【作者】 狄敏；

【导师】 薛整风； 徐平；

【作者基本信息】 扬州大学 ， 动物学， 2007， 硕士

【摘要】 随着生命科学的发展,作为研究生命科学基础条件之一的实验动物也在我国得到长足的发展。而小鼠在众多的实验动物中应用最为广泛。因此建立一套有效的小鼠种质资源保存方案显得尤为重要,胚胎工程学和低温生物学的联合应用在各种实验动物的保种中已获得成功。小鼠精子冷冻技术成为胚胎保种的有效方法之一,复苏精子体外受精技术对于相当一部分品系小鼠来说已很成熟,但是对于一些特殊品系小鼠的冻融精子体外受精率仍然很低,卵母细胞胞浆内单精子注射技术成为解决这一问题行之有效的方法。本文采用R18S3和FERTIUPTM-CPA两种冷冻保护剂,对DBA/2、C57BL/6J、KM和B6.129S7 -Ldlr^tm1Her/J四个品系的小鼠精子进行冷冻,冷冻精子用三种方法复苏,以体外受精率来评价精子冷冻的效果。三种复苏方法分别为:（1）冷冻精子37℃水浴15min,解冻液为HTF;（2）冷冻精子60℃水浴6s, 37℃水浴15min ,解冻液为HTF;（3）冷冻精子60℃水浴6s, 37℃水浴15min,解冻液为FERTIUP TM-PM。以R18S3作为冷冻保护剂,DBA/2（73.3%,88.4%,55.6%）和KM（64.9%,60.2%,39.6%）品系小鼠冷冻精子复苏后体外受精率差异显著（P<0.05）,C57BL/6J（3.0%,10.3%,3.7%）和B6.129S7 -Ldlr^tm1Her/J（0%,5.0%,0%）结果差异不显著（P>0.05）;以FERTIUPTM-CPA作为冷冻保护剂,DBA/2（33.6%,14.1%,91.6%）、C57BL/6J（8.4%,21.0%,4.9%）和B6.129S7-Ldlr^tm1Her/J（8.2%,10.0%,28.9%）品系小鼠冷冻精子复苏后体外受精率差异显著（P<0.05）,而KM（48.1%,48.0%,48.1%）小鼠冷冻精子复苏后体外受精率差异不显著（P>0.05）。对于DBA/2和KM品系小鼠来说,用R18S3或FERTIUPTM-CPA冷冻精子,选择一种恰当的复苏方法,均可以得到较理想的体外受精率,而C57BL/6J和B6.129S7-Ldlr^tm1Her/J品系小鼠无论采用哪种冷冻保护剂,选择何种方法复苏精子,得到的体外受精率都较低。为了提高C57BL/6J和B6.129S7-Ldlr^tm1Her/J品系小鼠冷冻复苏精子体外受精率,本实验采用卵母细胞胞浆内单精子（ICSI ）注射技术。将C57BL/6J和B6.129S7-Ldlr^tm1Her/J品系小鼠的冷冻精子37℃水浴15min复苏,复苏精子利用Piezo装置分离精子头尾,将精子头注入卵母细胞胞浆。C57BL/6J与B6.129S7-Ldlr^tm1Her/J小鼠冷冻复苏精子体外受精率分别达到44.7%和50.0%,与冻融精子常规体外受精（3.5%,4.3%）相比,结果有明显提高。更多还原

【Abstract】 With the development of life science, as one of basis of life science, the laboratory animal has gained a degree in our country as well. Whereas mouse have the widest idioplasm using in many laboratory animals. So establishing a effective mouse idioplasm resources keeping protocol seems especially important. Embryo engineering and cryobiology using together has made other resources of kinds of laboratory animals keeped successfully . Mouse sperm cryopreservation is as one of the valid skills of keeping embryos. To a considerable part of strains mouse, In vitro fertilization of thawed sperms is very mellow, but to a part of particular strains mouse, the rate of IVF of thawed sperm is still low. As a result, Intracytoplasmic sperm injection（ICSI） in mouse is applied to resolve this situation.R18S3 and FERTIUPTM-CPA were used as cryopreservation protection solutions to freeze DBA/2、C57BL/6J、KM and B6.129S7 -Ldlr^tm1Her/J four strains mouse sperm. Three thawed methods were betaken, rate of IVF（In Vitro Fertilization） assessed the effect of frozen-thawed sperm. Three methods were: （1） Frozen sperm kept water bath at 37℃for 15min to thaw, thawed solution is HTF, （2） Frozen sperm kept water bath at 60℃for 6s then 37℃for 15min to thaw, thawed solution is also HTF, （3） Frozen sperm kept water bath at 60℃for 6s then 37℃for 15min to thaw, thawed solution is FERTIUP TM-PM. When R18S3 was used to freeze sperm,there were distinct difference in the rate of IVF of DBA/2（73.3%,88.4%,55.6%）and KM（64.9%,60.2%,39.6%）among three thawed methods （ P<0.05 ） ,but the results of C57BL/6J（3.0%,10.3%,10.3%） and B6.129S7-Ldlr^tm1Her/J （0%,5.0%,0%） were not notably different（P>0.05）; the ratio of IVF of DBA/2 （ 33.6%,14.1%,91.6% ）、C57BL/6J （ 8.4%,21.0%,4.9% ） and B6.129S7-Ldlr^tm1Her/J（8.2%,10.0%,28.9%）was remarkably different（P<0.05） while using FERTIUPTM-CPA, the rate of IVF of KM（48.1%,48.0%,48.1%）was not different（P>0.05）. For DBA/2 and KM mouse sperm ,frozen with either R18S3 or FERTIUPTM-CPA ,chosen a fit thawed method can both get a ideal rate of IVF. No matter what using any cryopreservation protection solution, choosing any thawed method,rate of IVF of C57BL/6J and B6.129S7-Ldlr^tm1Her/J thawed sperm were relatively low.In order to improve the rate of In Vitro fertilization of C57BL/6J and B6.129S7-Ldlr^tm1Her/J mice′s frozen sperm , Intracytoplasmic sperm injection（ICSI） was used in this experiment. Frozen sperm of C57BL/6J and B6.129S7-Ldlr^tm1Her/J mice thawed at 37℃water for 15min . The head of sperm was separated by Piezo-electric actuator,then injected into the cytoplasm of mouse oocytes. The rate of IVF of C57BL/6J and B6.129S7-Ldlr^tm1Her/J mice reached 44.7% and 50.0% respectively. Compared to the ordinary IVF of frozen-thawed sperm（3.5%,4.3%）, the result was enhanced obviously.更多还原