Effects Of Meiotic Inhibitors On The Meiotic Resumption And Embryonic Development Of Buffalo Oocyte

The low quality of buffalo oocyte in maturation stage is one of the factors that restrict the embryo production in vitro. This study aimed to improve the oocyte development capacity and detected the influence on microtubule and microfilament after a period of prematuration culture with a specific PDE3inhibitor Cilostamide.In the first experiment, the optimum effective dose of Cilostamide was determined. Buffalo oocytes were cultured in the IVM media with20μM and50μM, respectively, for24h, the oocytes were inhibited at GV stage effectively and the difference in inhibited efficiency among the treatment groups was not significant (75.56%VS74.11%, P>0.05). Therefore, the concentration of20μM Cilostamide was used in the subsequent experiments. Buffalo oocytes were inhibited following culture with the inhibitor for6h,12h, and24h.The inhibited efficiency was decreased at GV stage (88.11%,85.93%and64.39%for6h,12h,24h, respectively) and the inhibited efficiency was much lower (P<0.05) in 24h group than that in the other two groups, but the nuclear resumption was still arrested. Oocytes were resumed meiosis and reached maturation after culture in ⅣM media without Cilostamide for24h following prematuring for6h,12h,24h with inhibitor and the MⅡ rate was63.43%,62.83%and48.50%for6h,12h,24h, respectively, the maturation rate was significantly lower (P<0.05) in24h group than that in the other two groups.At germinal vesicle (GV) stage, microtubules could not be detected. At pro-metaphase Ⅰ, microtubules were found and surrounded the condensed chromatin. At metaphase Ⅰ, microtubules were seen in the meiotic spindle. In anaphase/telophase Ⅰ, microtubules were found in between the two parts of chromatin. At metaphase Ⅱ, microtubules were detected in the spindle and some were around the polar body. Microfilaments were observed with a thick area around the cell cortex and overlying chromosomes following germinal vesicle breakdown. There was no influence on morphological characteristics of microtubules and microfilaments in the MⅡ stage of buffalo oocytes by prematuring with inhibitor for6h,12h, and24h.Fertilization and embryo development in vitro of matured oocytes were performed after prematuring with inhibitor for6h.The rate of cleavage and blastocyst, and the number of blastocyst of oocytes treated were not significantly different compared with the control group (65.73%VS55.54%,30.71%VS22.56%,203.2VS205.5, P>0.05)These results indicate that PDE3inhibitor Cilostamide can reversibly arrest the meiosis resumption without adverse influence on the in vitro development capacity and without any dynamic change of microtubules and microfilaments of buffalo oocyte.