维生素C对藏猪精液4℃保存及其精子全基因组DNA甲基化的影响

Effect of Vitamin C on Tibetan Boar Semen Preserved at 4 ℃ and Levels of the Sperm Global Genome DNA Methylation

旨在研究维生素C对藏猪精液4℃保存效果及其精子全基因组DNA甲基化的影响。用手握法采集健康藏猪精液,在改进的猪精液低温稀释液中添加不同浓度的维生素C(0.0、2.5、5.0、7.5、10.0g·L^(-1)),4℃保存到第5天时,以藏猪精子活力、精子畸形率、精子顶体完整率和质膜完整率为评价指标,探讨维生素C对藏猪精液4℃保存的效果;用甲基化荧光定量法检测藏猪新鲜精液组、保存到第5天时未添加维生素C组、添加维生素C组(添加维生素C最佳效果组5.0g·L^(-1))精液DNA甲基化的水平,以探讨维生素C对藏猪精液4℃保存所造成的精子全基因组DNA甲基化的影响。结果显示:添加5.0g·L^(-1)维生素C组其精子活力、畸形率、顶体完整率和质膜完整率均显著好于其它组(P<0.05);鲜精组、未添加维生素C组、添加维生素C组的DNA甲基化水平分别为(0.604 7±0.040 3)、(0.920 2±0.016 9)、(0.656 9±0.048 3)ng,鲜精组与添加组均显著低于未添加组(P<0.05),而鲜精组与添加组间差异不显著(P>0.05)。综上表明,在改进的猪精液稀释液中添加5.0g·L^(-1)维生素C可以明显改善藏猪精液4℃保存的效果,并且可以明显降低4℃保存对藏猪精液精子全基因组DNA甲基化的影响,这将为藏猪精液低温保存的生产应用奠定基础。

This research was conducted to study the effect of vitamin C on Tibetan boar semen preserved at 4℃and the subsequent influence on global genome DNA methylation. The semen was collected from Tibetan boar and preserved at 4 ℃ in modified medium supplemented with vi- tamin C at 0.0,2. 5,5. 0,7. 5,10. 0 g · L-1. Sperm motility, abnormality, acrosome integrity and plasma membrane integrity were evaluated 5 days after preservation. And the global methylation level of the fresh and preserved sperm was detected by using MethylFlashTM. The results showed that a significantly higher sperm motility and plasma membrane integrity, acrosome integrity and less abnormality was observed in group supplemented with 5.0 g· L-1 vitamin C（P〈0.05）. DNA methylation levels of fresh sperm （0. 604 7±0. 040 3） ng and sperm preserved with vitamin C （0. 656 9 ±0. 048 3） ng were significantly lower than those preserved without vitamin C （0. 920 2±0. 016 9） ng （P〈0.05）,but no significant difference was found between fresh semenand sperm preserved with vitamin C（P〈0.05）. These results indicated that supplementation of vitamin C at 5.0 g ·L-1 semen extender could improve quality of Tibetan boar semen preservation at 4 ℃ and mitigate effect on DNA methylation of Tibetan boar sperm. The results in the current study would be helpful for further investigation of the freezing protocol for Tibetan boar semen.