Sources Of Bile Acids In Buffalo Follicular Fluid And Their Involvement In The Regulation Of Follicular Atresia

At present,the content of fat in the feed of animal husbandry enterprises is higher and higher,and the animal body cannot secrete enough bile to decompose fat,so bile acid is used as a new feed additive.It has been reported that the follicle fluid of mammals was rich in bile acids,suggesting that the addition of bile acids in feed may affect the reproductive function of female animals.In this study,the sources of bile acids in buffalo follicles were firstly analyzed.And then,subspecies and contents of bile acids in healthy follicles(HF)and atretic follicles(AF)were detected by targeted metabolomics method.Finally,the effects of differential bile acids on the apoptosis,proliferation and hormone secretion of granulosa cells were also studied.These studies will provide new viewpoints for exploring the physiological role and mechanism of bile acids in buffalo follicular atresia and provide more theoretical basis for the addition of bile acids in feed.The main results of the study are as follows:1.The source of bile acids in buffalo follicle fluid was investigatedThe immunohistochemistry and WB were used to verify whether the bile acid synthetase genes CYP7A1 and CYP27A1 and the bile acid transporter genes NCTP and ASBT were expressed in the ovarian tissues.The results showed that the bile acid synthetase gene was not expressed in ovarian tissue,but the bile acid transporter gene was expressed in ovarian tissue.Cholesterol was added into the culture medium of theca cells and granulosa cells of follicles,respectively,to detect the changes of cholesterol content in the cells and bile acid content in the culture medium.The results showed that the cholesterol content in the cells was significantly increased after the addition of cholesterol(P < 0.05),but the content of bile acid in the culture medium was not changed,suggesting that the follicle cells could not synthesize bile acids.2.The subspecies and content of bile acids in healthy follicles(HF)and atretic follicles(AF)were detectedHF and AF were distinguished by external characteristics and internal morphology of follicles,and the follicle classification was verified to correctly by detecting the contents of estradiol and progesterone as well as the relative expression of apoptosis-related genes.The results of targeted metabolite profiling showed that the standard curves of each analyte had good linearity,and R2 was greater than 0.999.Bile acids in HF and AF follicle fluid were quantified by standard curves.The types of bile acids in HF and AF are consistent,with CA and GCA being the major bile acid subtypes,but with different concentrations of each bile acid.There were 13 kinds of bile acids with significant differences,the content of conjugated bile acids in AF was significantly higher than that of HF,and the content of free bile acids was significantly lower than that of HF.The total bile acid content in HF was significantly higher than that in AF.3.The effect of glycodeoxycholic acid(GDCA)on the function of buffalo granulosa cells cultured in vitro was investigatedGDCA was the binding bile acid with the largest difference between HF and AF.0 M,200μM,300μM and 400μM GDCA were added into the granulosa cells cultured in vitro,then the changes of granulosa cell state were detected.The results showed that after the addition of GDCA,the apoptotic rate of buffalo granulosa cells was significantly increased(P < 0.01).The secretion of E2 was significantly decreased(P < 0.01).P4 secretion was significantly decreased(P <0.01)while there was no change in the EDU positive rate.It was found that the expression of apoptosis-related genes caspase3,BAX,and P53 in buffalo granulosa cells was promoted and the anti-apoptotic gene Bcl2 was inhibited by GDCA by detecting the relative expression levels of genes related to apoptosis,proliferation and steroid synthesis.GDCA was also found to inhibit the expression of steroid-related genes CYP11A1 and CYP19A1,but had no effect on cell proliferation-related genes PCNA and Cyclind1.Therefore,the preliminary results in this study indicated that bile acids in buffalo follicle fluid were derived from blood transport,and the follicle cells themselves could not synthesize bile acids.There were differences in the subtypes of bile acids in HF and AF.In AF,the conjugated bile acids increased while the free bile acid decreased.Among them,the differential conjugated bile acid GDCA could promote the apoptosis of follicular granulosa cells and reduce the secretion of steroid hormones,thereby regulating the function of granulosa cells and participating in follicular atresia.