Intratesticular injection of busulfan for producing recipient male pigs for spermatogonial stem cell transplantation

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and fertility in male animals. In agricultural perspectives, SSC transplantation holds potential in expanding the availability of gametes from elite livestock rapidly, thus offering a highly efficient reproductive technique for livestock production. A limiting factor in application of this technique is acquisition of non-endogenous-SSC recipients, especially from large domestic animals. To explore a simple method to ablate pig endogenous SSCs and prepare transplant recipients, we directly injected different doses of busulfan into the testis tissue of Duroc pigs. Following injection, no significant health problems and body weight loss were observed in pigs treated with 3 mg/kg busulfan. Morphological analysis and gene expression assays showed that busulfan treatment efficiently removed endogenous SSCs, but sustained an intact Sertoli cells structure in pig testis. Compared with 5 mg/kg busulfan treatment group, 3 mg/kg busulfan treatment resulted in a complete SSCs ablation with less toxicity, providing a suitable environment for transplantation of exogenous SSCs. This study showed that intratesticular injection of busulfan in pigs to prepare recipients was an efficient and safe method, which lays the groundwork for the application of pig SSC transplantation.