Abstract: To develop a series of practical culture methods of Hu-sheep spermatogonial stem cells (SSCs) in vitro for future studies on transgenesis, seminiferous tubule cells were isolated through a two step enzyme digestion from dissociated testis, and the cell suspension was classified by percoll gradient. The effects of different purification methods, FCS and GDNF on SSCs culture in vitro and alkaline phosphatase (AKP) staining were investigated. The results were as follows: (1) The time of colonies formation and colonies numbers from panning method were significantly lower than that from differential plating (P<0.05), in contrast, AKP positive rates from panning method significantly increased (P<0.05). (2) The colonies formation time, numbers and AKP positive rates significantly increased when the medium supplemented with 0.5% BSA and 1% FCS (P<0.05), however, there were no significant difference in the colonies formation time and AKP positive rates between 1% and 5% FCS (P>0.05). (3) The colonies number and AKP positive rates were significantly higher under 20 ng/mL GDNF than that under 20 ng/mL GDNF (P<0.05), but there were no significant difference in colonies formation time and AKP positive rates between 20 ng/mL GDNF and 30-40 ng/mL GDNF (P>0.05)．

Key words: spermatogonial stem cells (SSCs), culture in vitro, colonies, Hu-sheep