J.F. Asturiano

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.

Effects of Polyunsaturated Fatty Acids and Prostaglandins on Oocyte Maturation in a Marine Teleost, the European Sea Bass (Dicentrarchus labrax)

Abstract The effects of the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and prostaglandins (PGs) on oocyte maturation were investigated in a marine teleost, the sea bass (Dicentrarchus labrax). Follicle-enclosed postvitellogenic, preovulatory oocytes were cultured in vitro and maturation was verified by assessing volume increase, lipid droplet coalescence, yolk clarification, and germinal vesicle migration and breakdown. Human chorionic gonadotropin was administered as the maturation-inducing gonadotropin (GTH) and was capable of inducing maturation in a time- and dose-dependent manner. Free AA induced maturation in a dose- and time-dependent manner and enhanced GTH-induced maturation, while EPA, DHA, and oleic acid were ineffective. Maturation induced by GTH was significantly suppressed by a phospholipase A2 blocker, suggesting that mobilization of AA was involved in GTH-induced maturation. Moreover, EPA and DHA exhibited a significant, dose-dependent attenuation of GTH-induced maturation. Maturation induced by GTH was inhibited in the presence of a cyclooxygenase inhibitor, indomethacin, and this inhibition was reversed by addition of AA, PGE2, or PGF2α. PGE2 and PGF2α alone were both effective stimulators of maturation, while PGE1 and PGE3 were ineffective. The effect of PUFAs on oocyte maturation in vitro were corroborated with studies in vivo. Oocytes were obtained from females fed a commercial, PUFA-enriched diet (RD) and maturational behavior was compared with oocytes from females fed a natural diet (ND) with a higher EPA content and n-3:n-6 ratio. Although no significant difference was observed in the rate of spontaneous oocyte maturation, a higher percentage of GTH-induced maturation and lower percentage of atresia were observed in RD oocytes. Moreover, while basal PGE production from oocytes from both groups was the same, RD oocytes produced significantly higher levels of PGE in the presence of hCG. The results from this study provide evidence for the participation of AA metabolism in GTH-induced oocyte maturation, and suggest that other PUFAs and PGs may play important roles in the induction of maturation in a marine teleost.

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.

Molecular and physiological study of the artificial maturation process in European eel males: From brain to testis

European eel males can be artificially matured (1.5IU hCG/g fish), but the regulatory mechanisms of their reproductive development are practically unknown. Spermatogenic stages (S1-S6), biometric characters [eye index (EI), gonadosomatic index (GSI), hepatosomatic index (HSI)] and sperm quality parameters (motility, viability and head spermatozoa morphometry) were analysed. Moreover, the present study evaluated the expression of GnRHs (mammal and chicken II Gonadotropin Release Hormone I) and gonadotrophins (FSHbeta and LHbeta) during hormonal treatment, as well as 11-ketotestosterone (11-KT) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) plasma levels. One week was enough to observe the S2 of gonad development, but it was necessary to reach the 7th week of treatment to obtain animals that presented the most advanced stage of development (S6). Differential regulation of the two GnRH expressions was found, supporting the main role of mGnRH in the control of gonadotrophin release. One hCG injection was enough to dramatically decrease the FSHbeta expression, being close to zero during the rest of the treatment. LHbeta expression and 17,20beta-P registered a significant increase in the same stage of development, S3/4, confirming the role of this gonadotrophin in the last steps of maturation and 17,20beta-P in the spermatozoa maturation. The 11-KT increased with GSI, and the highest 11-KT values coincided with the advanced steps of spermatogenesis prior to spermiation. Being consistent with the known role of the steroid in these processes. Furthermore, this study supports a role for 11-KT in stimulating eye growth, presenting high values when EI increased. Sperm production was obtained from the 4th week of treatment, but it was in the 8th week when a significant increase was observed in sperm quality [viability, high motility (>75%)].

Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females

We examined the effect of temperature in European silver eels during their maturation induced by injections of carp pituitary extract on endocrine parameters: pituitary fshβ and lhβ expression, plasma 17β-estradiol (E2) and vitellogenin, estrogen receptor 1 (esr1), and vitellogenin 2 (vtg2) expression in liver. A variable thermal regime (T10) that increased from 10° to 14° and 17°C was compared with a constant 20°C regime (T20) during 12 weeks. T10 caused a faster development until week 8, higher fshβ, lhβ, esr1 expression, and higher E2 levels. The results strongly suggest that T10 is inducing a higher endogenous FSH level which increases the E2 circulating level during vitellogenesis. A variable thermal regime induced an fshβ expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.

