Improvement of the Efficiency of Gene Transfection in Frozen-Thawing Goat Sperm

Objective To improve the efficiency of gene transfection of exogenous DNA into frozen-thawing goat sperm,as well as maintain the preincubated goat semen quality.Methods An orthogonal experimental L-9(3~4) was designed to optimize four parameters: the diluent buffer,sperm concentrations,equilibration time and thawing buffer.The sperm quality of fresh and frozen-thawing semen was determined,and the positive goat spermatozoa were detected by in situ hybridization.Results The method of goat semen freezing and thawing was as follows: the washed spermatozoa were resuspended in 20% egg yolk diluted to 2×10~8/mL as the final sperm cell concentration,then followed by 4 h equilibration period at 4℃ before freezing,and goat semen frozen in pellet form and thawed at 40℃ by TALP buffer.The frozen-thawing influenced significantly the vigor of the spermatozoa(74.43%±13.78 % vs 46.25%±2.69%,P = 0.002).The spermatozoa after frozen-thawing resulted in the reduction of viability,sperm life span and fertility.Conclusion The efficiency of sperm picking up exogenous DNA was affected by frozen-thawing treatment and a cryopreservation protocol of goat sperm has been optimized in this study.