| Mammalian oocyte cryopreservation has been an important research topic in the conservation of rare animal species,the field of human assisted reproduction and embryos.In the process of vitrification and freezing of oocytes,the factors such as freezing volume,cryoprotectant concentration,freezing and cooling-thawing rate are the key factors that affect the freezing effect,and suitable carriers can reduce the freezing frozen volume,thereby improving the cooling thaw rate.In recent years,although a variety of carriers reported research,but different researchers reported the production methods and effects are not the same.Therefore,the aim of this study is to explore the research methods of different carriers and based on the comparative test of four kinds of frozen carriers,the best vitrification chilled carrier under our existing experimental conditions was screened and the freezing effect of the oocytes at different developmental stages was discussed.Thus laying a technical foundation for improving the cryopreservation effect of oocytes.1.Exploring the fabrication methods of cryopreservative carriersExperiment 1: Optimization of OPS carrier.A tubule is divided into two,With conventional alcohol lamp and simple alcohol lamp as heat source,respectively,rotate the end of the thin tube heating,to be the front end of the tube to the front of the expansion,the rapid use of small tweezers to the other part of the swelling to the other end,after waiting for cooling and fixing,the OPS frozen carrier can be obtained by cutting off the enlarged portion of the front end.Results: Only a simple small alcohol lamp for the heat source can be successfully optimized OPS carrier,and this method not only increases the uniformity of the heat pipe,easy to operate,but also can reduce the material loss.Experiment 2: To explore the method of GMP carrier preparation.Make the oocyte move the needle as raw materials(the inner diameter is about 1 mm,the wall thickness is about 0.09 mm,and the length is about 6 cm).With PC-10 micropipette puller,regular alcohol lamp and simple alcohol lamp as heat source,respectively,to explore the method of preparation of GMP carrier.Make the glass tube as raw materials(outside diameter is 0.8 cm),make the alcohol burner as the heat source,to explore the method of preparation of GMP carrier.Result: Alcohol burner as heat source,fine glass tube for raw materials can be successful GMP carrier is prepared.Experiment 3: Improvement of GMP cryopreservative carrier.he alcohol burner and conventional alcohol lamp as a heat source,respectively,the outside diameter is0.8 cm,0.4 cm and 0.3 cm glass tube rotary heating.Result : the GMP carrier prepared with glass tube with outer diameter of 0.8 cm had no difference with the preparation effect of 0.5 cm.The outer diameter of 0.4 cm and 0.3 cm of fine glass tube with alcohol torch as a heat source,the prepared carrier diameter between 0.43~0.5 mm,leaving its rear end of the load bar was measured from the front of the carrier about 4 cm can be used as GMP Carrier use,but the method of material loss is large;With regular alcohol lamp as heat source,the carrier of preparation by measuring the outer diameter of 0.4 mm to 0.5 mm,wall thickness of 0.03 mm,about 3 cm away from the back-end carrier can be used as carrier of GMP,and in the process of preparation of between the carrier and the carrier bar will have a small glass bubble,when frozen to siphon effect also to have certain role in promoting.The conclusion: The GMP carrier prepared with conventional alcohol lamp,the outside diameter is 0.3 cm and 0.4 cm glass tube was the best.The plastic tube is divided into two,so that one end of the heating method can be based on the original OPS preparation method greatly reduces the loss.2.Effect of different cryopreservative carriers on vitrification of bovine immature oocytesThe extracted COCs were randomly divided into 5 groups: OPS group,GMP group,frozen leaf group,homemade leaf group and control group.Four groups of frozen cells were frozen and thawed by liquid nitrogen to carry out IVM,IVF and IVC,respectively.The fresh control group was not subjected to cryopreservation and were directly subjected to IVM,IVF and IVC,with statistical maturation rates,cleavage rates and blastocyst rates.Result: The normal rate,maturation rate,cleavage rate and blastocyst rate of frozen group were significantly different from those of fresh control group(100%?78.1%±3.1%?64.4%±2.8%?34.7%±2.5%,P<0.05),and there was no significant difference in the normal rate of OPS group and GMP group(74.3%±1.8%,72.5%±2.6%,P>0.05),and both below the Cryoleaf group(82.1%±1.3%,P<0.05).But there was no significant difference in the maturation rate,cleavage rate and blastocyst rate between the three groups(P>0.05).The conclusion: OPS,GMP and Cryoleaf were used as the frozen carrier to successfully vitrify the frozen cow immature oocytes.3.Effects of vitrification of oocytes in different developmental stagesThe extracted COCs were randomly divided into 4 groups: cultured for 0 h,8 h,16 h,and 22 h in vitro.he oocytes were frozen at different times by GMP carrier,the clearage ratio of warmed oocytes cultured 24 h,16 h,8 h,2 h,respectively,after IVM,IVF IVC,statistical analysis on the rate of normal morphology,the maturation rate,cleavage rate and blastocyst rate.Result: The morphological normal rates of the rats were 72.7%,75.3%,77.7% and 78.0% at 0 h,8 h,16 h and 22 h,respectively.There was no significant difference between the four groups(P>0.05),and there were significant differences between the 0 h and 8 h(P<0.05),but the difference of culture development index of 16 h oocytes with the 0 h and 8 h were significantly(P<0.05),and in vitro development of oocytes in the index of 22 h group were higher than those of other developmental stages.The conclusion: the frost resistance of oocytes increased with the increase of maturity. |
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