Studies On The Micromanipulation Methods Of Oocytes For Somatic Cell Cloned In Yanbian Yellow Cattle

The aim of this experiment was to explore a simple, low-injury, to remove nuclear accuratemethods for Yanbian cattle and it's also to establish a suitable injection method for somatic cell cloned.In order to lay the foundation for improving the overall efficiency of somatic cell cloned.Methods: To compare two different oocyte's enucleation methods, the experimental results wereassessed for operating efficiency, cleavage rate and growth rate, thus verifying the reliability andviability of this enucleation method. Meanwhile, to confirm the time that was the best time for thismethod to remove nucleus. Removing the cytoplasm, as well as the best methods to conduct research, and to establish the best injection method for Somatic Cell Cloned Cell Cloned.Results:1.To compare with two different methods: The modified Willadsen's method, which was calledsqueezing method, in the operating time of removing nuclei (41s/one) or enucleation rate (80.70％)were significantly superior to blind adsorption (76s/one, 65.00％. P＜0.05). Moreover, the blindadsorption's cleavage rate was 42.15％, and cleaved rate of the extrusion method (modified Willadsen)was 5t.40％. The difference between them wasn't significant (P＞0.05). The squeezing method(modified Willadsen's method) is an efficient and simple enucleation method with the advantage ofsaving manipulating time and none-damage to Yanbian yellow cattle oocytes.2. To further determine the best time of removing nuclei for this enucleation method, byHoechst 33342 staining, Fluorescence microscopy observation, the results showed that the distance isincreasing between Nuclei and polar body with extending maturation time of oocytes in vitro forYanbian yellow cattle. The rate of removing nuclei for squeezing method, which is according to apolar body for signs of enucleation methods, maturation rate of Yanbian yellow cattle oocytesincreases as the IVM duration prolongs, while the enueleation rate decreases. The experimentdetermined that the best time of removing nuclei is 20～22h of maturation oocytes in vitro for nuclearextrusion by squeezing method.3. This experiment studied on removing cytoplasm. In the enucleation rate, removing one thirdand one fourth the cytoplasmic volume (82.54％and 81.05％) was significantly higher than one-fifth(64.75％), from tested results, we can see that removing cytoplasmic volume was increased, the enucleation rote was tend to higher, but It's bad for development of reconstructed embryo in futur(?)Table 8 to determine the removal optimum volume from the cytoplasm is 1/4～1/3 of total volume;icleavage rate of reconstructed embryos and the developed rate. The results showed that comparewith other groups, the cleavage rote, 4-Cell development rate in 1/4 of removal cytoplasm group is thhighest, which is 41.68％, 70.32％respectively (P＜0.05).From above results of the experiment, th(?)removing one-fourth of the total cytoplasmic volume is the best.4. In the pilot study of injection methods, the method of electroform fusion injection comparewith cytoplasm injection method, the results showed that the efficiency of the micro-manipulatiotime, the cleavage rate, the 4-cell development rate is 53s/one, 43.65％, 56.39％(P＜0.05) and 77one, 18.69％, 17.50％respectively. There are significant differences between method of electroforfusion injection and method of the cytoplasm injection (P＜0.05). So it identified the injection meth(?)of electroform fusion is suitable, effective injection methods of somatic cell cloned in Yanbian yellocattle.5. In the sixth experiment, the media of injection nuclear by adding 0.1％PVA solution, thooctye's cleavage rate, growth rate compared with the control group (49.34％, 63.16％and 52.14％61.67％) (p＞0.05), there is no significant difference. The results showed that it can't impro(?)cleavage rate, growth rate and not play the role of nuclear transplantation in Yanbian yellow cattle badding 0.1％PVA solution.Conclusion: The experiment for somatic, cell cloned in Yanbian cattle oocytes experimental researcon micromanipulation. The squeezing method (modified Willadsen's method) is an efficient ansimple enucleation method with the advantage of saving manipulating time and none-damage(?)Yanbian Yellow cattle oocytes.Meanwhile, the injection method of electroform fusion is a suitable effective injection meth(?)for somatic cell cloned Yanbian cattle.##原图像不清晰...