| In order to elucidate the interaction between the female steroid hormones(Progesterone,P4;Estradiol,E2)and the gut microbes,we firstlyanalyzed the changes of the fecal steroid hormones contents,bacterial flora and metabolites at d30 and d90 of gestation in Meishan sows.Further,the interactions betweenthe steroid hormones(P4 and E2)and fecal microflora were studied in vitro.1 The changes of the steroid hormones contents,microflora and metabolites in different gestation stages of Meishan sowsThe fecal concentrations of P4 and E2 in pregnant Meishan sows at d30 were significantly lower than d90(P<0.01).The sequencing profile of the fecal bacterial DNA indicated that the relative abundance of Burkholderiales and Selenomonadales in stool at d90 of pregnancy were increased signifcantly than that of d30 at the order level(P<0.05).At the genus level,the relative abundance of Lachnospiraceae UCG-007 and Pseudomonas in pregnancy d30 were significantly higher than that of d90(P<0.05),but the relative abundance of Anaerotruncus,Candidatus Soleaferrea,Phascolarctobacterium,Prevotellaceae UCG-004,Intestinibacter and[Eubacterium]coprostanoligenes group were significantly lower than that of d90(P<0.05).However,there were no signifcant differences in the richness estimators(ACE and Chao)and the diversity indices(Shannon and Simpson)between the d30 and d90 of gestation.Further,the metabolomics profle revealed 11 metabolites changed greatly from the d30 to d90 of gestation(P<0.01),involved in amino acid metabolic pathway(1 metabolite),lipid metabolic pathway(6 metabolites),carbohydrate metabolic pathway(3 metabolites)and Vitamin metabolic pathway(1 metabolite).2 The interaction between the pig fecal progesterone and the bacterial floraThe anaerobic culture trial of pig fecal bacterium was conducted with a series doses(0.2,2,4,8 ?g/mL)of P4,meanwhile with control group(Control I)and the control group containing Dimethyl Sulfoxide(DMSO)equal to the 8 ?g/mL P4 group(Control ?).After 8 hours' culture at 37?,the contents of SCFAs and the total number of bacteria of fecal culture medium in Control I and Control II did not show obvious differences,which indicated that the 27.84 ?L DMSO contained in the 87 mL culture system had no effect on the experiment.The OD630 in the 0.2?8 ?g/mL P4 groups were significantly higher than that of the Control II group(P<0.05),and the 2 ?g/mL P4 group was significantly higher than the other groups(P<0.05).The results of fluorescence quantitative PCR showed that the Lg(copies/mL)of 0.2?8 ?g/mL P4 groups were significantly higher than that of the Control II group(P<0.01),and the 2 ?g/mL P4 group was significantly higher than the other groups(P<0.01).The results showed that P4 could promote the proliferation of bacteria.When compared to Control ? group,the levels of acetic acid,iso-butyric acid,iso-valeric acid,valeric acid and total SCFAs in the 2 ?g/mL P4 group increased significantly(P<0.01),while the levels of propionic acid and butyric acid reduced significantly(P<0.05).The results showed that 2 ?g/mL P4 could affect the metabolic type of bacterial flora.The reduction rates of P4 in the culture medium from high to low were 2,4,8,0.2 ?g/mL P4 group,and the 2 ?g/mL P4 group was significantly higher than the other groups(P<0.05).The reduction rate of P4 in 4 pg/mL group was significantly higher than that of 0.2 and 8 ?/mL group(P<0.01).However,there was no significant difference between 0.2 ?g/mL P4 group and 8 ?g/mL P4 group.The results showed that the utilization and decomposition of P4 in fecal culture medicum were dose dependent.The sequencing profile of the cultured fecal bacterial DNA indicated that the diversity index(OTUs,Ace,Chao,Coverage)of 2 ?g/mL P4 group did not show obvious difference with Control II group.The Shannon index of the 2 ?