The Application of Busulfan to Inhibit the Spermatogenesis in Chicken Testis

Spermatogonia cells are considered as a valuable genetic material to produce individuals with desired traits in the framework of preservation of animal and poultry gene pool (cryobanks) and genetic modification of their genome (transgenesis). This technology is based on the introduction of donor spermatogonia into the testes of male recipients and the following obtaining the exogenous sperm to produce progeny with desired characteristics. A key step of this technology is the removal of own spermatogenic cells (spermatogenesis inhibition) in the male recipient. We have optimized the method to inhibit the spermatogenesis in chicken testes using alkylating agent busulfan. The multiple injections of busulfan at the concentration from 40 to 100 mg per 1 kg of body weight was performed directly into the parenchyma of testes of six month old chicken males (n=16). In order to detect the presence of spermatogenic cells in seminiferous tubules, we carried out the histological studies of testes in one month after injection. We observed the decrease in diameters and the number of spermatogenic cells at the cross sectional area of the seminiferous tubules in the experimental group as compared to the control. When using busulfan at concentration of 40 mg/kg the number of cells averaged 35±3 cells per seminiferous tubule, which was 32.8 times less as compared to the control (1149±95). At higher concentrations of busulfan the number of cells decreased and averaged 20±2, 12±0.5 and 9±0.4 cells at concentration of 60, 80 and 100 mg/kg, repectively. Analysis of cell types of the seminiferous tubules revealed that mainly the Sertoli cells and spermatogonia represented the cell population. Moreover, with increasing concentrations of busulfan the ratio of spermatogonia was significantly reduced. The ratio of Sertoli and spermatogonia cells after introduction of busulfan into the testes at a concentration of 40, 60, 80 and 100 mg/kg was 57 and 43%, 75 and 25%, 95 and 5 % and 96 and 4%, respectively. However, the application of busulfan at a concentration 100 mg/kg led to high mortality of chicken. Thus, we demonstrated that the optimal concentration of busulfan to kill the spermatogonial cells was 80 mg/kg. The complete inhibition of spermatogenesis was observed in 9-10 months after introduction of busulfan at this concentration.

The study was supported by the Russian Science Foundation within Project no.16–16–10059.