Study On Identification Of Developmental Ability Of Bovine Oocyte By Brilliant Cresyl Blue (BCB) Staining In The Future

Cumulus oocyte complexes (COCs) , the important element for embryo production in vitro, the IVM quality of which is the key factor affecting in vitro embryo production (IVEP, affecting the ability of oocyte activation, fertilization and embryo growth. In the current, several influencing factors such as collecting ovaries and screening oocyte rashly, especially asynchronization of nuclear and cytoplasm growth for oocytes, would result in low efficiency of embryo production in vitro. The technology of oocyte maturation in vitro was harnessed to obtain plenty of oocytes, which would not only circumvent the limitations of the superovulation but also meet the need of the embryo engineering. In this experiment, TCM199 was used as basic culture medium and immature bovine oocytes were chosen through brilliant cresyl blue(BCB) staining, then the effects of BCB staining on in vitro mature-in vitro fertilization nuclear transplantation(IVM-IVF-NT) of oocytes were studied, and concentration of glutathione in mature and immature oocytes was measured in order to find an optimal method of screening oocytes with high quality, the effect of epidermal growth factor(EGF),β-mercaptoethanol(β-ME) and亚硫磺酸(HTAU) on IVM-IVF-NT of oocytes was also investigated to realize optimization of culture system in vitro and meet the need for embryo engineering and embryo transplantation, to realize industrialized production of elite domestic animal embryo and to increase the developmental speed of animal husbandry. The results were as follows:1. After immature bovine oocytes were stained with 13,26,39,52μmol/L BCB for 90 min, the percentages of BCB+ oocytes stained with 26μmol/L (27.7%), 39μmol/L (27.6%) and 52μmol/L BCB (31.9%) were higher than those stained with 13μmol/L (2.1%) BCB (p<0.05). And there were no significant differences in oocyte maturation between different BCB+ groups (p>0.05), BCB+ oocyte showed a higher maturation rate (77.4%) than BCB-(51.3%) and control oocytes (60.2%)(p<0.05), the optimal concentration of BCB for COCs was 26μmol/L; After the COCs were stained with 26μmol/L BCB for 90 min, blastula rate and number of blastular cells for COCs was the best and significantly higher than BCB- group and control group (P <0.05). It was demonstrated that activity of immature bovine oocytes can be identified by the BCB staining. BCB staining did not improve the mature rate and in vitro fertility rate of oocyte in MII phase but help to choose the oocytes with developmental ability before in vitro maturation.2. After the COCs were stained with 26μmol/L BCB for 90 min, the result showed that there was significant difference in concentration of glutathione between experimental group(BCB-group, 3.51179, 7.291422 pmol/cell; BCB+ group, 5.044247, 11.26002 pmol/cell) and unstained group(3.801034, 7.418366 pmol/cell) for mature and immature COCs, respectively.(P<0.05); and concentration of glutathione in BCB+ group was significantly higher than unstained group and BCB-group(P<0.05). It was found that the concentration of GSH in mature oocyte was double in immature oocyte, and BCB+ oocytes had better maturation ability and embryo development ability.3. BCB stained oocytes and unstained oocytes had different status of apoptosis. There was significant difference in maturing rate and apoptosis rate between BCB+ group(80.9% , 6.4%)and control group(60.2% , 27.6%), BCB-group(51.9% ,38.6%)(P<0.05). With the decrease of oocyte growth potency, the apoptosis rate was increased.4 Ion+6-DMAP, A23187+6-DMAP and ethanol+6-DMAP all can realize parthenogenetic embryo activation to make the embryo grow, upon three activation method, it was not good for the embryo to had too long activation time, and a nice effect was obtained by 5 min activation, the result showed that cell was dead after activation for a long time and a nice activation effect was obtained by treating with Ion+6-DMAP.5. Effect of BCB staining on the development of oocyte and effect of culture system on SCNT were studied in this experiment. In conclusion, bovine oocytes with different development ability can be identified though BCB staining before mature, so nice COCs can be provided for the study of IVM-IVF and nuclear transplantation of somatic cell ; On nuclear transplantation, oocytes were stained with BCB which were used as recipient oocytes, blastula rate of the embryo(32.26%) was higher than the control group without BCB staining and optimal culture system for 79.22%, which culture in the mature culture medium with 50 ng/mL EGF and 100μmol/Lβ-ME and recombination embryo culture medium adding with 0.5 mmol/L HTAU, respectively, it was demonstrated that mature culture medium and recombination embryo culture medium were optimal culture system for improving the efficiency of IVM-NT.