Abstract:

Objective To investigate the effects of vapor nitrogen freezing method and direct liquid nitrogen freezing method on the motility, morphology and ultrastructure of post-thaw human sperm. Methods Twenty-five semen samples from semen donors were selected, and glycerol-yolk-sodium citrate (GYC) was added as cryoprotectant with the proportion of 1∶1. Each semen sample was equally distributed to two cryovials, and were cryopreserved with vapor nitrogen freezing method and direct liquid nitrogen freezing method respectively. The total motility rate, progressive motility rate and motility parameters of post-thaw sperm were examined by IVOS sperm analyzer. The morphology of post-thaw sperm was observed with papanicolaou staining, and the rate of post-thaw sperm with normal morphology was calculated. Besides, the ultrastructure of post-thaw sperm was observed by transmission electron microscope. Results It cost 468 s to drop in temperature from 28 ℃ to -185 ℃ by vapor nitrogen freezing method, and an hour later the temperature went to -196 ℃. However, it only cost 90 s to drop in temperature from 30℃ to -196 ℃ by direct liquid nitrogen freezing method. The total motility rates, progressive motility rates, motility parameters and rates of post-thaw sperm with normal morphology in two cryopreservation methods were significantly lower than those before cryopreservation (P＜0.05). The revival rate, total motility rate and progressive motility rate in vapor nitrogen freezing method were significantly higher than those in direct liquid nitrogen freezing method (P＜0.05). There was no significant difference in motility parameters and rates of post-thaw sperm with normal morphology between two cryopreservation methods （P＞0.05）. Transmission electron microscopy indicated that no obvious damage was found on the ultrastructure of serosa and acrosome membrane at the head of post-thaw sperm in two different cryopreservation methods by using GYC as cryprotectant. Conclusion GYC has a protective effect on the ultrastructure of sperm. Vapor nitrogen freezing method may yield better post-thaw results than direct liquid nitrogen freezing method in cryopreservation of human sperm.

Key words: sperm, cryopreservation, vapor nitrogen freezing method, direct liquid nitrogen freezing method, sperm motility, ultrastructure