诱导牛精子体外获能的培养基及方法

Media and Methods to Induce Bull Sperm in Vitro Capacitation

用去透明带金黄地鼠异种精子体外穿透试验，检测冻精复苏经各种处理后的精子穿卵率。发现在４种基本培养基（ＢＯ、ｍＢＷＷ、ＴＹＨ、ＨａｍＦ１０＋ＲＰＭＩ－１６４０）中不加亚牛磺酸时，牛精子不能耐受较高浓度（４μｍｏｌ／Ｌ）钙离子载体（ＩＡ）的刺激（以１０ｍｉｎ计）；而低浓度ＩＡ（低于４μｍｏｌ／Ｌ）处理精子不能诱导获能。生物活性物质，如肝素、咖啡因、亚牛磺酸、肾上腺素单独或联合低浓度ＩＡ处理精子，短期（６ｈ）内也不能诱导获能。当基本培养基中含有亚牛磺酸时，牛精子能耐受高浓度ＩＡ（１０μｍｏｌ／Ｌ）的刺激，并使牛精子获能；亚牛磺酸与肾上腺素、咖啡因联合使用，并在获能液中添加透明质酸酶时，穿卵率进一步提高，获能效果显著改善。以ＨａｍＦ１０＋ＲＰＭＩ１６４０为主的混合培养基，在牛精子的存活时间、穿卵率等方面优于ＢＯ、ｍＢＷＷ、ＴＹＨ培养基。结果表明，用Ｆ１０和１６４０的混合培养基，添加亚牛磺酸、肾上腺素、咖啡因、透明质酸酶，经１０μｍｏｌ／ＬＩＡ处理牛精子１０～１５ｍｉｎ。

In order to develop more efficient media and methods for inducing bull sperm in vitro capacitation the interspecific sperm penetration assay of zona free hamster ova was used for evaluating the efficacy of in vitro capacitation of the frozen thawed bull sperm under some controlling conditions. The results showed that bull sperm can not tolerated higher than 4 μmol/L ionophore A23187 (exposing 10 minutes) and even die in the basic media (BO, mBWW, TYH, Ham F10+RPMI 1640) and not capacitate in the basic media when sperm exposed for 10 minutes in lower than 4 μmol/L ionophore A23187 (IA). Life active substances, including heparin, caffein, hypotaurine and epinephrina, neither induce bull sperm capacitation in short period, singly of jointly with low concentration IA. In basic media supplemented with hypotaurine, bull sperm can resist 10 μmol/L IA for 10 minutes exposure and preceed capacitation. When basic media supplemented with hypotaurine epinephrine and caffein, and the hyaluronidase added in capacitation droplet the penetration rate of zona free hamster ova rise further and the efficiency of capacitation improve remarkably. The mixed medium of Ham F10 and RPMI 1640 appears to be better than BO, mBWW and TYH medium in survival time and penetration rate of bull sperm. These results suggest that the mixture medium of Ham F10 and RPMI 1640 supplemented with hypotaurine, epinephrine, caffein and hyaluronidase and the treatment of 10 μmol/L IA for 10 to 15 min should be an efficient pathway for inducing bull sperm in vitro capacitation.