| Significant progress has been made in the bovine embryos in vitro production and other assisted reproductive technologies.However,IVP of embryos is still facing some problems that limit the wider utilization of industrial production of quality embryos,including the decline of embryo quality and deterioration of oocyte quality after oocyte maturation in vitro,effect of oxidative stress,one of important mechanisms,on oocyte quality and embryonic development.The study is aimed at investigating the effect of broad-spectrum plant growth regulator Chlormequat chloride(CCC)on bovine oocytes,the effect of melatonin(MLT)supplementation on oocyte quality,and the effect of subsequent oocyte development on embryo.The study mainly includes the following two experiments:Experiment 1:Effect of MLT on the quality of injured oocyte during CCC meiosisCCC is a broad-spectrum plant growth regulator,and it is widely used in farmland in China.Residual CCC are ingested by cattle through straw feed and takes effect in a complete form.In this experiment,th e related indexes of meiosis in maturation of bovine oocyte were analyzed,to explore the compensation strategy of injured oocyte.The results showed that 1.5 mM of CCC significantly decreased the maturation rate of oocyte of Yanbian yellow cattle,while 10-9 M of MLT significantly increased the maturation rate of oocyte injured by CCC.During the in vitro maturation,CCC resulted in abnormal meiotic process by increasing the proportion of abnormal oocyte,stagnation in MI phase,GV phase in the process of meiotic maturation(P<0.05).The expression of antioxidative stress and related genes was also significantly decreased by the CCC,while adding MLT to the culture medium could effectively improve the abnormal proportion of meiotic process(P<0.05)and increase the maturation rate of oocyte nuclei(P<0.05).Above results showed that CCC could lead to stasis of oocyte meiosis and decrease the maturity ratio of nucleus during in vitro maturation,and that proper amount of MLT could increase the maturity ratio of oocyte injured by CCC and enhance the expression of antioxidative stress related genes(P<0.05).CCC may lead to non-synchronous maturation of oocyte nucleus and cytoplasm.Therefore,the oocyte quality-related indexes were analyzed in this experiment,and the results showed that CCC significantly decreased the level of glutathion(GSH)(P<0.05),and increased the level of reactive oxygen species(ROS)of oocyte(P<0.05);and the level of mitochondrial membrane potential was decreased(P<0.05);however,with supplement of MLT,the level of ROS of injured oocyte decreased significantly(P<0.05),the level of GSH increased significantly(P<0.05)and the level of mitochondrial membrane potential restored significantly(P<0.05).The above results showed that although 1.5 mM of CCC could not cause death of oocyte,it would aggravate the oxidative stress of oocyte and decrease the oocyte quality;supplement of MLT can partly improve the injury caused by CCC.Experiment 2:Effect of MLT on embryonic development of oocyte injured by CCCThe embryonic development ability is the best indication of oocyte quality;in this experiment,the oocytes of control group,1.5mM CCC group and MLT group were fertilized and developed to blastocyst stage respectively,and the quality of blastocyst was analyzed.The results showed that CCC had no significant effect on the division rate of bovine oocyte(P<0.05),but it significantly decreased the rate of 8 cell and the blastocyst rate on the sixth,seventh and eighth day(P<0.05);in addition,the expressions of embryo totipotency related genes Sox2,Cox2 and Oct4 in CCC treatment group were also significantly decreased(P<0.05),the expression of apoptosis-promoting gene caspase-3 was significantly increased(P<0.05),and the transcription level of anti-apoptosis gene Bax and Bcl-xl was significantly decreased(P<0.05);CCC reduced the number of blastocyst cells significantly,and the expression of apoptin Annexin-V was significantly increased on the seventh day(P<0.05);the probability of quality embryos produced by CCC cultured oocytes was significantly lower than that of the control group(P<0.05),and the above indexes of oocytes were improved by adding MLT(P<0.05).These results suggest that CCC has an effect on the subsequent development ability of bovine oocytes,and the supplement of MLT can compensate for the loss of embryonic development ability caused by CCC.In conclusion,CCC may cause obstacles in the meiotic process of bovine oocytes,lower the quality and weaken the late development ability during embryo culture in vitro.However,adding MLT in the process of culture can effectively repair the injury caused by CCC to bovine oocytes. |
