| It is well documented that the cryopreservation process induces spindle disorganization leading to disrupted chromosomes and microfilaments. This problem may be in part due to the complex structure of matured buffalo oocytes, which consequently impact cell structure and the physical and chemical properties during vitrification. Previous research has shown that addition of cytoskeleton stabilizers greatly decrease injury to oocytes during vitrification. The purpose of this study was to investigate the effects of cytochalasin B (CB) on vitrification of matured buffalo oocytes, with the aim of understanding the vitrification damage mechanism and giving directions to improve the efficiency of vitrification in buffalo oocytes.In the first experiment, optimization of CB concentration used in vitrification of bufalo oocytes was investigated. In vitro matured buffalo oocytes were randomly assigned to cultured in the IVM media with different concentrations of 0 u g/mL,4 u g/mL,8 u g/mL and 12 μg/mL, for 30 min. The results showed that the oocyte survival and embryo development rates were significantly lower for the four vitrification groups compared to the fresh control group (P<0.01). However, the cleavage rate and blastocyst formation rates with 8 μ g/mL CB group were significantly higher compared to 0 μ g/mL CB groups (59.18% vs 43.89%,17.28% vs 10.27%, P<0.05), but the effect in development of oocytes with 4 u g/mL and 12 μ g/mL treatment were not obvious. As the result,8 u g/mL CB was chosen for the following experiments.In the second experiment, whether the treatment of buffalo oocytes with CB at the optimum concentration prior to vitrification would stabilize the cytoskeleton system and affect the integrity of the cytoskeleton expression was determined. At metaphase II stage of buffalo oocyte, microtubules were observed around the first polar body and formed the meiotic spindle encompassed the chromosome, oriented perpendicularly or parallelly. In addition, microfilament-rich domain were located in the polar body. The percentage of normal spindle organization, chromosome alignment and actin filaments distribution of oocytes pretreated with 8 u g/mL CB before vitrification were significantly higher than those from the vitrified groups without CB (48.99% vs 38.66%,52.14% vs 42.11%,54.59% vs 43.54%, P<0.05), such that percentages were different to those obtained from control oocytes (70.57%,77.03%,81.47%, P<0.01) and CB fresh group (74.72%, 76.76%,80.60%, P<0.01). Besides that, the expression of tubulin and actin of vitrified-warmed buffalo matured oocytes were examined. The results showed that the group pretreated with 8 μg/mL CB before vitrification had higher expression of tubulin comparing with the group of vitrification without CB treated, but effects of actin were neglectable.In the third experiment, investigation of embryonic development in vitro of vitrified buffalo matured oocytes treated with CB following ICSI was carried out. The 8-cell cleavage and blastocyst formation rates of the 8 μ g/mL CB groups were significantly higher than that of the vitrified groups without CB (54.50% vs 34.90%,17.08% vs 10.21%, P<0.05), but these two groups’ rates of the second PB extrusion,2-cell cleavage and blastocyst were significantly lower than the control group. There was no significant difference in total cell numbers among the three treatment groups (84.0% vs 81.0% vs 74.4%).These results from the present study indicate that addition of 8 μg/mL CB has positive effect on the spindle microtubules, chromosome, microfiliment configuration and cytoskeleton protein along with development capacity of vitrified buffalo oocytes. |
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