酶解制备日本沼虾游离精子方法的研究

Trypsinase Digestion in Acquiring Free Sperms from Macrobrachium nipponensis

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p H 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。

The purpose of this paper was to acquire free sperms with high quantity from spermatophores of Mac- robrachium nipponensis by tryptic digestion. Viable sperms of spermatophore per gram were identified by nor- mal morphology, intact structure and not being colored by trypan blue, the optimum enzyme processing condi- tions were determined according to the results of sperm morphology observing and trypan blue dyeing. The re- search 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 （5 min, 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 prima- ry factor, followed by reaction temperature and enzymatic reaction time. The best effect of trypsinase digestion was at trypsinase concentration 0.8%, time 10 rain and temperature 40℃. This method of acquiring free sperms is useful for further study on sperms of Macrobracbiu nipponensis.