Abstract: Objective To optimize the method of separation of mouse sperm head and tail by treating with different concentration of NaOH so as to improve the efficiency of ICSI. Methods The sperm were collected from the epididymis of male mice at the age of 2-3 months and diluted to an appropriate density. The sperm head and tail separation rate was determined under the microscope after incubating with different concentration of NaOH at the range of 0-37°C. Flow cytometry was utilized to evaluate both of membrane and DNA integrity of the sperm cells. Results The sperm head and tail separation rate was positively correlated with NaOH concentration, incubation time and temperature. However，DNA integrity of the sperm cells was negatively correlated to NaOH concentration and incubation temperature and time. The lower NaOH concentration and temperature as well as the shorter incubation time led to less serious DNA damage. The results indicated that the condition of 10 mmol/L NaOH for 2 h at 0°C was the optimal treatment to achieve 55% of sperm head and tail separation rate without injury of their DNA. Conclusion The low concentration NaOH treatment of sperm with the optimized condition in our study is a simple and convenient methodwhich can effectively separated the tail from sperm while remain the high DNA integrity.

Key words: ICSI, DNA damage, Flow cytometry, NaOH