Abstract: Objective To establish a more suitable method to frozen sperm of C57BL/6J by comparing in vitro fertilization rate of post- thawed C57BL/6J mice spermatozoa in various cryoprotectants. Methods To R18S3-based fluid, adding with the different concentrations of egg yolk and glycerine as cryoprotectants to frozen sperm from C57BL/6J and Y3F mice and using the same method of sperm recovery with the rate of in vitro fertilization to evaluate the effect of frozen sperm. Results R18S3 with liquid-based, add with the concentration of 5%，10%，15%，20%, 25% of yolk and centrifuged, then the supernatant as cryoprotectants, the rate of in vitro fertilization and frozen-thawed sperm of C57BL/ 6J mice is 18.7%, 15.6%，36.0%, 40.0%，29.7% respectively with a significant different. R18S3 with liquid-based,add with the concentration of 1%, 2%, 3%, 4%, 5%, 6% glycerol as cryoprotectants, the rates of in vitro fertilization of freeze-thawed sperm of C57BL/6J mice were 7.8%, 10.0%, 16.9%, 6.0%, 10.3%, 18.1% respectively with a significant difference.The supernatant of R18S3 +5%yolk after centrifugation +6% glycerol; the supernatant of R18S3 +15% yolk after centrifugation +2% glycerol; the supernatant of R18S3 +20% yolk after centrifugation +1% glycerol as cryoprotectants, the rates of in vitro fertilization of freeze-thawed sperm of C57BL/6J mice were 16.7%, 36.0% and 22.9% respectively with a significant difference. R18S3 as cryoprotectants, the rate of in vitro fertilization of freeze-thawed sperm of C57BL/6J mice is 25%, the rate of abnormal cleavage is 2.6%; the rate of in vitro fertilization of freeze-thawed sperm of Y3F mice is 52.2%, the rate of abnormal cleavage is 5.1%. Conclusion The supernatant of R18S3 +15%/20% yolk after centrifugation as cryoprotectants can raise the rate of in vitro fertilization of freeze-thawed sperm of C57BL/6J mice but adding with the different concentrations of glycerol can reduce the rate. The frozen effect of adding with both yolk and glycerol less than the effect of adding only yolk, but better than the effect of adding only glycerol.

Key words: Yolk, Glycerol, Sperm, in vitro fertilization