An efficient and convenient method for the preservation of germplasm resources: Cryopreservation of mature testis of large yellow croaker (Larimichthys crocea)

Abstract

Large yellow croaker (Larimichthys crocea) is one of the most important marine economic fish. However, due to overfishing and long-term artificial breeding, the germplasm has degraded in recent years, and the wild germplasm has been listed as critically endangered. It is urgent to protect the germplasm resources of Larimichthys crocea. Cryopreservation may be an alternative way, however, the toxicity of cryoprotectants and the complicated preservation procedure that uses a programmable freezer has greatly increased the difficulty of cryopreservation. Therefore, in this study, we aimed to explore an efficient, convenient, inexpensive and low toxicity method for cryopreservation of mature testes in this species. The cryoprotectants of dimethyl sulfoxide (Me₂SO), ethylene glycol (EG), propylene glycol (PG) were selected to cryopreserve the mature testes with liquid nitrogen in simple style. Finally, the testes were cut into blocks (1.0 cm³) with cryoprotectants of 20 % PG and 15 % Me₂SO, placed at a position 10 cm above the liquid nitrogen for 10 min, then plunged into liquid nitrogen, resulting in high survival rate of 64.17 % at 3 days post cryopreservation. And the recovery rate of spermatogonia was more than 70 %. Subsequently, the cells viability were further detected by qRT-PCR, in situ hybridization (ISH) and germ cell transplantation. These results suggested that the spermatogonia from cryopreserved testes had high viability, which could migrate and colonized after transplantation. The present study first established cryopreservation protocols of mature testis in Larimichthys crocea, which will serve as a promising tool to protect Sciaenidae and other fish germplasm resources.