Successful transplantation of cryopreserved spermatogonia in Sebastes schlegelii: A simple and suitable alternative approach for conservation of viviparous fish

Black rockfish (Sebastes schlegelii) is one of the most important marine economic viviparous fishes. Recently, germplasm degradation and genetic diversity reduction have occurred due to overfishing and long-term artificial breeding. Germ cell transplantation combined with cryopreservation may be an alternative way to protect genetic resources. However, in viviparous fish that undertake fertilization and embryo development in vivo, transplantation is more difficult than in oviparous fish, including selection of transplantation stage, isolation of germ stem cells, and preparation of sterile recipients. This seriously restricts the development of viviparous transplantation. Therefore, in this study, we aimed to explore a transplantation method suitable for these species. Donor cells were isolated from cryopreserved whole testes of 300-400 g male Sebastes schlegelii in May, labeled by PKH26, and intra-peritoneally transplanted into allogeneic larvae at 5-10 days post-birth. Subsequently, the development of donor-derived cells in recipients were continuously detected by fluorescence labeling, histology, microsatellite markers, and fecundity tests. The results showed that donors were rich in spermatogonia (75%) and recipients maintained a high survival rate after transplantation, with a rate of >20% at sexual maturity. Further, donor-derived cells successfully migrated (100%), colonized, and incorporated into the developing recipient gonad (93.33%). Finally, transplanted recipients could normally develop and differentiate into male and female individuals, with donor-derived gametes found in 65.38% of mature recipients. In the present study, we first establish a simple and suitable transplantation method for Sebastes schlegelii using immature males and specific larvae, which will serve as a promising tool in the protection of germplasm resources for this transplantationrestricted marine viviparous species.