In Vitro Maturation Of Porcine Oocytes And Optimization Of Embryo Culture Medium

Obtaining high-quality in vitro mature oocytes is the first step of embryo engineering technology such as in vitro fertilization,somatic cell cloning and transgenic animal preparation.However,compared with in vivo mature oocytes,maturation rate,maturation quality and development potential of mature oocytes in vitro are significantly different.Therefore,improving the oocyte maturation system in vitro is the key problem to be solved urgently in embryo engineering technology.Add in system this study analyzed the cultures of follicular fluid,protein,carbohydrate,protein and hormone on pig oocyte in vitro maturation,and found in the process of study the relationship between estrogen and leukemia inhibitory factor,and preliminary discussion on the estrogen（E₂）and leukemia inhibitory factor（LIF）in the oocyte maturation and embryo development coordination promote relations,The research contents and results are as follows:（1）The effects of exogenous proteins and their analogues on oocyte maturation and subsequent embryonic development were explored.M199 was selected as the medium,in which 10%FBS,0.1%PVA and 4 mg/m L BSA were added as the exogenous proteins and their analogues,and no proteins and their analogues were selected as the control group.The experimental results showed that the maturation rate of oocytes added with 10%FBS was higher than that of the control group,but significantly lower than that of the mature culture medium added with 0.1%PVA and 4 mg/m L BSA（p<0.05）.In terms of cleavage rate,the cleavage rate of 10%FBS group was higher than that of 0.1%PVA group and the control group,but there was no significant difference with 4 mg/m L BSA group（p>0.05）,while the blastocyst rate of 10%FBS group was significantly higher than that of the other three groups,4 mg/m L BSA group was significantly lower than 0.1%PVA group（p<0.05）,but they were significantly higher than those in the control group（p<0.05）（2）The effects of different follicular fluids on the in vitro maturation and embryonic development of porcine oocytes were explored.The results showed that whether the follicular fluid with or without corpus luteum was added to the mature culture medium had no significant effect on the maturation rate（p>0.05）,but the expansion of single COCs in the follicular fluid with corpus luteum was significantly higher than that with the addition of follicular fluid without corpus luteum（p>0.05）.（3）LH and FSH were added as hormones in the mature culture medium,and LH was used as a variable.It was found that adding 0.02 IU/m L of LH to the culture medium had a significantly higher maturation rate than the other three groups（p<0.05）.As the concentration increased,the single COCs spread was further enhanced.（4）The cleavage rate and blastocyst rate of oocytes after electrical activation were significantly higher than those in the chemical alone group and the electrical activation plus chemical activation group（p<0.05）,while the combined electrical activation and chemical activation After use,the cleavage rate and blastocyst rate were significantly lower than the other two groups（p<0.05）.（5）The effect of glucose on parthenogenetic embryo development was explored.The results showed that in terms of cleavage rate,there was no significant difference between the3.05 m M group and the no glucose group,both of which were significantly higher than the11.00 m M group and 4.00 m M group（p<0.05）,while the 4.00 m M group was significantly lower than the 11.00 m M group（p<0.05）.In terms of blastocyst rate,the blastocyst rate in the non-glucose group was significantly higher than the other groups,the non-glucose group was lower than the 5.55 m M group,but significantly higher than the other three groups（p<0.05）,and the 4.00 m M group was higher than 3.05m M.There was no significant difference between the 3.05 m M and 11.00 m M groups.In terms of morula rate,the 3.05 m M group was significantly higher than the other four groups（p<0.05）,the 4.00 m M group was significantly higher than the 5.55 m M group and the 11.00m M group,and the glucose-free group was significantly lower than the other four groups（p<0.05）.It can be seen that at different stages of embryo development,embryos cultured in vitro have different requirements for glucose（6）The relationship between the interaction of different concentrations of estrogen and LIF on porcine oocyte maturation and embryonic development was explored.The results showed that in terms of maturation rate,there was no significant difference in the maturation rate of the LIF-added group（p>0.05）,but the maturation rate of adding 1 u M estrogen was significantly lower than that of adding 1μg/m L estrogen（p<0.05）,and the expansion degree of COCs to a single oocyte,the addition of LIF+0.05μg/m L E₂ was significantly higher than the other groups（p<0.05）,while the addition of LIF+1μg/m L E₂ group was significantly lower than the other groups（p<0.05）,the ROS level of adding E₂ and LIF alone was significantly higher than that of the other groups（p<0.05）,while there was no significant difference in the ROS level between the LIF+E₂ groups（p>0.05）.The results showed that the co-addition of LIF and E₂.It was able to reduce ROS levels between mature oocytes,but the ability to reduce ROS was independent of the added dose.（7）The interaction between estrogen and LIF on embryonic development under different concentrations of glucose was explored.According to the results,in the 0 m M glucose group,the cleavage rate,blastocyst rate and the number of blastocyst cells,estrogen and LIF on embryo development were significantly higher than those in the other groups（p<0.05）,but in the 5.55 m M glucose group Among them,the promoting effect of estrogen and LIF on embryo development was higher than that of 3.05 m M glucose group and 11.00m M glucose group,while the blastocyst rate and the number of blastocyst cells in the 3.05m M glucose group and 11.00m M glucose group were significantly lower than those in the other groups（p<0.05）.In terms of the number of apoptotic blasts,0 m M glucose group was significantly lower than the other groups,indicating that the interaction between estrogen and LIF was most significant in the addition of 0 m M glucose group（p<0.05）.The above results showed that the maximum number of blastocysts and blastocyst cells could be obtained by electric stimulation of 1.2 k V/cm,60μs and electric pulse twice.The maximum number of blastocysts and blastocyst cells could be obtained by culture medium without glucose,and 20 ng/m L LIF and 20 ng/m L IGF-1 and 40 ng/m L FGF-2 and 1μg/m L E2 were added into mature medium could significantly decrease the ROS level of mature oocytes,decrease the apoptotic number of blastocyst cells,and increase the development rate of blastocyst（p<0.05）.