| Compared with other domestic animals,the sperm motility,oocyte utilization rate and embryo survival rate of sheep are generally low during in vitro embryo production.Intracytoplasmic sperm injection(ICSI)technology can effectively solve the above problems,make full use of sperm and oocytes,and effectively improve the survival rate of embryos.However,there is still a problem of low blastocyst rate.In order to optimize the in vitro production system of sheep ICSI embryos and improve the blastocyst rate.In this study,oocytes were cultured at different time to determine the best time for ICSI.Then determine the optimal concentration of Estrus sheep serum(ESS)in embryo culture medium.Finally,the development of embryos after ICSI was compared between conventional sperm and sex-controlled sperm,and the feasibility of injecting sex-controlled sperm was proved by sex identification using enamel gene(AMEL).The research mainly includes the following three aspects :(1)In the first experiment,in order to study the effect of in vitro maturation of sheep oocytes at different time points on ICSI embryo culture.In this study,sheep oocytes were matured for 22 h,23h,24 h,25h,26 h,immediately degranulation cells,followed by ICSI operation,compared the blastocyst rate.The results showed that the highest blastocyst rate(33.71 %)was obtained when sheep oocytes were matured for 24 h by ICSI,which was significantly higher than that of 22h(25.92 %)and 26h(27.14 %)(P<0.05).However,there was no significant difference in blastocyst rate between ICSI and 23h(33.32 %)and 25h(32.38 %)(P>0.05).This indicated that sheep oocytes could effectively improve the blastocyst rate after ICSI after maturation for 24 h.(2)In the second experiment,to optimize the culture effect of sheep ICSI embryos.In this study,different concentrations of ESS(0 %,5 %,10 %,15 %,20 %)were added to ICSI embryo culture medium,and the blastocyst rate was compared after ICSI.The results showed that the blastocyst rate(38.45 %)was significantly better than that of 5 %(34.08 %),15 %(34.77 %),0 %(30.94 %)and20 %(31.16 %)(P<0.05)after the sheep oocytes were cultured in ICSI embryo culture medium supplemented with 10 % ESS.It shows that adding 10 % ESS in sheep ICSI embryo culture medium can effectively improve the blastocyst rate.(3)In the three experiment,to study the effect of injection of sex-controlled sperm on ICSI embryo culture in sheep.In this study,conventional sperm,sex-controlled X sperm and sex-controlled Y sperm were used for ICSI operation,and the rate of blastocysts obtained from late culture was compared,and the sex of embryos was identified,so as to determine the feasibility of using ICSI technology to produce controlled embryos.The results showed that there was no significant difference in the rate of blastocysts obtained by injecting conventional sperm,X sperm and Y sperm(40.78 % vs 38.54 % vs 39.39 %)(P>0.05).AMEL was used to identify the sex of embryos.It was found that the sex of embryos was consistent with the characteristics of injected sex-controlled sperm,and no parthenogenetic activation occurred.It is feasible to produce sheep sex-controlled embryos by ICSI.In summary,this study determined the optimal time of in vitro maturation of sheep oocytes and the optimal concentration of ESS in embryo culture medium,optimized the in vitro production system of sheep ICSI embryos,and revealed that there was no significant difference in the blastocyst rate of sex-controlled embryos between conventional,X,Y sperm ICSI(P>0.05).It provides further technical support for the development of sheep in vitro embryo production system and intracytoplasmic sperm injection technology,and lays a foundation for later experimental research. |
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