Study On Sperm Crypreservation And Intracytoplasmic Sperm Injection In C57BL/6J Mice

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%.