【作者】 贾青；

【导师】 芮荣； 徐平；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2008， 硕士

【摘要】 随着生命科学研究的日益发展,实验动物科学也在我国得到快速的发展,小鼠是应用最为广泛的实验动物之一。C57BL/6J品系小鼠作为应用最广泛的一种近交系小鼠,因其遗传背景稳定,是研究基因突变的重要工具;多种转基因小鼠均以C57BL/6J品系小鼠为遗传背景。如何开展这些基因工程小鼠的保种工作,建立一套有效的小鼠种质资源保存方案显得尤为重要。胚胎生物技术和低温生物学技术的联合应用在小鼠保种中已获得成功,冷冻精子和胚胎业已成为建立冷冻库、保存特定品系最有效的策略。精子冷冻的技术操作方便、快速、无需特殊的设备,应用前景十分广阔。小鼠精子冷冻已成为小鼠种质资源保存的有效方法之一,其冷冻精子体外受精技术对于部分品系的小鼠来说已很成熟,但对于一些特殊品系如应用最广的C57BL/6J近交系小鼠及以C57BL/6J为遗传背景的转基因小鼠,冷冻精子体外受精卵裂率仍然很低。本实验以C57BL/6J小鼠为研究对象,旨在通过对该品系小鼠精子冷冻及冷冻精子的体外受精、单精子胞浆内注射技术的研究,全面提高该品系小鼠体外受精卵裂率,为C57BL/6J小鼠的资源保存奠定基础。1 C57BL/6J小鼠精子冷冻技术研究本试验主要以C57BL/6J品系小鼠为研究对象,系统比较冷冻稀释液、解冻方法、获能时间和获能液、体外受精液、胚胎体外培养液等对该品系小鼠冷冻精子体外受精卵裂率与胚胎发育率的影响。结果表明:（1）以R₁₈S₃为基础液添加15%或20%卵黄离心上清液作为冷冻稀释液,可有效提高C57BL/6J小鼠冷冻精子体外受精卵裂率（分别为36%和40%）,但添加不同浓度的甘油或乙酰胺却会降低冻精体外受精卵裂率,混合添加卵黄和甘油冷冻效果不如单独添加卵黄,但比单独使用甘油效果要好;（2）采用37℃水浴15min和先60℃水浴6s再37℃水浴解冻15min 2种方法解冻精子,C57BL/6J小鼠冻精体外受精卵裂率无显著差异（P＞0.05）,因一步解冻法操作简便,可作为常规解冻程序;（3）分别采用30、50和60min为C57BL/6J小鼠冻精的获能时间,体外受精卵裂率分别为17.3%、25%和19.4%,以50min获能效果最佳;（4）在100μl HTF体外受精液中分别添加1.0 IU/ml FSH和0.1ug/ml E₂,冷冻精子体外受精卵裂率分别为24.1%和27.3%,与不添加对照组（25%）相比没有明显提高（P＞0.05）;（5）在100μl HTF中分别添加45μmol/L肾上腺素、75μmol/L亚牛磺酸、5 IU/ml肝素、7.5 mmol/L牛磺酸,以及不含BSA的TYH中添加0.75 mmol/L MBCD作为精子获能液,结果只有添加5 IU/ml肝素可提高冷冻精子体外受精卵裂率,但无统计学差异（P＞0.05）;用R₁₈S₃+15%卵黄上清液冷冻精子时,添加5 IU/ml肝素的体外受精卵裂率最高（49.3%）;若用R₁₈S₃+20%卵黄离心上清液冷冻,添加45μmol/L肾上腺素组所获体外受精卵裂率和囊胚发育率最高;（6）体外培养实验表明,KSOM更适合桑椹胚前的发育,而CZB更适合桑椹胚向囊胚的发育;（7）采用R₁₈S₃+20%卵黄离心上清冷冻C57BL/6J小鼠精子,并配合使用100μL HTF+45μmol/L肾上腺素作为精子获能液,体外受精所获2-细胞胚胎20枚移植至1只受体小鼠,产仔4只,产仔率为20%。2 C57BL/6J小鼠ICSI技术的研究以C57BL/6J品系小鼠及DBA♂×C57BL/6J♀（B6D2FI）杂交鼠为研究对象,以Piezo操作系统为技术支撑;通过小鼠卵母细胞胞浆内精子注射（ICSI）技术,系统比较不同注射针内径、不同显微操作液、不同采卵时间对ICSI结果的影响,并比较鲜精、冻精体外受精和ICSI显微受精结果。结果表明:（1）使用内径为4～6μm的注射针,C57BL/6J小鼠ICSI胚胎卵裂率为88.5%,显著高于8～10μm内径组（85.7%对53.8%,P＜0.05）;（2）C57BL/6J小鼠注射hCG后19h采卵进行ICSI操作,卵子存活率虽比注射后12或14h采卵组降低,但其卵裂率最高（92.7%）,囊胚发育率也最高（36%）;（3）C57BL/6J和B6D2F1小鼠卵使用Hepes-CZB显微操作液,ICSI操作后的卵子存活率分别为36.1%和61.2%,卵裂率为82.4%和63.1%,囊胚发育率为50%和45.8%;当操作液中添加3%蔗糖时,卵存活率分别为59.6%和80.2%,卵裂率为90.3%和82.1%,两品系小鼠存活率和卵裂率较对照组都有显著提高（P＜0.05）;囊胚发育率为35.7%和26.3%,有显著降低（P＜0.05）;当操作液中添加0.5mg/L细胞松弛素B,ICSI操作后的卵子存活率为49.1%和73.0%,卵裂率为90.0%和82.0%,两品系小鼠存活率、卵裂率都得到显著提高（P＜0.05）;囊胚发育率为37.0%和33.0%,显著下降（P＜0.05）;（4）C57BL/6J小鼠鲜精常规体外受精的卵裂率为90.4%,与其冻精体外受精后的卵裂率（25.0%、36.0%、40.0%）相比差异显著（P＜0.05）,与冻精单精注射后的卵裂率（53.8%）也差异显著（P＜0.05）;但冻精ICSI卵裂率与冻精常规体外受精后的卵裂率相比,明显提高（P＜0.05）。冷冻精子ICSI后囊胚发育率显著低于新鲜精子常规体外受精后体外培养的囊胚率（56.3%对71.6%,P＜0.05）;（5）用添加3%蔗糖的Hepes-CZB作为显微操作液,经R₁₈S₃冷冻稀释液冷冻保存的C57BL/6J小鼠精子解冻后进行ICSI注射,所获40枚2-细胞胚胎移植给ICR受体假孕小鼠,获得着床8d胚胎13枚,妊娠率为32.5%。更多还原

【Abstract】 With the increasing development of life science,laboratory animal science was also developed rapidly in our country.Mice are one kind of laboratory animals which were most widely used.C57BL/6J strain mice,a widely used strain,become an important model animal for gene mutation research due to its stable genetic background and various of gene transfer mice did originate from this strain of mice.It became more important for how to carry out genetic resource protection of these genetic engineering mice and to establish an efficient conservation protocol of murine resources.Combination of embryo biotechnology with cryobiologic techniques succeeded in murine breeder conservation.Frozen spermatozoa and embryos became the most efficient strategy to establish the freezing bank to keep