Effect Of Ooplasmic Transfer On Early Embryonic Development Of Rabbit

Effect of ooplasmic transfer on early embryonic development of rabbitDiscipline: Special economic animal breedingDirective: Control of animal reproductionSupervisor: Prof. Zhang jiahuaCandidate: LijunfengThe fertility of women declines gradually as they get older. After women reach the later phase of fertile age, the environment of body endocrine or ovarian endocrine which is necessary for oocytes growth and maturing is not at optimum state and it is easy to cause ooplasmic defect which can lead to chromosomes abnormality that will cause fertilization failure and early embryonic development stop. Therefore, the oocytes quality decline induced by ooplasmic defect may be the main reason that cause older female sterility and pregnancy failure (abortion). In recent gears, the teachnology of ooplasmic transfer has been used to mend ooplasmic defect and satisfactory results are visible in clinic. In order to get a further sight of the roles ooplasmic content play in fertilization and early embryonic development, we studied the influences of ooplasmic content and heterogenous ooplasm by increasing or decreasing a small amount of the cytoplasm in pronuclear embryo of rabbit or transferred a small amount of homologous or heterogenous ooplasm into rabbit metaphase-II oocyte which were then treated intracytoplamic sperm injection.Early embryos in vitro culture system in one of the basic technologies in embryonic engineering. So, firstly we studied pronuclear embryo of rabbit by sequence culture that is a more developed in vitro culture system. Rabbit zygotes were cultured in sequence culture system that contains: R1 culture medium which is with low concentration of glucose (1.0 mmol/L) and then R2 culture medium which is with high concentration of glucose (3.5 mmol/L). The state of rabbit early embryonic development in sequence culture was appraised by comparing it to that cultured in the conventional RD single culture. The result were followed: after sequence culture, the ratio of morula, shedding zona pellucida, attachment and outgrowth of rabbit pronuclear embryo were 100.0%, 33.33%, 59.72% and 43.06%, and they were all significantly higher (P<0.05) than the result of RD single culture group in which the ratio were 89.65%, 5.71%, 41.38% and 22.41%, but the ratio of blastocyst development and the number of blastocyst cell have no significant difference between the two systems (P>0.05). The result showed that to culture early embryos in Rl culture medium (low in glucose) and R2 culture medium (high in glucose) respectively in sequence can effectively overcome the hold-up of rabbit early embryo development and enhance the abilities of hatch and implantation of embryos. We can conclude, that sequence culture method can meet the physiological need of early embryo and it is more effective than conventional single culture method in accelerating in vitro embryonic development.In the research of ooplasmic transfer, the preparation of DNA templet for individual embryo is one of the key technologies, which are used to determine cytoplasm genetic material in early embryo. So, we did much work on preparation of DNA templet for individual embryo and PCR amplification of mitochondrial DNA. KOH/DTT degraded and DNA extraction were used to prepare DNA templet of individual oocyte and/or early embryo in different development stage, and 3 pairs of primers were used in the general PCR amplification of mtDNA, a set of PCR amplification result of mouse somatic cell mtDNA which was extracted by differential centrifugation was used as positive control. We compared the impacts of differently prepared DNA templets had on PCR amplification efficiency and here's the result: the PCR amplification success rate of single oocyte and KOH/DTT degraded early embryo was 100% (70/70), while the rate of single oocyte treated by DNA extraction buffer was 92.9% (65/70), when we used same 3 pairs of primers in these two experiments. The difference between the two results was significant (P<0.05), and the PCR false positive...