Production of Chinese rosy bitterling offspring derived from frozen and vitrified whole testis by spermatogonial transplantation.

Bitterling is a small cyprinid fish facing an increasing risk of extinction owing to habitat destruction and decreasing freshwater mussel population that are used as their spawning substrates. Owing to their large size and high yolk contents, methods for cryopreservation of their eggs or embryos, which is a promising method for long-term preservation of their genetic resources, are still not available. We conducted this study to evaluate the feasibility of gamete production by transplanting cryopreserved testicular cells into germ cell-less recipients that were produced by knockdown of dead end gene. Immature testes isolated from recessive albino Chinese rosy bitterlings were cryopreserved by slow freezing or vitrification. Approximately 3000 slow-frozen or vitrified cells were transplanted into the peritoneal cavity of 4-day-old germ cell-less wild-type Chinese rosy bitterlings. We observed no significant differences in the incorporation rates of the slow-frozen and vitrified cells into the genital ridges of recipients compared with those of freshly prepared cells. When the recipients matured, almost half of the male or female recipients that received freshly prepared, slow-frozen, or vitrified cells produced gametes derived from donor cells, with no significant differences in their fecundity among the 3 groups. Moreover, fertilization of the resulting eggs and sperm produced donor-derived offspring exhibiting the albino phenotype. Therefore, the abovementioned methods could be used as a powerful and practical method for long-term preservation of bitterling genetic resources for biotic conservation.