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.

The objective of the current study was to identify an optimal time period for donor cell transplantation after irradiation in sheep. The testes of recipient rams were treated with a single dose of 15 Gray (Gy) irradiation followed by germ cell transplantation either 3 or 6 weeks later. Transplantation of donor cells at 6 weeks after irradiation resulted in production of donor sperm by all five recipient rams compared with 4 of 11 rams transplanted at 3 weeks. Rams transplanted 3 weeks post-irradiation appeared to show reduced libido and fertility. Two rams produced sperm with low motility (< 20%) and two other rams were azoospermic. More than 1 year after cell transfer, there were heavy infiltrates of CD45-positive cells and more fibrous tissue in 9 of 14 recipient testes (seven rams) that received cells 3 weeks after irradiation. Taken together, these results suggest that the interval between irradiation of recipients and germ cell transplantation affects the success rate of the procedure, with a 6-week interval preferable. The elevated inflammatory/immune reaction may be responsible, at least in part, for the reduced fertility and low libido observed in the rams that received cells 3 weeks post-irradiation.

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