Improvement of time-dependent decrease of sperm motility following freezing and thawing with the addition of N-acetyl L-cysteine

The low motility of sperm after thawing has been a problem in artificial insemination (AI) using frozen-thawed boar semen. A cause of low functionality of post-thawed sperm, sperm oxidative stress is induced by freezing and/or thawing stimulus. Thus, in this study, we examined whether the sperm motility parameters after freezing and thawing improved with the addition of N-acetyl L-cysteine (NAC), an antioxidant, to the thawing solution. Firstly, frozen semen was incubated in Modena solution with (0.5 or 5.0 mM) or without (control) NAC for up to 3 hr, and the sperm motility parameters (motility rate and progressive velocity at 0, 1 and 3-hr) and membrane status (membrane and acrosomal integrity at 3-hr) of each group were measured. Furthermore, in order to investigate the effect of the addition of NAC on the sperm ROS (reactive oxygen species) activity, its level was analyzed by flow cytometer using Total ROS/Superoxide Detection Kit. The motility rate of sperm treated with 5.0 mM NAC was significantly higher than that of the control at 3-hr incubation (P<0.05, 24.2 vs. 42.9%), and the NAC treatment improved sperm progressive velocity as compared with the control after 1-hr incubation (P<0.05, 1hr; 22.5 vs. 39.0microm/s, 3hr; 16.7 vs. 35.0microm/s). For the motility parameters, it was only in the control that the values decreased with time (P<0.05). On the other hand, the additional of NAC did not affect the membrane status such as plasma membrane and acrosomal membrane integrity (P>0.05). Because the addition of 5.0 mM NAC improved the sperm motility parameters after thawing, we examined whether 5.0 mM NAC would actually suppress sperm ROS activity with flow cytometer. In the result, intense fluorescence signal of the total ROS activity was detected in the control at 3-hr incubation, as the NAC treatment significantly suppressed it (P<0.05). It was suggested that this suppression was due to the direct antioxidant activity of NAC or indirect influence with stimulation of glutathione synthesis by NAC. From these results, we concluded that the addition of 5.0 mM NAC to the thawing solution protected sperm from ROS activity, and improved its motility parameters.