts. In conclusion, this paper presents the optimal conditions for triploidy induction in the sea bass using cold shock and indicates that it is possible to mass-produce triploids of this commercially important species.

Short- and long-term dietary effects on female sea bass (Dicentrarchus labrax): seasonal changes in plasma profiles of lipids and sex steroids in relation to reproduction

Abstract Sea bass were fed a natural diet (ND) (trash fish) or two commercial diets ( CD 1 = 56% (P)rotein, 11% (L)ipids; CD 2 = 47% P, 7% L) during two consecutive reproductive cycles in order to assess the effects on the reproductive performance. CD1 and CD2 had lower energetic and PUFA (polyunsaturated fatty acids) content, especially n -3 fatty acids, with respect to ND. During the first spawning, the levels of sex steriods (17β-estradiol [E2] and testosterone [T], the percentage of good quality (i.e. buoyant) eggs and the hatching rates were similar between the ND and CD1 groups. Females fed CD2 did not show significant variations in E2 or T, and had few spawnings, low egg quality and no egg survival. At second spawning, the fish fed the commercial diets had reduced fecundity and egg viability, together with lower levels of E2 and T, altered patterns of plasma lipids and early presence of atresia in the ovary, with respect to the females fed ND. The present study provides evidence for the importance of dietary lipid for the reproductive processes in the sea bass. The data suggest that long-term dietary deficiences in n -3 fatty acids may affect the patterns of plasma lipids and may induce early gonadal atresia. For example, follicular atresia could reduce the production of gonadal steroids, the fecundity and the subsequent egg survival.

Seasonal and daily plasma melatonin rhythms and reproduction in Senegal sole kept under natural photoperiod and natural or controlled water temperature.

Abstract:  The melatonin daily rhythm provides the organism with photoperiod‐related information and represents a mechanism to transduce information concerning time of day. In addition, the duration and amplitude of the nocturnal elevation gives information about duration and thus the time of year. In this study, we investigate the existence of an annual rhythm of plasma melatonin in the Senegal sole. Differences in plasma melatonin levels between fish kept at a controlled temperature (17–20°C) and those exposed to the environmental temperature cycle (11.5–25°C) were also examined throughout the year. Spawning was registered in both groups to determine the time of year in which reproductive rhythms occurred. Our results pointed to the existence of an annual rhythm of plasma melatonin at mid‐darkness (MD), with the highest levels (203 ± 44 pg/mL) observed when water temperature reached 25°C. Water temperature influenced nocturnal, but not diurnal melatonin. Daily melatonin rhythms showed seasonal differences, with higher mean nocturnal levels during the summer solstice (138 ± 19 pg/mL) and autumn equinox (149 ± 49 pg/mL). When animals were kept at a constant temperature throughout the year, plasma melatonin levels differed from those observed in fish exposed to the environmental temperature cycle. Regarding the reproductive rhythms, spawning was observed at the end of spring in sole kept under natural temperature conditions, whereas no spawning at all was registered in sole reared at a constant temperature. In short, both photoperiod and temperature affected melatonin production in the Senegal sole, transducing seasonal information and controlling annual reproductive rhythms.

Incubation of embryos and larvae of marine fish using microtiter plates

Egg quality can be defined using various parameters, but in the aquaculture industry it is defined as the potential of an egg to hatch into a viable larva (Kjorsvik et al., 1990; Brooks et al., 1997). The decision to stock a batch of eggs for production is firstly based on the fertilization % of the particular spawn, and then on the hatching % and survival to first feeding, i.e. when the mouth has opened (Bromage, 1995). When carrying out reproduction experiments, the ultimate success of the broodstock management or hormone treatment is determined by the production of good quality eggs. Evaluation of the fertilization % of different batches of eggs is easy, can be carried out within a few hours from spawning and does not require special facilities to keep the eggs. Evaluation of hatching % and survival to first feeding, however, requires maintaining the eggs for at least 5 d in the case of marine species such as the gilthead seabream (Sparus aurata) (Zohar et al., 1995) and the European seabass (Dicentrarchus labrax) (Carrillo et al., 1995). Maintaining a large number of egg batches produced from multi-factorial experiments in commercial-type incubators is unfeasible, due to space and cost constraints. Development of an incubation method which does not require extensive facilities will enable monitoring of egg quality from many different spawns or