不同冷冻保护剂用于C57BL／6J小鼠精子冷冻的比较研究  

The Comparative Studies on Spermatozoa Cryopreservation in C57BL/6J Mice by Various Cryoprotectants

作　　者：贾青 刘丽均[2] 高娟[1,2] 杨博[2,3] 郁丽丽[2] 徐平[2] 芮荣[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]中国科学院上海实验动物中心,上海201615 [3]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《实验动物与比较医学》2009年第3期147-152,共6页Laboratory Animal and Comparative Medicine

基　　金：国家十一五科技支撑项目（2006BAI23B04）.

摘　　要：目的通过对不同冷冻保护剂用于C57BL／6J小鼠精子冷冻后冻融精子体外受精率的比较，建立一套更适合C57BL／6J小鼠精子冷冻的方法。方法以R18S3为基础液添加不同体积浓度的卵黄和甘油作为冷冻保护剂对C57BL／6J和Y3F小鼠精子进行冷冻，并采用同种方法复苏精子，以体外受精率来评价精子冷冻的效果。结果以R18S3为基础液分别添加体积浓度为5％、10％、15％、20％、25％卵黄离心后获得的上清液作为冷冻保护剂，C57BL／6J小鼠冻融精子体外受精率分别为18.7％、15.6％、36.0％、40.0％、29.7％，差异显着（P〈0.05）；以R18S3为基础液分别添加体积浓度为1％、2％、3％、4％、5％、6％的甘油作为冷冻保护剂，C57BL／6J小鼠冻融精子体外受精率分别为7.8％、10.0％、16.9％、6.0％、10.3％、18.1％，差异显著（P〈0.05）；以R18S3＋5％卵黄离心后获得的上清液＋6％甘油、R18S3＋15％卵黄离心后获得的上清液＋2％甘油、R18S3＋20％卵黄离心后获得的上清液＋1％甘油作为冷冻保护剂，C57BL／6J，小鼠冻融精子体外受精率分别为：16.7％、36.0％、22.9％，差异显著（P〈0.05）：以R18S3作为冷冻保护剂，C57BL／6J小鼠冻融精子体外受精率为25％，异常卵裂率为2.6％。Y3F小鼠冻融精子体外受精率为52.2％，异常卵裂率为5.1％。结论以R18S3为基础液添加15％或20％的卵黄离心后获得的上清液可以有效提高C57BL／6J，小鼠冻融精子的体外受精率但添加不同浓度的甘油却降低C57BL／6J小鼠冻融精子体外受精率。添加卵黄和甘油冷冻效果不如卵黄但好于甘油。Objective To establish a more suitable method to frozen sperm of C57BL/6J by comparing in vitro fertilization rate of post-thawed C57BL/6J mice spermatozoa in various cryoprotectants. Methods To R18S3-based fluid, adding with the different concentrations of egg yolk and glycerine as cryoprotectants to frozen sperm from C57BL/6J and Y3F mice and using the same method of sperm recovery with the rate of in vitro fertilization to evaluate the effect of frozen sperm. Results R18S3 with liquid-based, add with the concentration of 5%, 10%,15%, 20%, 25% of yolk and centrifuged, then the supernatant as cryoprotectants, the rate of in vitro fertilization and frozen-thawed sperm of C57BL/ 6J mice is 18.7%, 15.6%, 36.0%, 40.0%, 29.7% respectively with a significant different. R18S3 with liquid-based,add with the concentration of 1%, 2%, 3%, 4%, 5%, 6% glycerol as cryoprotectants, the rates of in vitro fertilization of freeze-thawed sperm of C57BL/6J mice were 7.8%, 10.0%, 16.9%, 6.0%, 10.3%, 18.1% respectively with a significant difference.The supernatant of R 18S3 ＋5%yolk after centrifugation ＋6% glycerol; the supernatant of R18S3 ＋ 15% yolk after centrifugation ＋2% glycerol; the supernatant of R18S3 ＋20% yolk after centrifugation ＋ 1% glycerol as cryoprotectants, the rates of in vitro fertilization of freeze-thawed sperm of C57BL/6J mice were 16.7%, 36.0% and 22.9% respectively with a significant difference. R18S3 as cryoprotectants, the rate of in vitro fertilization of freeze-thawed sperm of C57BL/6J mice is 25%, the rate of abnormal cleavage is 2.6%; the rate of in vitro fertilization of freeze-thawed sperm of Y3F mice is 52.2%, the rate of abnormal cleavage is 5.1%. Conclusion The supernatant of R18S3 ＋ 15%/20% yolk after centrifugation as cryoprotectants can raise the rate of in vitro fertilization of freeze-thawed sperm of C57BL/6J mice but adding with the different concentrations of glycerol can reduce the rate.The frozen effect of adding with both yolk and glycerol less than the effect of adding only

关 键 词：卵黄 甘油 精子 体外受精 

分 类 号：R714.8[医药卫生—妇产科学] R-332[医药卫生—临床医学]