Testicular Germ Cell Depletion and Transplantation in Cattle and Sheep

Germ cell transplantation has been shown to be a useful technique for studying spermatogenesis in rodents. In livestock species, the germ cell transplant technique could be used to produce transgenic animals and as a reproductive technology in extensive production systems. One of the contributing factors to poor success thus far has been poor recipient preparation, such as depletion of endogenous germ cells. This thesis investigated four depletion methods known to affect germ cells; heat, cold, chemotherapy (Busulfan) and irradiation. ... This thesis has found that testicular irradiation is the most successful method tested to deplete endogenous spermatogonia in the sheep testis. In addition, irradiated testes can support both endogenous and donor derived spermatogenesis. It seems that irradiation at high doses (15Gy) at the peri-pubertal to pubertal stage of testicular development can produce suitable recipients for germ cell transplantation and therefore is a potential methodology to increase the efficiency of germ cell transplants in livestock species

Spermatogonia survival in young ram lambs following irradiation, Busulfan or thermal treatment

Many alternatives to surgical castration have been explored to induce short or long term infertility in male animals. Comparatively few have been carefully evaluated in very young production animals. A comparison of four treatments known to deplete testicular cells in rodents (heating, cooling, chemotherapy, radiation) was undertaken using ram lambs. Neither testicular cooling (0 °C) nor heating (45 °C) affected testis weights, tubule diameters or germ cell numbers. Low dose chemotherapy (Busulfan 4 mg/kg) treatment caused dramatic falls in white blood cells and platelets numbers which recovered within 3 weeks. Spermatogonia numbers were not significantly reduced (27% change). The impact of irradiation doses (0–15 Gy) delivered with high precision to the testis by a 6 MV photon beam was assessed by serial biopsies. Sertoli cell numbers were depleted by 90% at 3 weeks in 15 Gy treated testes. Spermatogonia were depleted 8 weeks after irradiation with 9 Gy, 12 Gy and 15 Gy. By 13 weeks, only in the 15 Gy treated testes were spermatogonia and Sertoli cell numbers lower. At 13 weeks testis atrophy resulted in 3/6 lambs irradiated with 12 Gy or 15 Gy. Irradiation of very young lambs clearly compromised testis function, thermal treatment was ineffective and busulfan treatment resulted in minimal effects

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