The successful use of busulfan to deplete endogenous spermatogonia in ram testes

Abstract

Our research into germ cell transfer builds upon murine research1 and is aimed at using this technique in livestock species. To increase the efficiency of colonization of transplanted germ cells, the recipient testes must be depleted of endogenous spermatogonia, without affecting the supporting cells. Three depletion methods were investigated, heat, cold and chemotherapy. Our first investigation looked at the direct cooling (0°C) and heating (45°C) of the testes of 4-6 week old ram lambs. Testes were collected 7 days post treatment. The second investigation involved the systemic injection of busulfan to ram lambs aged 3-4 months. Busulfan is used for preparing recipient mice for testes cell transfer. At doses affecting the stem cells of the testes, busulfan will result in mylosuppression. Therefore a preliminary dose response trial was conducted at dose rates 4, 8 and 16mg/kg to determine the most effective dose, without threatening the survival of the animal. Testes were recovered after 3 and 6 weeks. All testes sections followed routine histology and immunochemistry with PGP 9.5 (Table 1). For the heat and cold study, only gonocytes were present and there were no differences in testes weights, tubule diameters or gonocyte numbers in any of the treatment groups. For the busulfan study, dose rates of 8 and 16 mg/kg resulted in severe mylosuppression and euthanasia of 7 out of 8 animals between day 12 and 18, whereas animals in the 4mg/kg group showed only mild clinical effects, that were not life threatening. These results indicate that busulfan reduced endogenous spermatogonia in the pre-pubertal ram. This effect is observed at systemic doses of 4 mg/kg or higher; however, doses of 8 mg/kg and above are lethal to the survival of the animal. The use of direct heat (45°C) or cold (0°C) to the testes does not affect gonocyte numbers in ram lambs; however, effects on more mature stages was not studied.