Effective production of recipient male pigs for spermatogonial stem cell transplantation by intratesticular injection with busulfan

Germ cell transplantation has facilitated spermatogonial stem cell (SSC) and spermatogenesis research and shown great potential in the seed-breeding of domestic livestock. However, little progress has been made in large animals, primarily reflecting the difficulties in preparing sterile recipients. Here, we developed a novel protocol to prepare recipient pigs through the direct injection of busulfan into the cavum vaginale of the scrotums of Landrace-Large bi-crossbreeding male pigs and Seghers male pigs, two economically-important types of pigs, to eliminate endogenous spermatogonia. No severe diseases or weight loss was observed in either pig type after the injection with busulfan. Histologic analysis showed an advanced and dose-dependent germ cell loss, with complete germ cell loss observed in the highest dose group, 3.0 mg/kg in the Landrace-Large bi-crossbreeding pigs and 2.0 mg/kg in the Seghers pigs. A smaller seminiferous tubule diameter, a vacuolized seminiferous epithelium and the overproliferation interstitial cells, frequently observed in mouse germ cell deficiency models, were present in the most of the high-dose busulfan-treated groups. Molecular markers detected in Seghers pigs further confirmed the depletion of endogenous germ cells, providing an accessible niche for exogenous SSCs. This study provides a basis to prepare the transplantation recipients of SSCs in pigs.