| The formation and development of follicles is directly related to the number of follicles available throughout the reproductive stage of female mammals,which is an important factor affecting reproductive ability.Meishan pig is one of the native breeds of taihu pigs in China.Meishan pig is famous for its high ovulation rate and litter size.Previous studies showed that the number of follicles recruited in the follicle development period of Meishan pig was significantly higher than that of the introduced breeds.This is the reason why the reproductive performance of Meishan pigs is better than that of introduced breeds.Meanwhile,the native pig breeds have strong crude fiber feedinds and gut stress resistance than that in introduced breeds.However,Most of the large-scale pig farms in China are mainly introduced varieties,which have high lean meat rate and growth rate,but the overall fertility is low and intestinal health problems are serious.Studies have found that intestinal microbes play an important role in regulating host intestinal health,immunity and metabolism.It has been proved in human medicine that intestinal microorganisms can regulate estrogen metabolism and ovarian development.However,there is no report on the effect of intestinal microorganisms on the development of ovarian follicles in sows.Here,we transferred fecal microbiota from healthy Meishan sows through fecal microbiota transplantation(FMT)to Landrace×Yorkshire gilts to investigate the effects of FMT on age of puberty,serum hormone levels and ovarian tissue development in gilts.Through 16SrDNA gene amplicon sequencing and iTRAQ technology,To compare the effects of FMT on intestinal microflora and ovarian differential protein expression in Landrace×Yorkshire gilts.Experiment 1:The effect of FMT on the age of puberty,serum hormone levels and ovarian tissue development in giltsEight Meishan gilts(Meishan,MS)with age(107 d)were selected as FMT donor pigs.Another 28 Landrace×Yorkshire gilts with(90±4 d)were selected as FMT recipient pigs.Landrace×Yorkshire gilts were randomly divided into 4 groups,each group of 7heads,the specific grouping is as follows:control group(Ctrl,20mL 0.9%NaCl solution),sterilized-dose fecal microbiota transplantation(SFMT,20 mL Sterilized Meishan pig fecal microbiota suspension),low-dose fecal microbiota transplantation(LFMT,20 mL10~7 CFU/mL Meishan pig fecal microbiota suspension),high-dose fecal microbiota transplantation(HFMT,20mL 10~8 CFU/ml Meishan pig fecal microbiota suspension).The bacteria suspension was fed once every other day,and was taken after eating at 8:00am every day.Until the first estrus,the age of puberty was recorded.The day of the second estrus cycle occurrence was designated as d 1,and blood is collected of d1,d3,d5,d7,d9,d11,d13,d15,d17,and d19 to detect serum follicle stimulating hormone(FSH).Lutein(LH),progesterone(P4)and estradiol(E2)levels.Landrace×Yorkshire sows were slaughte in the third estrus cycle of standing heat.The main results are as follows:(1)Compared with the Ctrl group,the FMT treatment group(SFMT,LFMT,and HFMT)had significantly earlier age of puberty(P<0.05),There was no significant difference in age of puberty between the three groups of SFMT,LFMT and HFMT(P>0.05).(2)When counting the number of large follicles(diameter?5 mm)on the ovary at slaughter,the LFMT group was significantly higher than that in the Ctrl group(P<0.05),and the other FMT group and Ctrl group were not significantly different(P>0.05).(3)When detecting serum hormone changes in the estrus cycle,it was found that the level of luteinizing hormone in the serum of LFMT group was significantly higher than that of SFMT and HFMT group at D3(P<0.05),and there was no significant difference at other time points.On d3 and d9,the serum progesterone level in the LFMT group was significantly lower than that in the Ctrl group(P<0.05),and there was no significant difference between the HFMT and MS groups.There was no significant difference in serum estradiol content and follicle stimulating hormone levels between the LFMT group and the other groups during the whole estrus cycle(P>0.05).The above results indicate that low-dose fecal microbiota transplantation(LFMT)can significantly promote the initiation of puberty sows,affect serum hormone levels,and promote ovarian tissue development.Experiment 2:Effect of FMT on the expression of intestinal microbes and ovarian protein in giltsThis study used 16S rDNA gene amplicon sequencing technology to compare the differences in microbial flora between Meishan pigs and Landrace×Yorkshire sows.The iTRAQ technique was used to compare the ovarian differential protein levels between Ctrl group and LFMT group with the best effect.The main results are shown as follows:(1)16Sr DNA gene amplicon sequencing sample produced 1120 OTUs,and the five groups of identical OTUs were 696.Ctrl,SFMT,LFMT,HFMT and MS groups did not show significant difference in alpha diversity index.The PLS-DA analysis based on OTU abundance showed that the LFMT group and the MS group were closest to each other,and the microbial composition was changing toward the MS group.(2)A total of 6804 proteins were identified by iTRAQ proteomics.A total of 446differentially significant proteins were screened by LFMT/Ctrl differential multiples>1.20 and P values<0.05.194 were significantly down-regulated and 252 were significant upregulate the protein.(3)GO enrichment analysis,KOG annotation,subcellular localization and KEGG metabolic pathway integration analysis of differential proteins,we found that differential proteins were mainly enriched in PI3K-AKT signaling pathway,steroid biosynthetic pathway,which may act as microbial regulation follicles Candidate signaling pathways for development.In summary:(1)Treatment of Landrace×Yorkshire gilts by low-dose fecal microbiota transplantation(10~7 CFU/mL)from Meishan pig can advance age of puberty of gilts and increase serum estradiol concentration to promote follicular development.(2)We transferred fecal microbiota from Meishan sows through FMT to Landrace×Yorkshire gilts,the microbial flora of theLandrace×Yorkshire gilt was converted to Meishan pigs.(3)The differential protein enriched PI3K-AKT signaling pathway and steroid biosynthesis pathway may serve as candidate signaling pathways for microbial regulation of follicular development. |
PDF Full Text Request