Effect Of Different Factors On In Vitro Fertilization Of Cryopreserved Sperm In Mice

Recently,for the reason that the strains of mice were increased fast,and the transgenic mice used for different research needs were appeared in mass,traditional conservation methods have been overwhelmed.Therefore,sperm cryopreservation has been widely used in laboratory animals which is an effective means of animal germplasm resources conservation.The method is convenient,low cost,which have been widely used by the researchers.However,there were still many problems in this method,for example in the process of cryopreserved sperm in vitro fertilization after recovery,sometimes the fertilization rate is unstable,embryonic development is blocking,and the success rate of produced offspring is lower.In this paper,the factors affecting in vitro fertilization of frozen sperm in mice were studied,and the mechanism of the factors affecting in vitro fertilization of frozen sperm was preliminarily discussed in order to establish the best system of in vitro fertilization of frozen sperm.In this research,Nakagata modified sperm cryopreservation methods was used to conduct the experiments,R18S3 was used as cryopreservation solution for mouse.After recovery,TYH was used as capacitation fluid,HTF was used as fertilization medium,HTF/KSOM was used as the early embryo culture medium.The principles of control variables were used to conduct the fertilization in vitro experiments of different density,different capacitation time,whether added L-GSH to the fertilization medium or not,different strains,different age and genotypes.The results of early embryo development(fertilization rate,cleavage rate,high-quality 4-cell embryo rate and blastocyst rate)were compared and analyzed.The effect of in vitro fertilization was evaluated,and the mechanism of various factors was preliminarily explored in order to establish the best system of in vitro fertilization of frozen-thawed sperm.Results show:The density of frozen sperm was different in mice,the development of in vitro fertilization and early embryos was significantly different.When the sperm density was too low,the effect of in vitro fertilization was very poor.When the sperm density is too high,the fertilization rate decreases slightly,but the cleavage rate is low.And the density was in the range of 0.8-2.0×10~6?/mL,which could obtain better in vitro fertilization results.The effect of in vitro fertilization of frozen sperm at different capacitation time is different.Fertilization rate,cleavage rate,high quality4-cell embryo rate and blastocyst rate were the worst when the capacitation time was too short(5minutes)or too long(90 minutes).When the capacitation time was 20-40 minutes,in vitro fertilization efficiency was the highest.The addition of L-GSH in fertilization medium could significantly increase the fertilization rate and cleavage rate of frozen sperm in vitro,but had little effect on the rate of high-quality 4-cell embryos and blastocysts.The development of early embryos in vitro fertilization of frozen sperm from 8 different strains of mice was significantly different.Different strains of mouse sperm have different biological characteristics at low temperature.Mice in hybrids and closed groups were better than inbred lines.In vitro fertilization of frozen sperm was significantly worse than that of fresh sperm,and the decrease was disproportionate.The results of in vitro fertilization of frozen sperm in mice of different age were also different.Sperm cryopreservation and in vitro fertilization are not suitable for mice under 8 weeks of age.The blastocyst rate of mice over 30-34 weeks of age decreases greatly.The effect of frozen sperm in vitro fertilization and early embryo development is better in mice aged 12-30 weeks.Among them12-24 weeks is best.The in vitro fertilization rate,cleavage rate,high quality 4-cell embryo rate and blastocyst rate of frozen-thawed sperm of different genotypes of mice were significantly different.In summary,the in vitro fertilization of frozen sperm and early embryo development of mouse affected by sperm density,capacitation time,whether L-GSH was added to fertilization medium,strain background,age,genotype and other factors,The optimum density of in vitro fertilization of frozen sperm was 0.8-2.0×10~6?/mL,and the optimum capacitation time is 20 min-40 min.The addition of L-GSH in fertilization medium can effectively improve fertilization efficiency.In order to select the appropriate freezing method to achieve the best recovery effect,attention should be paid to its strain background,age and genotype before cryopreservation of mouse sperm.