Abstract: To isolate spermotogonia using two combinatorial enzyme three step wises digest the testicular tissues from 19 d-old-embryo after hatching, the cryopreserve effect of dimethylsulphoxide (DMSO), glycerol (GLY) and ethylene glycol (EG) cryoprotectants, each at (5%, 10%, and 15%) concentration on the 19 d chicken embryonic SSCs was compared with fast freeze method. The results indicated that： (1) When SSCs frozen in 5%,10% and 15% DMSO medium, the viability rate of after thawed SSCs were 73.1%,88.6% and 74.8% respectively, the difference of three concentration were significant(P<005) When SSCs frozen in 5%,10% and 15% EG medium, the viability rate of after thawed SSCs were 69.4%,83.1% and 65.2% respectively, and the difference of three concentration were significant(P<0.05). When SSCs frozen in 5%,10% and 15% glycerin medium, the viability rate were less than 15%, and difference of three concentration were not significant (P>0.05); (2) The SSCs were cryopreservated in 10% DMSO for 1-2 weeks, then thawed and seeded on the feed cells to culturing, the chicken embryonic fibroblast cells as feed cells. The results found that the SSCs had formed the colonies which were positive after AKP staining. On the contrary, when the SSCs were cultured without feed cells, the colonies couldn’t form The results showed that the 10% DMSO was suitable freezing media for the 19 d SSCs from chicken embryo