Spermatogenesis depletion in diploid and triploid yellowtail tetra (Astyanax altiparanae) for germ cell transplantation.

The piracanjuba (Brycon orbignyanus) is a species threatened with extinction due to human activities, highlighting the need for genebanking and alternative propagation methods for its conservation. The objective of this study was to obtain a completely sterile yellowtail tetra (Astyanax altiparanae) and to transplant the stem spermatogonia (SSCs) of B. orbignyanus. Complete depletion of spermatogenesis in adult diploid males was successfully achieved through five applications of busulfan, using dosages of 15 mg kg-1 and 40 mg kg-1 at a high temperature of 35°C. One month after completing the busulfan treatment, spermatogenesis returned, occurring more rapidly in the 15 mg kg-1 treatment, with spermatozoa visible in the testicular lumen. In the treatment with 40 mg kg-1, only SSCs and some spermatid cysts were observed. Depletion of spermatogenesis in triploid juveniles was observed after two applications of busulfan (50 mg kg-1). One month after the end of the treatment, only a few SSCs cells were observed in the testes. The B. orbignyanus SSCs were isolated using a Percoll density gradient and identified by alkaline phosphatase activity, resulting in a cell suspension of 2.6 × 106 SSCs/mL with 93% cell viability. The cells were labeled with PKH26 and transplanted via the urogenital papilla into recipients (triploid juvenile males) previously treated with busulfan. PKH26-positive cells were visualized up to 80 days after transplantation. The combination of two sterilization methods appears to be more effective for cell transplantation after the endogenous germ cells have been eliminated from the recipient testes of triploid juveniles.