| By research on apoptosis of oocytes and embryos, It would help us to understand the law of embryo development, then regulate the process of apoptosis, and make it develop towards to our needs. Especially in the process of in vitro culture, we can acquire more fine embryos, accelerate the process of animal breeding and provide theoretical and experimental basis to treatment of deseases such as infertility, aging, et al.There are numbers of factors influencing apoptosis of bovine oocytes and early embryos. In this experiment, we mainly analyzed the two factors of ethanol and temperature, detected the apoptosis of NT donorcells, and compared the apoptosis rate of NT embryos with IVF embryos. The results are as follows:1.In the experiment, it's the first time we studied the effect of alcohol on apoptosis of oocytes and embryos by soaked in it. It's very effective of using 75% ethanol to wash the ovaries to prevent contamination. When using this method, we should wash quickly and leave the ovaries exposed to ethanol as shortly as possible. In the treatment of ovaries exposed 10min to ethanol compared to the control, there were no significant differences between the oocytes soon after collected (P>0.05), but significant differences between matured oocytes and parthenogenetic embryos(P<0.05). Apoptosis was involved in normal oocytes and embryos, ovaries exposed to ethanol would promote apoptosis, and accelerated following with time. The cleavage and blastocyst rate was also decraesed.2.Also the first time we studied the effect of temperature of 4℃and RT on apoptosis of matured oocytes. Changes of temperature (heat shock) would induce apoptosis in matured oocytes. Compared to the control, oocytes exposed 1h to 4℃and room temperature(RT; 25℃) were both significant differences(P<0.01). This indicated that it's harmful of oocytes and embryos manipulated in the environment out of incubator, at least increased the apoptosis rate.3.Changes of temperature could also induce apoptosis in IVF embryos. Compared to the control, all the physiological heat shock (36.5℃~40.5℃) induced apoptosis to embryos, and the apoptosis rate elevated following with the temperature and stage of embryo development. The apoptosis almost appeared after 4-cell stage and rare before 4-cell stage. Capacity of apoptosis is a essential mechanism in embryo development, which can eliminate cells demaged by heat shock.4.Donorcells play an important role in NT, so it's necessary to check out the quality of donorcells. In this experiment, we choosed Simmental fibroblasts that were shortly cultured (4 passages) as donorcells. Key biological characteristics analysis and apoptotic detection indicated that, cells of P3 and P4 were all eumorphism, had motility rate above 90% and low apoptosis rate below 2%.5.Apoptosis rates between NT embryos and IVF embryos, there were no significant differences before 4-cell stage. It became significant (P<0.05) at 8-cell stage that the NT apoptosis rate was higher and lasted to blastula stage. It suggested that the low NT efficiency was relevant of the high apoptosis rate. And the induction of apoptosis were possibly because of the complex process, long time of NT and the selected reagents and fluid. On the whole, every step of NT may bring about adverse effect to oocytes and embryos.
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