| To investigate the effect of Boron on sow granulosa cells and the possibility as the feed additives used in the production of the mature healthy sow ovaries,cultured granulosa cells,through the identification and toxicity test preliminary determination of boron addition were 0,0.5,1,5,40 and 80 mmol/L,respectively by CCK-8,HE staining,flow cytometry,Western Blot and immunofluorescence method for related indicators detection;for the beneficial effect on the reproductive performance of boron preliminary verification of the female animal,dosage conversion through the test,determine the mice drinking water with low dose boron addition of 0,5,10,20 mg/L,96 healthy.Mature mice fed 30 d artificial insemination,pregnancy of 0 d and 14 d groups were killed 8,ovaries,uterus;each group of 8 mice breed,detection and reproductive performance of ovary and uterus index.The results are as follows:1.Effect of Boron on apoptosis of follicular granulosa cells:Compared with 0 mmol/L boron treatment group,mmol/L treatment group B 0.5 porcine granulosa cell and normal cell rate increased 5.3%,the apoptosis rate decreased by 5.7%;5,40 and 80 mmol/L of boron treatment group of normal cell ratio decreased 6.7%,14.45%and 15.01%respectively,the ratio of apoptotic cells were increased by 5.7%,11.6%,14.8%.2.Effects of Boron on follicular granulosa cell cycle:Compared with 0 mmol/L boron treatment group,0.5 and 5 mmol/L of boron treated G1/G0 cells decreased,the percentage of cells were decreased by 3.77%and 2.72%,the ratio of G2/M phase cells and cell proliferation index increased,the ratio of G2/M phase cells were increased by 1.13%and 1.57%,the cell proliferation index were increased by 1.75%,2.63%;10.48%mmol/L boron treatment decreased by 1 G1/G0 cell ratio,S cell ratio and cell proliferation index were increased by 5.65%,4.53%;40 mmol/L boron treatment group G2/M cell proliferation rate and cell index were decreased by 4.39%,2.97%;80 mmol/L boron treatment group G1/G0 cell ratio increased 6.65%,S,G2/M,the ratio of cell proliferation and cell index were decreased by 3.93%5.5%,9.43%.3.Effects of G-1 and G-15 on apoptosis of granulosa cells treated with boron:Compared with without G-1 and G-15 treatment group,low concentration boron inhibited the apoptosis of porcine follicular granulosa cells.The high concentration of boron(boron added above 5 mmol/L)could promote the apoptosis of cells.The apoptosis rate of G-1 treatment group were lower than those of the non treatment group,showed that the inhibitory effect of G-1 on apoptosis of porcine granulosa cells;apoptosis rate of G-15 group is higher than that of treatment group,showed that G-15 has a promoting effect on the apoptosis of porcine granulosa cells.4.Effects of G-1 and G-15 on cycle of granulosa cells treated with boron:Similar to the G-15 treatment group,the low concentration boron promoted the proliferation of granulosa cells treated with G-1 and G-15.The boron concentration was higher than 5 mmol/L,which inhibited the proliferation of follicular granulosa cells(G-1).Compared with the G-1 test without addition,the number and rate of S phase cells in the G-1 treated group were generally reduced.Compared with the no G-15 test,the number and rate of S phase cells decreased and the number of G2/M cells increased in the low concentration boron group treated with G-15.5.Effects of Boron on reproductive performance in mice:With 0 mg/L boron group compared to 5,10 and 20 mg/L of boron addition mice litter were significantly increased 3.83,3.16,4.33(P<0.05),birth weight were significantly decreased by 0.26,0.44,0.46 g(P<0.05);mice litter weight change was not statistically significant(P>0.05).6.Effect of Boron on mouse ovarian tissue structure:Compared with 0 mg/L of boron addition,pregnancy 7 d ovarian cortical follicle number with boron addition increased first and then decreased,10 and 20 mg/L of boron addition group significantly increased the number of follicles(P<0.05),primordial follicles decreased,secondary follicle number primary oocytes,large round nuclei and a small amount of follicular atresia;5,10 and 20 mg/L of boron addition group ovarian index with boron increase decreased,but the difference was not significant(P>0.05);pregnancy 14 d,ovarian follicles in cortex of normal structure,secondary follicle number,10 mg/L boron added group follicle number increased,but the difference was not significant(P>0.05),adding 20 mg/L Boron group significantly reduced the number of follicles(P<0.05),the number of atretic follicles compared with pregnancy in 7 d increased significantly;5,10 and 20 mg/L of boron addition group refers to the ovary The number of boron increased first and then decreased with the increase of boron content,but there was no statistical significance(P>0.05).7.The effect of Boron on the structure of uterus in mice:The structure of uterus in each treatment group was distinct,and there was no obvious pathological change.On the same day at the age of 0 and mg/L of boron addition group compared the pregnancy of 7 d,5,10 and 20 mg/L of boron addition group uterus index increased,but the difference was not significant(P>0.05);5 and 20 mg/L of boron addition group of uterine wall thickness was significantly increased(P<0.05);14 d of pregnancy,uterine pregnancy index was significantly higher than 7 d,5,10 and 20 mg/L of boron addition group uterus index increased significantly(P<0.05),adding 5 mg/L B group of uterine wall thickness was significantly increased(P<0.05),20 mg/L boron significantly reduced the uterine wall thickness added group(P<0.05);pregnancy endometrium is 14 d thick,significantly higher than the uterine wall thickness of 7 d in pregnancy.Conclusion:Low dose boron(?5 mmol/L boron)can regulate porcine granulosa cell cycle in vitro,promote granulosa cell proliferation;high dose boron(?5 mmol/L boron)can accelerate the apoptosis of granulosa cells,and showed a clear dose dependence.Agonists of GPR30 cell apoptosis in G-1 group showed a remission trend,the proliferation coefficient increased;GPR30 specific inhibitor of cell apoptosis in G-15 group showed increasing trend,the proliferation coefficient decreased,showed that the proliferation and apoptosis of boron can be through the GPR30-GPER1 pathway to regulate follicular granulosa cells.5-20 mg/L drinking boron improve mouse litter size,lower birth weight,but the litter weight at birth,ovary and uterus index had no significant effect;5,10 mg/L water boron promoted the follicle number increase,improve the development of follicles and endometrial glands within the ovarian cortex,which to add 5 mg/L of boron is the most effective;14 d index of uterus and uterine wall thickness was significantly higher than that of 7 d pregnancy. |
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