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Cryopreservation is an effective method for the long-term storage of fish sperm. However, because of a lack of methods for cryopreserving fish eggs and embryos, a technical challenge remains in preserving maternally inherited cytoplasmic compartments. Here, we aimed to generate functional eggs and sperm in sterile rainbow trout hatchlings by transplanting cryopreserved ovarian germ cells into the peritoneal cavity. Immature ovaries were isolated from 9-month-old transgenic rainbow trout, whose ovarian germ cells expressed green fluorescent protein (GFP), and cryopreservation was optimized by testing several different cryoprotective agents. Dominant orange-colored, pvasa-Gfp transgenic rainbow trout females served as donors, and wild-type triploid rainbow trout served as germ cell recipients. Viability and transplantation efficiencies of frozen ovarian germ cells were stable for up to 1,185 days. Ovarian germ cells frozen for 8months efficiently differentiated into eggs and sperm in the ovaries and testes of recipient fish. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor phenotypes (orange body color, green fluorescence, XX ovaries, and matching karyotype). This study represents the first success in generating functional gametes from cryopreserved female germ cells in any fish species, which should facilitate the conservation of protogynous fishes, female heterogametic fish, and currently endangered fish.
