| Neonicotinoid compounds are chemical insecticides with neurotoxic effects on biological acetylcholinergic receptors(ACh Rs).Among these,imidacloprid(IMI)is the most widely used during the past three decades in the world.IMI is famous for killing pests in agriculture,forestry,and horticulture,resulting in widespread residues in soil,air,water,and crops all over the world.So far,many studies have focused on the specificity of IMI for insect acetylcholine receptors,while its toxicity to mammals,especially reproductive toxicity,has been ignored.In this experiment,porcine oocytes were used as a toxicological research model of IMI.In an in vitro culture system,the effect of IMI on meiosis and maturation of porcine oocytes was investigated.In the same time,we also looking for a solution to the decline of female reproductive ability caused by IMI.The specific research results are as follows:1.62.5 μM,125 μM,and 250 μM concentrations of IMI were added to porcine oocyte in vitro maturation medium.The results showed that IMI would affect porcine oocyte maturation.With the increase of IMI concentration,the expansion of the cumulus cell became worse,and the emission rate of the polar body became lower.The average emission rates of 62.5 μM,125 μM,and 250 μM IMI were 40.05±4.42%,28.91±1.86%,and 15.32±4.00%.It was found that Vc could relieve the damage of IMI to porcine oocytes and restore cumulus cell expansion.The emission rate of the first polar body in the Vc+IMI treatment group(47.97±1.99%)was significantly higher than the IMI treatment group(28.91±1.86%),and P<0.01.2.Immunofluorescence staining of IMI-treated oocytes showed that the proportion of abnormal morphological spindles in the experimental group(54.40±2.44%)was significantly increased compared to the control group(23.36±3.42%),and P<0.01.It was found that Vc reduced the abnormal spindle assembly rate caused by IMI(29.86±3.80 %)compared to the IMI-treated group(54.40±2.44%),and P<0.01.The proportion of oocytes with abnormal distribution of cortical granules(32.25±6.24%)was significantly higher than that of the control group(12.01±3.00%),and P<0.01.Vc can restore the cortical granule distribution of IMI-treated oocytes.In the Vc+IMI treatment groups,the proportion of oocytes with abnormal cortical granule distribution(21.92±1.80%)was significantly lower than the IMI experimental group(43.96±1.24%),and P<0.001.Reactive Oxygen Species(ROS)levels in IMI-treated oocytes were increased(69.93 ± 5.87%)compared to the control group(21.94±3.50%),and P<0.01.Vc reduced the ROS level of IMI-treated oocytes.In the Vc+IMI treatment groups,the ratio of oocytes with high ROS levels(40.17±4.49%)was compared with the ratio of oocytes with high ROS levels in the IMI experimental group(69.93± 5.68%)significantly reduced,and P <0.01.The proportion of oocytes with impaired lipid droplet morphology was also increased(32.25±6.24%)compared to the control group(12.01 ± 3.00%),and P< 0.01.The ratio of oocytes with abnormal lipid droplet morphology(20.55±2.25%)in the Vc+IMI treatment groups was significantly lower than the IMI experimental group(35.68±1.55%),and P<0.01.3.Parthenogenetic activation of IMI-treated oocytes showed that the cleavage rate of the experimental group(31.37±0.92%)was significantly lower than the control group(36.05±1.65%),and P<0.05.The blastocyst rate of the experimental group(27.99±3.33%)was lower than the control group(35.37±2.52%),and the blastocyst cell number(39.17±12.84)of the experimental group was significantly lower than the control group(53.93±12.26),and P<0.01.The blastocyst rate of the IMI+Vc treatment group(33.47±1.45%)was higher than the IMI treatment group(27.99±3.33%).The number of embryonic cells in the IMI+Vc treatment group(49.00±9.33)was significantly higher than the control group(39.17±12.84),and P<0.01.In summary,our findings demonstrate that IMI adversely influences the porcine oocyte maturation by the excessive ROS.Adding vitamin C has a better protective effect on the in vitro maturation of porcine oocytes induced by imidacloprid. |
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