中间球海胆精子超低温冷冻保存方法的研究

对中间球海胆Strongylocentrotus intermedius精子的超低温保存技术进行了研究。以煮沸消毒海水为基础液,添加体积分数为10%的DMSO、体积分数为6%的甘油以及25 mmol/L海藻糖配制成冷冻保护液,与鲜精液按体积比以1∶1混合,在4℃下平衡15 min,于液氮面上方15、3 cm处分别停留3 min和5 min,然后浸入液氮保存。结果表明：用此方法保存的精子解冻后其存活率可达58.2%,受精率达24.7%;解冻后精子受精时,在受精海水中分别添加0、28、56、112、280 mmol/L的葡萄糖,56 mmol/L组的受精率高于其他组,受精率达26%;精子解冻后受精时,在受精海水中添加适量葡萄糖有助于提高受精率。本试验表明,冷冻过程中降温方式、冷冻保护液、平衡时间对精子的冷冻保存效果均有较大影响,各个因素通过优化组合可以大大提高解冻后精子的存活率和受精率。

Cryopreservation of spermatozoa in sea urchin Strongylocentrotus intermedius

The spermatozoon cryopreservation were conducted in sea urchin Strongylocentrotus intermedius.The higher pose-thawed sperm survival rate（58.2%）and fertilization rate（24.7%）were observed in the cryoprptectant comprised of natural sea water diluted with 10% DMSO,6% glycerin and 25 mmol/L trehalose as the cryoprotector and mixed with sperm at a ratio of 1∶1,equilibration at 4 ℃ for 15 min,swung at 15 cm above LN2 for 3 min and then 3 cm above LN2 for 8 min,finally transferred into LN2 for storage.When the post-thawed sperm was fertilized,the seawater were added to the sperm at a rate of 0,28,56,112 mmol/L,and 280 mmol/L glucose.There was higher fertilization（26%） in the sperm supplemented with 56 mmol/L glucose than that in the other groups,indicating that freezing method,cryoprptectant and equilibration time all had influence on cryopreservation of the spermatozoa in the freezing process.The survival rate and fertilization rate of the pose-thawed sperm were greatly improved through various factor optimization such as addition of appropriate glucose.