Reproduction of cultured Senegalese sole (Solea senegalensis): Protocol for hormonal inducted ovulation, stripping of eggs and artificial fertilization

Abstract

The Senegalese sole, Solea senegalensis (Kaup, 1858), is one of the leading candidates for the diversification of the Southern European pisciculture industry. However, there are still several problems that restrict the consolidation of this species at an industrial level. One of the primary problems associated with culturing Senegalese sole is the extent of reproductive dysfunctions detected in individuals hatched and reared in captivity (generation F1). Spontaneous spawns are scarce and generally not fertilized. So far, it has not been able to close the farming cycle nor draw genetic improvement plans. The aim of this work was to set a protocol for an hormonal inducted ovulation with synthetic analogs of gonadotropin releasing hormone (GnRHa) and the artificial fertilization of eggs obtained by stripping. Females in stage III of ovarian development according to the classification of Anguis and Cañavate (2005) were selected for all experiments. First, the most suitable method of administration of the hormone was determined. To induce ovulation, two different methods, weekly injections versus a single GnRHa implant, were tested (Rasines et al., 2012). The number of spawns per female and the relative fecundity were significantly higher in females which received hormonal therapy through repeated injection, 6.3 ± 0.8 spawns per female and 574.9 ± 67.2 x 103 eggs kg -1 body weight, than those treated with a single implant, 2.7 ± 0.6 spawns per female and 134.6 ± 40.9 x 103 eggs kg -1 body weight. Second, the influence of temperature on the lapsed time to ovulation (number of hours between hormonal injection and ovulation) and changes in fertilization and hatching rates of eggs retained in the ovarian cavity after ovulation were determined. The lapsed time of ovulation was inversely correlated with temperature and was 53.2 ± 0.5, 43.4 ± 1.1 and 39.5 ± 0.7 h at 14 ºC, 16 ºC and 18 ºC respectively. At 14 ºC there were no significant differences in the fertilization and hatching rates from eggs retained in the ovarian cavity 0, 3 or 6 h. At 16 °C, the highest fertilization and hatching rates were obtained from the eggs retained 0 and 3 h, but at 6 h these rates decreased significantly. At 18 ºC the highest rates of fertilization and hatching were obtained from freshly ovulated eggs and decreased significantly at 3 and 6 h. These results indicated that temperature influence both the lapsed time to ovulation and the viability of eggs retained in the ovarian cavity and overriping in this species is a quick process, happening in a few hours. The next step was to determine whether the time of the day for the GnRHa injection influences the lapsed time to ovulation or egg quality. Hormonal treatments were administered at 06:00, 12:00 and 19:00 h (Rasines et al., 2013). The lapsed time to ovulation was 42.5 ± 0.6 h and there were no significant differences between groups. However, it was noted that the lapsed time to ovulation was the most homogeneous in Groups 06:00 and 12:00, and most females in these groups ovulated at 41-44 h post-injection. Fertilization rates were lower in fish injected at 19:00 h than those injected at 06:00 or 12:00 h, and the largest number of larvae per kg female was obtained when the treatment was administered at 06:00 h. For artificial fertilization, it is important to assess the quality of the eggs by using indicators that are quick and easy to measure. Three possible quality indicators, the percentage of apparent viability based on the external appearance of the eggs (transparency, lack of perivitelline space and homogeneous distribution of oil droplets), egg buoyancy and pH of the ovarian fluid were determined in 57 spawns, obtained by a standard protocol of hormonal induction of ovulation and stripping. These spawns were fertilized artificially and fertilization and hatching rates determined. Results showed no correlation between the egg quality parameters and the resulting fertilization and hatching rates, indicating that none of the examined parameters can be used as reliable egg quality indicators to predict the success of artificial fertilization. Moreover, to optimize the performance of broodstock, the number of hormone injections and the time interval between them were determined, in order to produce the greatest number of good quality eggs per female. Three GnRHa administration protocols were tested: (A) two injections with a 7 - day interval, 15 - day break and another two injections with a 7 - day interval, (B) two injections with a 7- day interval, one month break and another two injections with a 7 - day interval and, (C) four injections with a 15 - day interval. In treatment A there was response only to the first two injections, whereas females in treatments B and C responded to the four injections. The largest number of eggs per kg female was obtained with treatment B. There were no differences in fertilization rates by the serial number of injections. Another group of females was treated with injections on Days 0, 7 and 43 and showed that the quantity of stripped eggs and fertilization and hatching rates were similar in all three injections. Finally, to validate the method for obtaining F2 Senegalese sole by artificial fertilization, the obtained F2 larva were further followed through larval culture and ongrowing to market and broodstock size. The results indicated that growth of the F2 larvae and juveniles was similar to that of F1´s. Furthermore, the F2 broodstock exhibited similar reproductive problems than those of the F1 broodstocks. In conclusion, for a protocol of hormonal induction of ovulation and artificial fertilization in Senegalese sole, injection should be used as a method of GnRHa administration, the influence of temperature on the time of ovulation and egg viability must be taken into account and also the influence of the time of day of administration of the hormone. Furthermore, several hormonal inductions by female are possible and it is not possible to predict the success of artificial fertilization by external parameters such as morphology, buoyancy or ovarian fluid pH. The following protocol is proposed: hormonal induction of females in maturity gonadal estate E III, GnRHa injection (25 µg kg-1 body weight) in the morning, temperature 16 ºC and stripping of the eggs 41-44 h after treatment. Seven days after it is possible a second induction and after a one month break a new one. References Anguis, V., J. P. Cañavate. 2005. Spawning of captive Senegal sole (Solea senegalensis) under a naturally fluctuating temperature regime. Aquaculture 243, 133–145. Rasines, I., M. Gómez, I. Martín, C. Rodríguez, E. Mañanos, and O. Chereguini, 2012. Artificial fertilization of Senegalese sole (Solea senegalensis): Hormone therapy administration methods, timing of ovulation and viability of eggs retained in the ovarian cavity. Aquaculture 326-329: 129-135. Rasines, I., M. Gómez, I. Martín, C. Rodríguez, E. Mañanos, and O. Chereguini. 2013. Artificial fertilisation of cultured Senegalese sole (Solea senegalensis): Effects of the time of day of hormonal treatment on inducing ovulation. Aquaculture 392-395: 94-97. Acknowledgments This work was funded by the Spanish Ministry of Agriculture, Fisheries and Food (MAPA) (Projects JACUMAR 2006 and 2009, II and III National Plan for the Cultivation of Sole).