Introduction: Transplantation of germ cells restores the male fertility. Nevertheless, a lot of questions remain incompletely resolved. The aim of this study was to evaluate in vitro colonization efficiency of germ cells and sperm production capacity of spermatogonial cells before and after culture by sperm number assay in epididymis of recipient mice. Materials and Methods: We developed a Sertoli cell feeder in a co-culture system with spermatogonial cells and the cells were co-cultured for 2 months. The cells were isolated from mouse neonates. Colony assay was performed during culture using light microscopy. The transplanted cells were traced using BrdU incorporation. Sperm parameters were assessed 2 months after transplantation. Results: Our findings showed that spermatogonial cells created colonies during culture. Transplantation of fresh spermatogonial cells at a concentration of 2×105 cells/ml did not show significant differences. However, after transplantation of 2×105 cells/ml cultured for 2 weeks, the number of epididymal sperms in recipients increased significantly in groups with more fresh cells. Conclusion: Epididymal sperm number in recipient mice can be increased by enrichment of type A spermatogonial cells using an in vitro co-culture system. Other important factors include the source of donor cells and the number of transplanted cells.