Ionic composition and physio-chemical parameters of the European eel (Anguilla anguilla) seminal plasma

Looking for good sperm diluting media, physio-chemical parameters and ionic composition of the eel seminal plasma were studied in relation with sperm motility.

Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software

The development of powerful computer-assisted sperm analysis software has made kinetic studies of spermatozoa possible. This system has been used and validated for several species, but some technical questions have emerged regarding fish sample evaluations (i.e., frame rate, sperm dilution, chamber model, time of analysis, magnification lens, etc.). In the present study, we have evaluated the effects of different procedural and biological settings with the aim to correctly measure sperm quality parameters of the European eel. The use of different chambers did not affect the sperm motility parameters. However, regarding lens magnification, 10× was the most accurate lens, showing the least variation in the acquired data. Similarly, the frame rate setting resulted in a dramatic effect in some sperm kinetic parameters, primarily in terms of curvilinear velocity; we therefore recommend using the cameras highest available frame rate setting. Finally, the reduction in sperm motility over postactivation times suggests that sperm analysis should be performed within the first 60 seconds after activation of the European eel sperm. In conclusion, some protocol variables of sperm analysis by computer-assisted sperm analysis software can affect the measurement of eel sperm quality parameters, and should be considered before directly comparing results obtained by different laboratories. Moreover, because marine fish species show relatively similar features of sperm kinetic parameters, these results could be considered in the evaluation of the motility of sperm from other fish species.

Improvement of European eel sperm cryopreservation method by preventing spermatozoa movement activation caused by cryoprotectants.

Sperm production has been obtained from European and Japanese eels, but its quality and quantity tend to be changeable. So, its cryopreservation has been tried in both species. Dimethyl sulfoxide (Me(2)SO) is the best cryoprotectant for European eel sperm, but increases the medium osmolality, inducing the activation of spermatozoa motility. To avoid this, different combinations of pH (6.5 and 8.5) and NaHCO(3) concentrations (20, 40 and 80mM) were tested with two Me(2)SO concentrations (5% and 10%). Foetal bovine serum (FBS, 25%v/v) was added as a membrane protector to all the freezing media used in the different experiments. The highest Me(2)SO and NaHCO(3) concentrations at pH 6.5 caused the best post-thawing motility (26+/-4%). A second experiment was carried out testing media with Me(2)SO 10% with additional NaHCO(3) concentrations (100 and 120 mM). The highest post-thawing motility (38+/-3%) was found in the media containing NaHCO(3) 100mM, but no significant difference was observed compared with the best in the previous experiment (NaHCO(3) 80 mM). In a parallel experiment, aiming to improve the protection against the cryopreservation process, bovine serum albumin (BSA, 5%w/v) was added instead of FBS. Lower motilities were registered with BSA as membrane protector. Spermatozoa activation caused by addition of Me(2)SO can be prevented using high NaHCO(3) concentrations, improving the cryopreservation process. This effect seems be based on some of the products dissociated from NaHCO(3) in aqueous solution, affecting the intracellular pH, essential in the sperm motility.

Effects of pH, Sodium Bicarbonate, Cryoprotectants and Foetal Bovine Serum on the Cryopreservation of European Eel Sperm

The main objective of the present study was to evaluate the influence of pH and bicarbonate concentration in the activation or inhibition of European eel (Anguilla anguilla) spermatozoa and to evaluate the effect of different cryoprotectants: dimethyl sulphoxide (DMSO), acetamide, ethylene glycol, propanol, glycerol and methanol (MeOH). The effect of these factors was evaluated comparing the percentage of motile cells, the percentage of alive cells (by Hoechst staining) and the spermatozoa morphometry pre- and post-cryopreservation (by computer-assisted morphology analysis). Based on the above findings, three cryoprotectants (DMSO, MeOH and glycerol) were chosen and evaluated in two media (P1 and P1 modified) with different concentrations of NaHCO(3) and in the presence or absence of foetal bovine serum (FBS). The effect of these factors was evaluated comparing the percentage of alive and motile cells post-cryopreservation. DMSO was the cryoprotectant showing better results in relation to the percentage of spermatic alive cells post-freezing and caused a smaller modification of the head spermatozoa morphology. The combination of P1-modified medium with DMSO and containing FBS increased slightly but significantly the percentage of motile spermatozoa post-cryopreservation.

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen

There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90s), the thermal regimen (i.e. 10°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30s is recommended as the standard time for motility acquisition. When eels were kept at 20°C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8±1.3%; mean ± s.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0±1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time.