With the expansion and diversification of global aquaculture, efforts continue to develop new bio-technologies for assisted reproduction in species that present reproductive dysfunctions. Flathead grey mullet (Mugil cephalus) males held in intensive conditions in the Mediterranean region do not produce fluent milt and most females are arrested at previtellogenesis. The weekly injections of recombinant follicle stimulating hormone (rFsh) and luteinizing hormone (rLh) induced and completed vitellogenesis in treated females (n = 21), and treated males produced fluent sperm (n = 9). The application of a priming dose of 30 µg kg⁻¹ rLh and resolving dose of 40 mg kg⁻¹ Progesterone, or priming and resolving doses of 30 µg kg⁻¹ rLh, resulted in the induction of maturation, ovulation, and spontaneous spawns with a spawning success of the 85% (8 of 9 females) and 100% (n = 6), respectively. The eggs collected had 63 ± 21% fertilization with embryo development and 58 ± 23% hatching. In comparison, control individuals did not show advances in gonadal development and did not produce fluent sperm. The present results confirm the possibility of controlling oogenesis from previtellogenesis to the completion of maturation and fertilised tank spawning using exclusively rFsh and rLh in a teleost species.

Background: Treatment with recombinant gonadotropin hormones (rGths), follicle-stimulating hormone (rFsh) and luteinizing hormone (rLh), was shown to induce and complete vitellogenesis to finally obtain viable eggs and larvae in the flathead grey mullet (Mugil cephalus), a teleost arrested at early stages of gametogenesis in intensive captivity conditions. This study aimed to investigate the transcriptomic changes that occur in the ovary of females during the rGths-induced vitellogenesis.Methods: Ovarian samples were collected through biopsies from the same five females at four stages of ovarian development. RNASeq libraries were constructed for all stages studied, sequenced on an Illumina HiSeq4000, and a de novo transcriptome was constructed. Differentially expressed genes (DEGs) were identified between stages and the functional properties of DEGs were characterized by comparison with the gene ontology and Kyoto Encyclopedia. An enrichment analysis of molecular pathways was performed.Results: The de novo transcriptome comprised 287,089 transcripts after filtering. As vitellogenesis progressed, more genes were significantly upregulated than downregulated. The rFsh application induced ovarian development from previtellogenesis to early-to-mid-vitellogenesis with associated pathways enriched from upregulated DEGs related to ovarian steroidogenesis and reproductive development, cholesterol metabolism, ovarian growth and differentiation, lipid accumulation, and cell-to-cell adhesion pathways. The application of rFsh and rLh at early-to-mid-vitellogenesis induced the growth of oocytes to late-vitellogenesis and, with it, the enrichment of pathways from upregulated DEGs related to the production of energy, such as the lysosomes activity. The application of rLh at late-vitellogenesis induced the completion of vitellogenesis with the enrichment of pathways linked with the switch from vitellogenesis to oocyte maturation.Conclusion: The DEGs and enriched molecular pathways described during the induced vitellogenesis of flathead grey mullet with rGths were typical of natural oogenesis reported for other fish species. Present results add new knowledge to the rGths action to further raise the possibility of using rGths in species that present similar reproductive disorders in aquaculture, the aquarium industry as well as the conservation of endangered species.

Cultured Senegalese sole (Solea senegalensis) breeders fail to spawn fertilized eggs. The implantation of large-scalein vitrofertilization protocols, to solve this problem, has been frustrated by low production of poor quality sperm. Cultured females were induced to ovulate with a 5 µg kg⁻¹single injection of gonadotropin-releasing hormone agonist (GnRHa) and viable eggs (82.6 ± 9.2% fertilization) were stripped 41:57 ± 1:46 h after the injection. Sperm was collected from cultured males, diluted in modified Leibovitz and used fresh to fertilize the eggs. Males were not treated with hormones. A nonlinear regression, an exponential rise to a maximum (R= 0.93,p< 0.0001) described the number of motile spermatozoa required to fertilize a viable egg and 1617 motile spermatozoa were sufficient to fertilize 99 ± 12% (±95% CI) of viable eggs. Similar, spermatozoa egg⁻¹ratios of 592 ± 611 motile spermatozoa egg⁻¹were used in large-scalein vitrofertilizations (190 512 ± 38 471 eggs). The sperm from a single male (145 ± 50 µl or 8.0 ± 6.8 × 10⁸spermatozoa) was used to fertilize the eggs. The mean hatching rate was 70 ± 14% to provide 131 540 ± 34 448 larvae per fertilization. The viability of unfertilized eggs stored at room temperature decreased gradually, and the sooner eggs were fertilized 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 with other collection methods. The spz egg⁻¹ratios for Senegalese sole were at the lower end of ratios required for fish. Senegalese sole have a pair-spawning reproductive behaviour characterized by gamete fertilization in close proximity with no sperm competition. The provision of a large-scalein vitrofertilization protocol (200 µl of sperm per 100 ml of eggs) will enable the industry to operate sustainably and implement breeding programmes to improve production.

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 µg 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 µL or 8.0 ± 6.8 × 10 ⁸ 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 µL of sperm per 100 mL of eggs) will enable the industry to operate sustainably and implement breeding programs to improve production.