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Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22 days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.

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