| In recent years,OPU(Ovum Pick Up)and in vitro embryo production technology have developed rapidly,and they have become an indispensable technical means in the breeding and production of improved cattle breeds.However,the problem of low blastocyst rate of embryos produced in vitro still exists.At present,most embryos adopt ultra-low temperature cryopreservation methods,and the pregnancy rate of frozen embryos is much lower than that of fresh embryos.It is because the freezing and thawing process caused significant damage to the embryo.This experiment mainly studies the optimization of the in vitro embryo production system of bovine OPU oocytes and the method of embryo cryopreservation,in order to improve the production efficiency of in vitro embryos and explore the method of embryo hypothermic storage.The research content and results are as follows:1.Bovine OPU oocytes undergo in vitro embryo production through C-type natriuretic peptide pre-maturation and conventional maturation.The results show that the cleavage rates of the two groups are 77.57 ± 9.18% and 74.91 ± 5.53%,respectively.No significant difference(P>0.05).The blastocyst rates of the two groups were 26.62±3.22% and 17.78±6.18%,respectively.The blastocyst rate of the C-type natriuretic peptide pre-mature group was significantly higher than that of the conventional maturation group(P<0.05).In vitro embryo production was carried out by adding ITS(ITS,Insulin-Transferrin-Sodium selenite)to the early embryo culture medium and conventional culture.The results showed that the cleavage rates of the two groups were 78.33±5.70% and 74.28±3.79%,respectively,and there was no significant difference(P>0.05).The blastocysts rate of the two groups were 28.23±3.64% and 17.90±5.28%,respectively.The blastocyst rate of the ITS group was significantly higher than that of the conventional control group(P<0.05).2.Embryos are stored at low temperature,using TCM199,25 mM HEPES,20%and 50% FBS storage solutions,respectively,to store the embryos at a low temperature of 4 ℃ for 24-168 h,each group contains 60 embryos.The results showed that the survival rate and hatching rate of the 50% FBS concentration group stored for 24 h were the highest,which were 63.33±7.64% and 36.67±2.89%,respectively.Embryos stored at low temperature for the same time,the embryo survival rate and hatching rate of the 50% FBS concentration group were higher than those of the 20% FBS concentration group.Within 48 h of cryopreservation,the survival rate and hatching rate of embryos were not significantly different(P>0.05).After hypothermic storage for more than 48 h,the survival rate and hatching rate of embryos were significantly reduced(P<0.05).Add 5mg/mL,10mg/mL,15mg/mLε-polylysine in TCM199,25 mM HEPES,20% and 50% FBS storage solution,and store the embryos at low temperature 4℃ for 72 h,120 h,168 h,each group contains60 embryos.The results show that ε-polylysine can have a certain effect but not obvious when stored at low temperature for 72 h.The appropriate concentration ofε-polylysine is 10 mg/mL.After storing at low temperature for more than 72 h,ε-polylysine has no effect.In summary,both C-type natriuretic peptide prematuration and addition of ITS can improve the production efficiency of in vitro embryos.The effect of adding ITS is slightly better.The embryos are stored at low temperature for 48 h without much impact.ε-polylysine has no effect on embryos.The effect of hypothermic storage is not obvious. |
