In Vitro Culture of Buffalo (bubalus Bubalis) Spermatogonial Stem Cells: Effect of Serum, Sertoli Cell Coculture and Single Growth Factors

The objective of this study was to develop a short-term culture system for buffalo spermatogonial stem cells (SSCs) in various culture conditions. Spermatogonial stem cells as well as sertoli cells were isolated from prepubertal buffalo calf testes and enriched to culture for two weeks in a serum-free medium. The serum-free medium was supplemented with various concentrations of serum (FBS) or four growth factors (bFGF, GDNF, SCF and LIF) to evaluate the spermatogonial cell viabilities every two days during the two weeks of culture. The SSCs were also co-cultured with the enriched population of sertoli cells with and without supplementation of serum in the culture medium. The identity of isolated spermatogonial cells was confi rmed with CD9 labeling and histological observations. The stem cell activity of buffalo spermatogonia was further confi rmed by transplantation in mouse testes. Inclusion of serum signifi cantly (P<0.05) improved the viabilities of buffalo SSCs in the culture system, however, addition of higher quantity (>3% v/v) only enhanced proliferation of somatic cells. Coculture with sertoli cells in the presence of 3% serum signifi cantly (P<0.05) improved viabilities of SSCs. GDNF and LIF were the two most effective growth factors in maintaining the viabilities of spermatogonial cells. Both freshly isolated as well as cultured CD9 positive buffalo spermatogonial cells induced spermatogenesis in recipient mouse testes on transplantation, thereby confi rming that the culture system maintained the viability and functionality of buffalo SSCs in the short-term culture system.