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中国农学通报

中国农学通报 ›› 2015, Vol. 31 ›› Issue (2): 107-111.doi: 10.11924/j.issn.1000-6850.2014-1743

所属专题： 生物技术； 水产渔业

• 水产 渔业 • 上一篇    下一篇

酶解制备日本沼虾游离精子方法的研究

颜跃弟1,傅洪拓1,2,乔 慧2,白鸿坤3,孙盛明2,蒋速飞2,   

  1. 熊贻伟2,龚永生2,金舒博2,张文宜2(1上海海洋大学水产与生命学院,上海 200000；2中国水产科学研究院淡水渔业研究中心/农业部淡水渔业和种质资源利用重点实验室,江苏无锡 214081；3南京农业大学无锡渔业学院,江苏无锡 214081）
  • 收稿日期:2014-06-21 修回日期:2015-01-14 接受日期:2014-07-18 出版日期:2015-03-19 发布日期:2015-03-19
  • 通讯作者: 颜跃弟
  • 基金资助:
    中央级公益性科研院所基本科研业务费专项资金项目“青虾性别决定机制及性别控制技术的研究”(2013JBFM15)；江苏省基础研究计划（自然科学基金）青年基金“青虾精巢发育调控基因的鉴别、克隆及表达研究”(BK2012091)；国家“十二五”支撑“青虾新品种选育技术研究”(2012BAD2604-05)；江苏省科技支撑计划-农业部分“优质高产抗逆青虾新品种的选育”(BE2012334)；江苏省三新工程工程项目“青虾新品种选育”(D2013-6)。

Trypsinase Digestion in Acquiring Free Sperms from Macrobrachium nipponensis

Yan Yuedi1, Fu Hongtuo1,2, Qiao Hui2, Bai Hongkun3, Sun Shengming2, Jiang Sufei2,Xiong Yiwei2, Gong Yongsheng2, Jin Shubo2, Zhang Wenyi2   

  1. (1Shanghai Ocean University, Shanghai 200000;2Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture/ Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi Jiangsu 214081;3Wuxi Fishery College, Nanjing Agricultural University, Wuxi Jiangsu 214081)
  • Received:2014-06-21 Revised:2015-01-14 Accepted:2014-07-18 Online:2015-03-19 Published:2015-03-19

摘要： 为了获得数量多且存活的游离青虾精子，首次采用胰蛋白酶消化日本沼虾精荚的方法，获得了游离日本沼虾的精子。通过观察游离精子形态，并经台盼蓝染色，以每克精荚所获得形态正常、结构完整且不被染色的存活精子数为依据，探究胰蛋白酶处理的最佳条件。在pH为7.1的条件下，设计三因素四水平正交试验，设置不同酶浓度(0.2%、0.4%、0.6%、0.8%)、处理时间(5 min、10 min、15 min、20 min)和处理温度(30℃、35℃、40℃、45℃)，根据正交试验结果确定酶处理最佳反应条件。结果表明，酶浓度对获得游离精子影响最大，其次是处理温度和处理时间。酶处理的最适条件为：酶浓度0.8%，处理时间10 min，处理温度40℃。经优化验证试验，表明上述理论组合确为最佳组合。本研究通过酶解法获得较多数量的存活游离精子，为进一步研究日本沼虾精子提供了获得游离精子的方法。

关键词: 营养液浓度, 营养液浓度

Abstract: The purpose of this paper was to acquire free sperms with high quantity from spermatophores of Macrobrachium nipponensis by tryptic digestion. ?Viable sperms of spermatophore per gram were identified by normal morphology, intact structure and not being colored by trypan blue, the optimum enzyme processing conditions were determined according to the results of sperm morphology observing and trypan blue dyeing. The research was conducted under the condition of pH 7.4 and different reaction temperatures (30℃, 35℃, 40℃ and 45℃, respectively), trypsinase concentrations (0.2%, 0.4%, 0.6% and 0.8%, respectively) and reaction times (5min, 10 min, 15 min and 20 min, respectively). The best reaction condition of trypsinase digestion method was studied by orthogonal tests and the sperm amounts which got from each gram of spermatophores were used as a criterion. According to the results of orthogonal tests, we found that trypsinase concentration was the primary factor, followed by reaction temperature and enzymatic reaction time. The best effect of trypsinase digestion was at trypsinase concentration 0.8%, time 10 min and temperature 40℃. This method of acquiring free sperms is useful for further study on sperms of Macrobrachiu nipponensis.