specific strains.Sperm freezing is quite convenient,quick to operate without special equipment,having wide foreground in future application and it is particularly suitable for the conservation of genetic resources in mice.The in vitro fertilization（IVF） using frozen sperm is a matural technique for partial strains of mice.However,for some special strains of mice such as C57BL/6J mice and their gene transfer mice,the cleavage rate of IVF embryos with frozen sperm is still very low.The purpose of this study is intended to improve the IVF rate of C57BL/6J mice via modification of murine sperm cryopreservation and IVF,ICSI techniques,so as to establish a basis for the conservation of genetic resource of C57BL/6J mice.1 Technological study on sperm crypreservation in C57BL/6J miceThe C57BL/6J strain mouse were used in present experiment in order to compare the effects of freezing extender,thawing procedures,sperm capacitation time and fluid,IVF medium and embryo culture medium on the resulting cleavage rate and subsequent developmental rate of embryos.The results were showed as follows.（1） R₁₈S₃ solution was the basic extender and addition of 15%or 20%yolk supernatants could improve the IVF rates efficiently（36%and 40%,respectively）.The addition of different concentrations of glycerol or acetamide would decrease the cleavage rates of IVF embryos.The addition of yolk+glycerol yielded worse freezing result than that from yolk addition alone but better than that using glycerol alone.（2） Two thawing methods of frozen sperm were used:37℃ water bath for 15min and 60℃water bath for 6s first followed a 37℃water bath for 15 rain.They didn’t result in different IVF cleavage rates（P＞0.05） and the one-step thawing method could be used routinely due to a simplified operation.（3） When the sperm capacitation time were 30,50 and 60min respectively,the cleavage rate of postthaw C57BL/6J sperm were 17.3%,25%and 19.4%and the best sperm capacitation time was 50 min.（4） Addition of 1.0 IU/ml FSH or 0.1μg/ml E₂ into 100μl HTF solution resulted in 24.1%and 27.3%of cleavage rates for cryopreservated sperm IVF and no difference with the control（25%,P＞0.05）.（5） When adding 45μmol/L Adrenaline,75μmol/L Hypotaurine, 5 IU/ml Heparin or 7.5 mmol/L Taurine into 100μl HTF solution used as sperm capacitation or adding 0.75 mmol/L MBCD into 100μl TYH solution without BSA,the IVF cleavage rate of only 5 IU/ml Heparin group could be increased slightly.When the freezing extender is the R₁₈S₃+15%yolk supernatant,the addition of 5 IU/ml Heparin yielded the highest cleavage rate of 49.3%.When the freezing extender is the R₁₈S₃+20% yolk supernatant,addition of 45μmol/L Adrenaline could resulted in the highest cleavage rate and developmental rate to blastocysts.（6） The IVC results showed that KSOM is more suitable for the development of embryos ahead of morula stage,while CZB is better for the development of morula to blastocysts.