251. Production of donor-derived live lambs following testis germ cell transplantation

Testes germ cell transplantation in livestock has the potential for amplification of transgenic genotypes and for use as an alternative to artificial insemination. This study investigated a workable protocol for testis germ cell transplantation in sheep between animals of the same breed and different breeds. Testes of two groups of recipients at the stage of pre-pubertal (transition from gonocytes to spermatogonia, n = 2) or peri-pubertal (spermatogenesis initiated, n = 2) were treated with a single dose of 9 grey (Gy) or 15 Gy with a 6MV photon beam irradiation, respectively. In the first experiment, using pre-pubertal irradiated animals, testis germ cell transplantation between the same breed was performed at 16 weeks post irradiation. The left testes of recipient rams were injected with donor cells labelled with fluorescent dye PHK26, while the right testes were given unlabelled cells. The left testes of recipients were removed by castration after 2 weeks following transplantation to evaluate the location of the transferred cells, while the right testes were kept in place for long-term assessment of sperm output. In cryosections of the left testes, PKH26 positive cells were found both on the basement membrane as single cells or in the interstitial area. In the second experiment, animals irradiated at the peri-pubertal stage, received donor cells at 5 weeks post irradiation and animals were kept intact for semen production. For a period of two years after transplantation, semen samples were collected routinely from two groups of rams and analysed using microsatellite markers. Two recipients (50%) demonstrated the presence of donor DNA in their ejaculates. In order to investigate the fertility of the donor-origin sperm in recipient ejaculates, 99 ewes were artificially inseminated with semen from two positive rams. Four lambs (8%) have been identified as being sired by donor-derived sperm produced in the recipient ram that received a Merino to Merino transplantation, while no donor-derived offspring was obtained from the recipient with Border Leicester to Merino transplantation. This study represents the first report of the production of live progeny following testis germ cell transplantation in sheep.