Sex differentiation and the effect of post-larval cannibalism on the sex ratio in Brycon orbignyanus assessed by gonadal morphology and gene expression analysis

Brycon orbignyanus is an endangered fish species that is utilized in conservation strategies, including restocking programs. However, ex-situ practices, as artificial breeding, cause sexual ratio deviation, resulting in predominantly male offspring, which is not adequate for conservation programs. In this work, the main aspects of sexual differentiation were studied by monitoring gonadal differentiation and gene expression. Furthermore, the impact of larval cannibalism on the sexual ratio in both individual and collective post-larval cultures was examined, with sexual ratios assessed in the 12th month. S ox9 , dmrt1 , foxl2 , ddx20 , and ddx4 gene expressions were analyzed in oocytes, larvae, juveniles, and adults. Additionally, the juvenile gonads from the 6th to 12th month were examined through histological observations to identify the sexual differentiation. The gene expression results showed that the genes ddx4 and dmrt1 are specifically expressed in the gonads, while the other examined genes were also expressed in various tissues. The genes dmrt1 , foxl2 , and sox9 displayed dimorphic expression patterns in male and female gonads, and dmrt1 was not detected in larvae. In juveniles, the ovary was first identified in the 6th month, while the testis was observed from the 10th month through histological analysis. This analysis also revealed the presence of intersex individuals during gonadal development. No sex deviation (1:1) was observed in the grouped and individual rearing treatments. Sex differentiation was detected around the 6th month, indicating that artificial rearing at this stage is unlikely to affect the sex ratio. These findings are important for future research on understanding the sexual differentiation process in this species and for ex-situ practices.