Ramos-Judez, S. | bioRxiv Science

bioRxiv2020

Low sperm to egg ratio required for successful in vitro fertilisation in a pair-spawning teleost, Senegalese sole (Solea senegalensis)

Cultured Senegalese sole (Solea senegalensis) breeders fail to spawn fertilised eggs and this bottleneck could be solved with the implementation of large-scale in vitro fertilisation protocols. However, low production of poor-quality sperm has frustrated the development of in vitro fertilisation protocols. Cultured females were induced to ovulate with a 5 {micro}g kg-1 single injection of gonadotropin releasing hormone agonist (GnRHa) and good quality eggs (82.6 {+/-} 9.2% fertilisation) were stripped 41:57 {+/-} 1:46 h after the injection. Sperm was collected from cultured males, diluted in modified Leibovitz and used fresh to fertilise the eggs. A non-linear regression, an exponential rise to a maximum (R = 0.93, P < 0.0001) described the number of motile spermatozoa required to fertilise a viable egg and 1617 motile spermatozoa were sufficient to fertilise 99 {+/-} 12% ({+/-} 95% CI) of viable eggs. Similar, spermatozoa egg-1 ratios of 592 {+/-} 611 motile spermatozoa egg-1 were used in large-scale in vitro fertilisations with 190,512 {+/-} 38,471 eggs. The sperm from a single male (145 {+/-} 50 {micro}L or 8.0 {+/-} 6.8 x 108 spermatozoa) was used to fertilise the eggs. The mean hatching rate of the large-scale in vitro fertilisations was 70 {+/-} 14 % to provide 131,540 {+/-} 34,448 larvae per fertilisation. When unfertilised eggs were stored at room temperature the percentage of viable eggs decreased gradually and indicated the sooner eggs were fertilised after stripping the higher the viability of the eggs. The collection of sperm directly into a syringe containing modified Leibovitz significantly increased the percentage of motile spermatozoa (33.4 {+/-} 12.2 %) compared to dilution in modified Leibovitz immediately after collection (6.6 {+/-} 4.9 %). Senegalese sole have a pair-spawning reproductive behaviour characterised by external gamete fertilisation in close proximity with no sperm competition. The low spermatozoa egg-1 ratio required for maximum fertilisation was consistent with this reproductive behaviour and strategy. The provision of a large-scale in vitro fertilisation protocol (200 {micro}L of sperm per 100 mL of eggs) will enable the industry to operate sustainably and implement breeding programs to improve production.

physiology↗

bioRxiv2020

Providing recombinant gonadotropin-based therapies that induce complete gametogenesis and produce viable larvae from an immature teleost, flathead grey mullet (Mugil cephalus)

Under intensive captive conditions, wild-caught flathead grey mullet (Mugil cephalus) females remained arrested at early gonad development and no sperm could be obtained from males. With the aim to induce and complete oogenesis, induce the release of sperm and obtain fertilized eggs, adult female and male grey mullet were treated with M. cephalus single-chain recombinant gonadotropins (rGths), follicle-stimulating (rFsh) and luteinizing (rLh) hormones. In Experiment 1, fish were treated with a weekly dose of rFsh (15 g kg-1), which in females significantly (P < 0.001) increased plasma concentration of 17{beta}-estradiol and induced vitellogenic oocyte growth up to a maximum mean diameter of 425 {+/-} 19 m after 9 weeks of treatment. In Experiment 2, fish were treated with weekly injections of both rFsh and rLh at different doses (from 2.5 to 12 g kg-1). Oocyte diameter reached 609 {+/-} 5 m, from which final oocyte maturation and ovulation was induced with 30 g kg-1 of rLh and 40 mg kg-1 of progesterone. Good quality sperm (> 75% motile spermatozoa) was obtained from males in both experiments, and in Exp. 2 the addition of rLh induced the production of higher quantities of sperm that were used to fertilise the eggs. Although fertilisation was low (0.4 %), these fertilized eggs with embryo development produced viable larvae (71% hatching rate). This is the first report in a teleost species, to obtain larvae from eggs that were from immature females induced through to maturation with rGths. In comparison, control females remained arrested as immature fish and control males did not produce sperm. The study demonstrated that both rGths are effective to induce the entire process of oogenesis in sexually immature female grey mullet and to obtain flowing sperm from males, adding more data to confirm the roles of the Gths in teleost gametogenesis. This advance provides the bases of a therapy for the use in the aquaculture of teleost of commercial interest or the conservation of endangered species.

physiology↗