Effect And Mechanism Of Gut Microbiota Intervention On The Development Of Sow's Ovary And Uterus

In recent years,studies have found that gut microbiota as the host's second genome have the function of endocrine organs,suggesting that they may be involved in regulating sow reproductive physiology.Our research team used high-reproductive Meishan sows,relatively low-reproductive Landrace×Yorkshire(L×Y)crossbred gilts and gene knockout mice as the research objects,and employed(Fecal Microbiota Transplantation,FMT)technology and multiple omics method to analyze the mechanism of gut microbiota involved in regulating the reproductive performance of sows.First,we compared the gut microbiota of Meishan sows with the same genetic background with high and low litter sizes.Subsequently,we studied the effect of Meishan sow FMT on the development of the uterus and ovaries of the recipient L×Y gilts and screened out the key different intestinal microbes.Finally,we use broad-spectrum antibiotic-treated and knock-out mice as models to study the mechanism of key different candidate bacteria regulating follicular development.The main findings are:Part 1.Litter sizes of Meishan sows with the same genetic background have a significant correlation with gut microbiotaWe compared the microbes and estradiol(estradiol,E2),Progesterone(P4),and short-chain fatty acids(SCFAs)in the fecal samples of high-fertility Meishan sows(HMS,litter size 15.9±2.6)and low-fertility Meishan sows(LMS,litter size 10.7±2.1).The main results are:1)The carbohydrate metabolism capacity of HMS gut microbes is stronger than LMS,while amino acid and lipid metabolism capacity is weaker than LMS;2)Correspondingly,the concentrations of HMS fecal SCFAs(including acetate,propionate acid and butyrate)were significantly higher than those of LMS;3)The concentrations of reproductive hormones P4 and E2 in the feces of Meishan sows with high litter size were significantly higher than those of Meishan sows with low litter size;E2,P4 and litter size were significantly correlated to gut microbiota(different species and diversity).Summary:The gut microbiota of HMS and LMS with the same genetic background are not only significantly different but also significantly related to the litter size and the concentrations of reproductive hormones in feces.Part 2.The fecal microbiota transplantation from Meishan gilts significantly changed the reproductive physiology of the recipient L×Y giltsWe used FMT technology to transplant the fecal microbiota of the gilts with similar ages into the gut of commercial L×Y gilts to study the effect of FMT on the development of uterus and ovaries of L×Y gilts,and to screen out key differences in gut microbiota.8Meishan(MS)gilts(95±7 days old)were used as FMT donor pigs.28 L×Y gilts(90±4 days old)were randomly divided into 4 treatment groups:control group(Ctrl),sterilized-dose fecal microbiota transplantation(SFMT),low-dose fecal microbiota transplantation(LFMT)and high-dose fecal microbiota transplantation(HFMT),fed 20 m L 0.9%Na Cl solution,autoclaved fecal bacteria suspension,10⁷ CFU/m L fecal bacteria suspension and 10⁸CFU/m L fecal bacteria suspension.Feed once every other day until 180 days of age.After slaughter and sampling,the following analyses were performed:histomorphological analysis of uterine horns and ovaries,16S r DNA amplicon sequencing analysis of fecal microbiota,non-targeted metabolome analysis of plasma and liver tissue metabolites,and i TRAQ quantitative proteomics of ovarian tissue.The results showed that:1)FMT changed the gut microbiota of recipient L×Y gilts(the LFMT group had the best effect),and changed them to the gut microbiota of FMT donor Meishan gilts;2)LFMT promotes the development of follicles and significantly increases the density of uterine glands;3)LFMT promotes the metabolism of steroid hormones in the circulation of the recipient L×Y gilts,and enhances the synthesis of ovarian steroid hormones.Summary:FMT may regulate the development of uterine glands and follicles by influencing the secretion of reproductive hormones of the recipient L×Y gilts and the metabolism of system steroid hormones.Part 3.Oral reproduction-related differential bacteria promoted the development of mouse folliclesCombining the intestinal microbial difference analysis of HMS and LMS in the first part and the FMT trial in the second part,we screened out 4 key differences related to the reproductive performance of sows including Fibrobacter?intestinalis(FI),Bifidobacterium?thermophilum(BT),Lactobacillus?mucosae(LM),and Ruminococcus?flavefaciens(RF).21-day-old ICR female mice treated with broad-spectrum antibiotics were orally administered with 0.2 m L of normal saline(Ctrl)or 0.2m L of microbial suspension,including FI,LM,BT,RF and their mixture(MIX),respectively.At the end of the third estrus period,ovarian tissue,hypothalamus,plasma,and gut feces were collected for analysis.The main results are:1)A single species of differential bacteria or MIX can significantly increase the density of secondary follicles and antral follicles,as well as the ratio of secondary follicles to primary follicles;however,only MIX increases the density of antral follicles relative to secondary follicles;2)Different bacteria have a regulatory effect on reproductive hormones,especially E2;3)Different bacteria significantly promote the development of gonadal white adipose tissue(g WAT)and leptin secretion;4)Different bacteria significantly promote the synthesis of steroid hormones.Summary:Oral administration of a single species or a mixture of four species of reproduction-related differential bacteria may promote the development of g WAT and leptin secretion by increasing the production of SCFAs,thereby regulating the secretion of reproductive hormones,and ultimately promoting the development of mouse follicles.Part 4.SCFAs signaling pathway mediates the promotion of reproduction-related differential bacteria on mouse follicular developmentThis experiment aims to further explore the relationship between SCFAs,the metabolites of gut microbiota,and follicular development.Considering that the activation of SCFAs receptor GPR43 is ligand-biased and concentration-dependent,we tested a series of SCFAs concentration gradients based on the concentration of SCFAs in the cecum of mice after mixed intervention of the four different bacteria to determine the optimal concentration of SCFAs for the promotion of mouse follicular development.Subsequently,we used Gpr43^-/-mice to further explore the role of GPR43 pathway in SCFAs in promoting mouse follicular development.The main results are:1)SCFAs increased the density of secondary follicles and antral follicles in a concentration-dependent manner;probably because the expression of Gpr43 gene in g WAT was significantly higher than other adipose tissues,SCFAs selectively promoted the development of g WAT;2)SCFAs simultaneously increased the concentration of kissin 1 and gonadotropin releasing hormone in hypothalamic tissue;SCFAs activated ovarian leptin receptors and promoted the steroid hormone synthesis pathway;3)These effects of SCFAs in Gpr43^-/-mice can not be observed.Summary:SCFAs promote mouse follicular development in a concentration-dependent manner;GPR43 mediates the promotion of SCFAs on mouse follicular development.In conclusion,gut microbiota is involved in regulating the development of sows'uterine glands and follicles.Before puberty,the gut microbial metabolites SCFAs promote g WAT adipogenesis and leptin secretion by activating GPR43 in a concentration-dependent manner.The elevated leptin in the body's circulatory system can regulate the secretion of follicle-stimulating hormone,luteinizing hormone and estradiol through the hypothalamic-pituitary-gonad axis,thereby promoting follicular development.