Introduction
The flathead grey mullet represents a potential candidate in the diversification of European aquaculture products mainly in the Mediterranean area. However, flathead grey mullet broodstock held in intensive conditions show reproductive dysfunctions; females remain arrested at previtellogenesis (Ramos-Júdez et al., 2021) or early stages of vitellogenesis (Aizen et al., 2005) and males with fluent milt are not usually observed (Aizen et al., 2005; De Monbrison et al., 1997; Ramos-Júdez et al., 2021). In a previous study, the application of a long-term treatment with species-specific single-chain recombinant gonadotropins (rGths), recombinant follicle-stimulating (rFsh) and luteinizing (rLh) hormones, produced in CHO cells, and the use of rLh and Progesterone (P4) at the later maturation stage was successful for obtaining eggs from females arrested at early stages of gametogenesis and sperm in males with no running milt that were used for in vitro fertilization. Nevertheless, low fertilization percentages (< 1%) were obtained (Ramos-Júdez et al., 2021). The present study aimed to demonstrate that long-term treatment with rFsh and rLh can result in eggs with higher fertilisation rates than the previously described.
Materials and Methods
The flathead grey mullet broodstock (mixed wild and from semi-extensive culture held for 1.5 to 3.5 years in intensive conditions) remained under natural light with 36 ‰ salinity water at 24ºC in one 10m³ tank in recirculation (IRTAMar®) during the experiment, timed to be in the natural spawning season in summer. Maturity was determined from ovarian biopsies and ease to obtain sperm. A total 21 of females and 9 males received weekly treatment with species-specific single-chain recombinant gonadotropins (rGths) while 9 control females and 6 control males received weekly saline injections. The pattern of application of rFsh and rLh aimed to mimic the physiological fluctuations of Fsh and Lh; rFsh during first stages of gametogenesis followed by a subsequent decrease with an increase of rLh to regulate late gametogenesis (Lubzens et al., 2010; Schulz et al., 2010). Doses ranged from 6 to 12 µg kg-1 for rFsh and from 2.5 to 12 µg kg-1 for rLh. Females with completed vitellogenic growth (oocytes near to 600 µm in diameter) and males with running milt were selected for spawning induction. The individuals were stocked in a separate 10m3 tank per treatment with the same conditions than the holding tank. To induce maturation, ovulation and spawning, females were treated with either (i) a priming dose of 30 µg kg-1 of rLh and a resolving dose of 40 mg kg-1 of P4 as in Ramos-Júdez et al. 2021, (ii) priming and resolving doses of 30 µg kg-1 of rLh, or (iii) priming and resolving doses of 40 mg kg-1 of P4 given 24:05 ± 0:40 h apart. One male from each spawning tank received a dose of 24 µg kg-1 of rLh while the others followed the weekly treatment. Spawning events had a sex ratio of 1:2 or 1:3 (female:male). Eggs were collected by surface out-flow egg collectors. Fertilization and hatching were examined and larvae survival was monitored in 96-well plates at 21 ºC until all hatched larvae had died.
Results
At the beginning of the experiment, 12 of the females (57%) belonging to the treated group were in previtellogenesis and 9 (43 %) in early vitellogenesis. All these females (100% of n = 21) completed vitellogenic growth with 603 ± 8 µm diameter of the most developed oocytes after 6 to 13 weeks of rGths treatment. Fertilized spawns were obtained after inducing with rLh + P4 or rLh + rLh (priming and resolving injections) with a spawning success of the 85% (8 of 9 attempted) and 100% (n = 6), respectively. All treated males were spermiating. The eggs obtained after both treatments presented 64 ± 22 % embryo survival and 58 ± 25 % hatching percentages. The mean fecundity of the females with fertilized spawns was 1,245,600 ± 552,117 eggs kg-1 bw (~ 1,700,000 eggs female-1). The percentage survival of larvae in 96-well plates was 67 ± 18 % at 9 days post hatching. The treatment P4 + P4 for maturation induction had a lower ovulation success (50%) and spawning success (17%) with no fertilized eggs. Success was independent of the initial gonadal stage of females (previtellogenesis or early vitellogenesis). In comparison, control females did not show any advance in gonadal development from initial stages (remained at previtellogenesis or developed atresia in some of the early vitellogenic oocytes) and control males did not produce fluent sperm.
Discussion and Conclusion
The present study indicates that oogenesis from previtellogenesis until spawning can be driven by the application of exogenous species-specific rFsh and rLh in a teleost species, as 100% of flathead grey mullet females completed vitellogenic growth, and maturation, ovulation and spawning were successfully achieved with the application of rLh as priming and resolving injections. The use of single-chain recombinant gonadotropins produced in CHO cells as a treatment to induce oogenesis from previtellogenesis or stimulate vitellogenesis to the completion of oocyte growth with high spawning success and egg quality as described in the present study offers reliability and replicability to control reproduction of the flathead grey mullet. The obtention of tank spawning contrasts with the study by Ramos-Júdez et al. (2021) in which eggs were hand stripped as no spawning was observed. The presence of good quality males with very fluent sperm in the tank in the present study may have played an important role in the stimulation of spawning and the spawning success. The fecundities and larval survivals obtained in the present study from flathead grey mullet females, indicate that the induction of 6 - 7 females (~1 kg) per season could enable a hatchery production of ~ 1 million fry. In addition, it would be possible to apply the present protocol to develop out-of-season breeding programs in this species.
Acknowledgements: Thanks to Cristian Martínez Rodríguez and the IRTA staff for technical help. This study was funded by the Spanish Government, MINECO, project: RTI2018-094710-R-I00
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