Irradiation Enhances the Efficiency of Testicular Germ Cell Transplantation in Sheep

Testis germ cell transplantation in livestock has the potential for production of transgenic genotypes and for use as an alternative to artificial insemination in animal breeding systems. In a pilot experiment, we investigated a workable protocol for testis germ cell transplantation in sheep, including donor cell isolation, rete testis injection, and microsatellite detection of donor spermatozoa in recipient semen. In a second experiment, the effect of depletion of endogenous stem cells with a single irradiation dose of 9 Gy (n = 5) or 15 Gy (n = 5) on the outcome of germ cell transplantation was investigated. Irradiation of recipient testes with a single dose of 15 Gy, followed by transplantation 6 wk after depletion, may be most advantageous because it resulted in all recipients (five of five) producing donor-derived spermatozoa, while the 9-Gy and control groups had limited success rates (two of five and one of three, respectively). Using microsatellite markers to detect the presence of donor DNA, 10 rams were identified that produced spermatozoa of donor origin. The proportion of donor DNA was between 1% and 30% of total ejaculate DNA. When three of these positive rams were used in breeding experiments, four donor-derived offspring (four of 50 [8% of progeny])resulted from a recipient in Merino to Merino transplantation. Six lambs (six of 41 [15% of progeny]) were sired by donor-derived Border Leicester sperm produced in a Merino recipient ram; however, no donor-derived offspring were detected among 34 progeny from a second Border Leicester to Merino combination. These results confirm that preparation of recipient animals with a correct dose of irradiation not only enhances the success rate of the transplantation procedure but also increases the proportion of donor spermatozoa in recipient semen. This study represents the first report of the production of live progeny following testis germ cell transplantation using irradiated recipients in a livestock species.