华东师范大学学报(自然科学版) ›› 2006, Vol. 2006 ›› Issue (2): 82-87.

• 生命科学 • 上一篇    下一篇

酶消化法和匀浆法获得河蟹游离精子的比较研究

马 强, 王 群, 李 恺, 丁银娣   

  1. 华东师范大学 生命科学学院，上海 200062
  • 收稿日期:2005-04-14 修回日期:2005-07-08 出版日期:2006-03-25 发布日期:2006-03-25
  • 通讯作者: 王 群

Comparative Study of Trypsinase Digestion and Homogenization in Acquiring Free Sperms from Eriocheir Sinensis(Chinese)

MA Qiang, WANG Qun, LI Kai, DING Ying-di   

  1. School of Life Science, East China Normal University, Shanghai 200062,China
  • Received:2005-04-14 Revised:2005-07-08 Online:2006-03-25 Published:2006-03-25
  • Contact: WANG Qun

摘要：

在pH 7.4的条件下，以不同的温度（27 ℃,32 ℃,37 ℃,42 ℃和47 ℃）、酶浓度（0.05 %,0.10 %,0.15 %,0.20 %和0.25 %）和酶作用时间（5 min,10 min,15 min,20 min和25 min），通过正交试验，研究了胰蛋白酶消化中华绒螯蟹精荚获得游离精子的最佳反应条件.以每克精荚获得的精子数量为判据，发现酶作用时间是主要的影响因子，其次是温度和酶浓度.酶消化的最适反应条件是：温度为37 ℃，酶浓度为0.20 %，作用时间为5 min.酶消化法与机械匀浆法的比较研究发现，酶消化法从每克精荚中获得的精子量为7.41×108个，显著高于匀浆法，且精子的死亡率为4.75 %，显著低于匀浆法.此外，用人工诱导顶体反应法进一步检测酶消化法以及机械匀浆法所获得精子的顶体反应能力，酶消化法获得精子的顶体反应率为62.3 %，而匀浆法为64.5 %，两者无显著性差异.结果表明，酶消化获得游离精子的方法明显优于机械匀浆法.

关键词: 中华绒螯蟹, 河蟹, 精子获得, 胰蛋白酶消化法, 机械匀浆法, 中华绒螯蟹, 河蟹, 精子获得, 胰蛋白酶消化法, 机械匀浆法

Abstract:

The purpose of this paper was to compare two methods of acquiring free sperms from spermatophores of Chinese mitten-handed crab (Eriocheir sinensis). Under the condition of pH 7.4 and different reaction temperatures (27 ℃,32 ℃,37 ℃,42 ℃and 47 ℃, respectively), trypsinase concentrations (0.05 %, 0.10 %, 0.15 %, 0.20 % and 0.25 %, respectively) and reaction times(5 min，10 min，15 min，20 min and 25 min, respectively), the best reaction condition of trypsinase digestion method (TDM) was studied by orthogonal tests and the sperm amounts which got from each gram of spermatophores was used as a criterion. According to the results of orthogonal tests, we found that enzymatic reaction time was the primary factor, and the reaction temperature and the trypsinase concentration also functioned. The maximum effect of trypsinase digesting works at temperature 37 ℃, trypsinase concentration 0.20 % and time 5 min. By using TDM the amounts of free sperms acquired from each gram of spermatophore were 7.41×108 which was significantly higher than that using HM which was only 3.33×108 and the sperm mortality rate was 4.75 % by using TDM which was significantly lower than that using HM which was 27.00 %. Furthermore, the acrosome reaction ability was detected by artificially-induced methodology and the reaction rates were 62.3 % (TDM) and 64.5 % (HM), and there was no significant difference between them. These results suggest that TDM is more effective than HM in acquiring free sperms.

Key words: sperm, homogenization method, trypsinase digestion method, Eriocheir sinensis, sperm, homogenization method, trypsinase digestion method

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