An in vitro system that supports spermatogonial stem cells survival and proliferation is useful for enhancement of stem cell number and efficient transplantation. The purpose of this study was to assess the effect of different concentrations of foetal bovine serum on survival of bovine spermatogonial stem cells and Sertoli cells after cryo-preservation. Samples were surgically obtained from testes of 3-5 months old calves and digested twice in enzymatic media to get the isolated cells. These cells were co-cultured for two weeks to obtain the adequate amount of cells. In the freezing step, the purified spermatogonial stem cells were frozen in four different freezing media including bio-freeze medium^® as Control and 3 other groups containing: Dulbecco’s modified Eagle’s medium (DMEM) and 50%, 70% and 90% fetal bovine serum (FBS) plus 10% dimethyl-sulfoxide (DMSO) as treatments. After 2 weeks of preserving in liquid nitrogen, to assess the viability, the cells were rapidly thawed by swirling in 38°C water bath for two minutes. The viability of the cells was assessed by using flow cytometry technique. The results showed that the frozen cells in 50 and 70% FBS groups had significantly (P<0.05) higher viability rates than control and 90% FBS groups. It is concluded that spermatogonial stem cells can be cryopreserved in DMEM plus 10% DMSO and 50 to 70% FBS.