PTD-FNK蛋白对水牛精子的冷冻保护作用

PTD-FNK蛋白可以穿透细胞膜进入细胞内发挥抗凋亡作用,抵抗各种不利因素造成的细胞死亡,但国内未见将其加入水牛精液稀释液抑制精子冷冻凋亡的报道。本试验中,在水牛精液冷冻稀释液中添加不同浓度(0、0.1、1、10、100 nmol/L)的PTD-FNK蛋白,冻后检测精子常规质量指标的同时检测精子凋亡相关基因(Caspase-3、Caspase-8、Caspase-9基因)mRNA表达和Caspase-3蛋白酶的活性;最后以最优PTD-FNK蛋白浓度批量生产冷冻精液,选择50头广西地方母水牛进行人工授精试验。结果显示:①1 nmol/L组精子质膜完整性和10 nmol/L组精子活力显著高于对照组(P<0.05),1 nmol/L组精子活力和1、10 nmol/L组精子活率极显著高于对照组(P<0.01),而100 nmol/L组精子活率却极显著低于对照组(P<0.01);②1 nmol/L PTD-FNK蛋白显著降低冻后精子Caspase-3 mRNA表达(P<0.05);③人工授精后第60天受胎率达60%,略高于其他研究者取得的受胎率(56.25%,P>0.05)。总之,PTD-FNK蛋白可以显著提高水牛精子冻后质量,其机制之一可能是它显著抑制了冻后精子Caspase-3 mRNA的表达;以PTD-FNK蛋白冷冻的水牛精液实施人工授精后可以取得比现有水平更高的受胎率。

Cryoprotection of PTD-FNK protein on buffalo spermatozoa

PTD-FNK protein can penetrate the cell membrane into the cell to play an anti-apoptotic role and resist cell death caused by various adverse facts.However,no domestic report about adding PTD-FNK protein to the buffalo sperm diluents to inhibit cryoapoptosis of sperm has been reported.In this study,different concentrations(0,0.1,1,10 and 100 nmol/L)of PTD-FNK protein was added to the freezing diluent of buffalo semen.Then after freezing,routine quality indexes of sperm as well as mRNA expression of apoptotic-related genes(Caspase-3,Caspase-8 and Caspase-9)and Caspase-3 activity were detected.Finally,50 local female buffalo in Guangxi were selected for artificial insemination(AI)test with semen in small quantities frozen in the optimal concentration of PTD-FNK protein.The results showed that sperm plasma membrane integrity in 1 nmol/L group and sperm motility in 10 nmol/L group were significantly increased(P<0.05).Sperm motility in 1 nmol/L group and viability in 1,10 nmol/L group were also significantly increased(P<0.01),while sperm viability in 100 nmol/L group was significantly decreased(P<0.01).1 nmol/L PTD-FNK protein significantly reduced Caspase-3 mRNA expression of frozen-thawed spermatozoa.Pregnancy rate(60%)on day 60 after AI was slightly higher than pregnancy rate(56.25%)reported by other investigators(P>0.05).In conclusion,PTD-FNK protein can significantly improve the quality of buffalo sperm after freezing,and one of potential mechanisms might be that it significantly inhibits the expression of Caspase-3 mRNA in post freezing sperm.AI of buffalo semen frozen with PTD-FNK protein can achieve fertility higher than current levels.