（7） Using R₁₈S₃+20%yolk supernatant,addition of 45μmol/L Adrenaline into the capacitation solution,twenty 2-cell stage embryos harvested from fertilization in vitro were transferred into the oviducts of one recipient mice,giving birth 4 offspring and the birth rate was 20%.2 Technological study on the ICSI in C57BL/6J miceIn this experiment,the Piezo operating system was used as the technical support of ICSI. On the basis of ICSI operation,C57BL/6J strain mice and DBA♂×C57BL/6J♀（B6D2FI） hybrid mice were used as experimental animals to systematically compare the effects of different internal diameter of injection pipette,micromanipulation solution and oocyte collection time on ICSI results in B6D2F1 and C57BL/6J mice,as well as to compare the IVF rate and the ICSI result derived from fresh and frozen-thawed sperm of the mice.The results were showed as follows:（1） When the internal diameter of injection pipette was 4～6μm,the cleavage rate of C57BL/6J mouse ovum in ICSI was 88.5%,which is obviously higher than that from 8～10μm group.（2） When oocytes were collected at 19h after iniection of hCG,the survival rate of oocyte is lower than that of 12h group but the cleavage rate was the highest（92.7%）,so was the blastocyst rate.（3） Hepes-CZB was used for the micromanipulation solution on ICSI in C57BL/6J and B6D2F.When injection 1h later,the survival oocyte rate respectively were 36.1%and 61.2%,the cleavage rates were 82.4%and 63.1%,the blastocyst rate which respectively were 50%and 45.8%.When added 3%sucrose in operation fuild,the oocyte surxival percentage of C57BL/6J and B6D2F1 were 59.6%and 80.2%,the survival percentage of two strain mouses were obviously enhancesd and the difference extremely remarkable（P＜0.01）,moreover the cleavage rate of two strain mouses respectively were 90.3%and 82.1%,also had the distinct enhancement,the difference were remarkable（P＜0.05）,the blastocyst rate of two strain mouses respectively were 35.7%and 26.3%,which were obviously reduced that the differences were remarkable（P＜0.05）.When added 0.5mg/L CB into Hepes-CZB which was used as operation fuild,injection 1h later,the oocyte surxival percentage of C57BL/6J and B6D2F1 respectively were 49.1%and 73.0%,the survival percentage of two strain mouses had remarkable enhancement,the differences were remarkable（P＜0.05）,moreover counted the cleavage rate which respectively were 90.0%and 82.0%on the next morning,two rates were obviously enhanced,the differents were remarkable（P＜0.05）,the blastocyst rate which respectively were 37.0%and 33.0%which were obviously reduced,the difference is remarkable（P＜0.05）.（4） When the fresh sperm of C57BL/6J mouse was used for convention fertilization in vitro,the Cleavage rate is 90.4%,compared with the Cleavage rate which used freezed sperm in fertilization in vitro,the difference was extremely remarkable（P＜0.05）,it compared with the cleavage rate of ICSI which was 53.8%,the difference was also extremely remarkable（P＜0.05）.The cleavage rate of ICSI compared with the Cleavage rate in vitro fertilization on post-thawed of C57BL/6J spermatozoa was distinct enhancement and the difference is remarkable（P＜0.05）,moreover the blastocyst rate of ICSI was obviously lower than the Cleavage rate in fertilization on fresh sperm the difference is extremely remarkable（P＜0.05）.（5） When Hepes-CZB supplemented with 3% sucrose was used as micromanipulation solution and R₁₈S₃ was the freezing extender,the frozen-thawed murine sperm was injected into ovum,by which forty 2-cell stage ICSI embryos were transferred into recipient and 13 fetuses were obtained at Day 8 of gestation. The pregnant rate was 32.5%.更多还原