/mL P4 group increased significantly than that of the Control II group(P<0.01),and the Simpson index decreased significantly than that of the Control II group(P<0.01).At the phylum level,the relative abundance of Firmicutes in the 2 p,g/mL P4 group decreased significantly than that of the Control ? group(P<0.01),while the relative abundance of Proteobacteria increased significantly than that of the Control II group(P<0.01).At the genus level,the relative abundance of Enterobacter,Escherichia-Shigella in the 2 ?g/mL P4 group were significantly higher than that of the Control II group(P<0.01),while the relative abundance of Achromobacter,Enterococcus,Pseudomonas,Ruminococcaceae UCG-005 and Streptococcus were significantly lower than that of the Control ? group(P<0.05).Results showed that 2 ?g/mL P4 could affect the structure of fecal flora.3 The interaction between the estradiol and the pig fecal bacterial floraThe anaerobic culture trial of pigs fecal bacterium was conducted with a series doses(20,40,60,120 ng/mL)of E2,meanwhile with control group(Control ?)and the control group containing DMSO equal to the 120 ng/mL E2 group(Control ?).After 8 hours' culture at 37?,the contents of SCFAs and the total number of bacteria of fecal culture medium in Control ? and Control ? did not show obvious difference,which indicated that the 0.42 ?L DMSO contained in the 87 mL culture system had no effect on the experiment.The OD630 in the 40,60,120 ng/mL E2 groups were significantly lower than that of the Control ? group(P<0.05),and the 120 ng/mL E2 group was significantly lower than the other groups(P<0.01).The results of fluorescence quantitative PCR showed that the Lg(copies/mL cultured feces)of 60,120 ng/mLE2 groups were significantly lower than that of Control ? group(P<0.01),and the 120 ng/mL E2 group was significantly lower than the other groups(P<0.01).The results showed that E2 could inhibit the proliferation of bacteria in feces,and the inhibition enhanced along with the increased concentration of E2.The levels of acetic acid,butyric acid,iso-butyric acid and total SCFAs in the 120 ng/mL E2 group were significantly lower than that in Control ? group(P<0.01),while the level of iso-valeric acid was significantly higher than that of Control ?group(P<0.01).The levels of propionic acid and pentanoate between the two groups had no significant difference.The results showed that 120 ng/mL E2 could affect the metabolic type of bacterial flora.The reduction rates of E2 in the culture medium from high to low were 120,20,60 ng/mL E2 group,and the 120 ng/mL E2 group was significantly higher than the other groups(P<0.01).But there was no significant difference between 20 ng/mL E2 group and 60 ng/mL E2 group.The results showed that there were differences in the utilization and decomposition of E2 in bacteria.The sequencing profile of the cultured fecal bacterial DNA indicated that the diversity index(Ace,Chao,Shannon,Simpson)of 120 ng/mL E2 group and Control ?group did not show obvious differences but the OTUs of 120 ng/mL E2 group was significantly lower than that of Control II group(P<0.05).Further,the relative abundance of Bacteroidetes in the 120 ng/mL E2 group was significantly lower than that of the Control ? group(P<0.01),the relative abundance of Proteobacteria was significantly higher than that of the Control ? group at the phylum level(P<0.01).Compared to Control ? group,the relative abundance of Macellibacteroides,Enterobacteriaceae_Unclassified and Kluyvera in the 120 ng/mL E2 group were significantly higher(P<0.05),while the relative abundance of Bacteroides were significantly lower at the genus level(P<0.01).The results showed that 120 ng/mL E2 could affect the structure of fecal bacterial flora.SUMMARY:(1)The content of P4 and E2 in feces of sows in late pregnancy was significantly higher than that of early pregnancy,and the structure and metabolites of bacterial flora were different significantly;(2)female steroid hormones could affct the structure and diversity of fecal bacteria flora,and the effects vary with hormone types and doses.INNOVATION:This study which were combined with in vivo and in vitro cultures for the first time revealed that the female steroid hormones(P4,E2)could impact on the structure and diversity of fecal bacteria flora and the effects vary with hormone types and doses. |
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