Detection Of Firmicutes And Bacteroidetes In The Pig Gut And The Correlation Between Their Abundance And Fat Deposit

The gut microbiota of mammals is mainly consisted of the Firmicutes and Bacteroidetes divisions,and their relative abundance is closely related to the body fat in humans and mice.The aim of this study was to investigate the correlation between the body fat storage and the relative abundance of Firmicutes,Bacteroidetes,Bacteroides,and the ratios of Firmicutes/Bacteroidetes(FD/BD) and Firmicutes/Bacteroides(FD/BS) of the gut microbiota in pigs.This study consists of five parts of experiments as follows:Exp.1 Development of a Real-Time PCR Method for detecting 16S rRNA gene of Firmicutes and Bacteroidetes in the gut microbiotaBased on the multialignment analysis of 200 16S rRNA gene sequences,two pairs of 16S rRNA gene-targeted group-specific primers were designed for the gut Bacteroidetes and Firmicutes divisions.The specificity of group-specific primers was confirmed by amplifying genomic DNAs from 8 representative bacterial strains with each of the two pairs of primers by conventional PCR and real-time PCR.The equal-fold dilutions of standards were used to test the two real-time PCR assays.The primer pairs were specific for their target strains and yielded the PCR products of the expected size only from their targets,and melting curves were analyzed to confirm the specific amplification.There was a linear relationship between the log of the standard DNA copy number and the calculated Ct value across the specified concentration range.So the real-time PCR assays developed for Firmicutes and Bacteroidetes could use to investigate the composition of gut microbiota.Exp.2 Differences of Firmicutes,Bacteroidetes and Bacteroides in the gut microbiota of obese and lean pigs from Banna mini-pig inbred lineTo investigate the relation between body fat and gut microbiota in pigs,the specific primers for Firmicutes and Bacteroidetes were used together with the previously developed group-specific primers to detect the copy numbers of the 16S rRNA gene of all Bacteria,Bacteroidetes division,Firmicutes division and Bacteroides spp.in the feces of obese and lean pigs from Banna mini-pig inbred line(BMI) by real-time PCR analysis,and their percentages based on all Bacteria(relative abundance) were calculated.The obese pigs had a～68%fewer percentage of Bacteroidetes division(P＜0.001) and a～65%fewer proportion of Bacteroides spp.(P＜0.001) than the lean pigs.The trends of quantitative changes of both Bacteroidetes and Bacteroides were similar in obese and lean pigs,and their percentages had a negative correlation with the body weight(P＜0.01).The percentage Firmicutes was not different significantly,although it was higher in numerical value in obese pigs than in lean pigs.The ratios of FD/BD and FD/BS in obese pigs were higher than in lean pigs(P＜0.05),and the ratio of FD/BS had a positive correlation with the body weight(P＜0.05).Exp.3 Correlation between the relative abundance of Firmicutes, Bacteroidetes and Bacteroides in the gut microbiota and fat deposit in Meishan and Landrance pigsTo determine whether body fat is relative to the gut microbial communities in the common breeds,the～40 kg Landrace and Meishan pigs were chosen and had ad libitum access to the same diet in this study.When body weight reached to 90 kg,6 pigs from each breed were selected randomly and subjected to exsanguinations to measure backfat depth, diameter and area of fat cells,copy numbers and abundance of Firmicutes,Bacteroidetes, Bacteroides in cecal content.Results indicated that:Meishan pigs had higher backfat depth, diameter and area of fat cells comparing with the Landrace pigs.Landrace pigs had higher copy numbers of all Bacteria,Firmicutes,Bacteroidetes,Bacteroides in their cecal contents than Meishan pigs(P＜0.05).Lower percentages of Bacteroidetes(P＜0.01) and Bacteroides spp.(P=0.013) were in Meishan pigs than in Landrance pigs,and Firmicutes percentage of Meishan was higher than Landrance(P=0.290),although the difference was not significant.The ratios of FD/BD(P=0.055) and FD/BS(P＜0.05) in Meishan pigs were higher than in Landrance pigs,and these ratios had a positive correlation with the backfat depth(P＜0.05).The trends of quantitative changes of both Bacteroidetes and Bacteroides were similar,their percentages had a negative correlation with the backfat depth(P＜0.05), and the ratio of Bacteroides/Bacteroidetes in Meishan was lower than in Landrance (P=0.089).These results confirmed that Meishan pigs are typical genetically obese and Landrace pigs are lean,the percentages of Bacteroidetes and Bacteroides had a negative correlation with the body fat,and the ratio of FD/BS had a positive correlation with the body fat.Exp.4 Changes of Firmicutes,Bacteroidetes and Bacteroides in the gut microbiota of obese and lean pigs in different phasesTo investigate the effect of dynamic changes of body fat on the gut microbial communities in pigs,we detected the populations of all Bacteria,the Bacteroidetes,the Firmicutes and the Bacteroides spp.in the feces of Meishan(obese) and DLY(lean) pigs at 3,4,5,7 months old.The results indicated that:the abundance of Firmicutes in Meishan was higher than in DLY at different phases,and the difference reached a significant level at 5 months old.With the age increasing,the copy numbers of Bacteroides were reduced (P＜0.05).With the age increasing,the abundance of Firmicutes in the both breeds raised (Meishan P＜0.05,DLY P＜0.01),the proportion of Bacteroidetes trended to diminish (Meishan P=0.104,DLY P＜0.05),the Bacteroides percentage was also reduced(Meishan P＜0.05,DLY P＜0.01),and FD/BD(Meishan P=0.101,DLY P＜0.01) and FD/BS(P＜0.01) correspondingly increased.These data suggested that the changes in gut microbiota were fit in with the dynamic changes of body fat;the decreased amplitude of the Bacteroides percentage was bigger than the Bacteroidetes percentage accompanied with increase of body fat,so the Bacteroides spp.may be a good biomarker correlative closely with body fat of host.Exp.5 Ratio of carbohydrate and protein in diets on fat deposit,gut microbiota and expression of gene related to fat storage in pigsTo further investigate the relation between body fat and the gut microbiota in pigs with dynamic changes of body fat,the diets with different carbohydrate/protein(CHO/CP) were used to feed obese and lean pigs with the same ages,and detected the content of subcutaneous fat and intramuscular fat,gut microbial communities and gene expression relative to fat deposit.Twelve Meishan(obese) and twelve DLY(lean) pigs at 5 months old were assigned randomly to high carbohydrate/protein(HCHO,CP:10.1-11.2%) or low carbohydrate/protein(LCHO CP:22.7%) diet for 3 months.The backfat depth in vivo and fecal microbial communities were detected at the start and 2 months of experiment.When the experiment was end,5 pigs from each treatment were selected randomly and subjected to exsanguinations to measure backfat depth,abdominal fat weight,intramuscular fat content,the microbial communities in cecal content,ANGPTL4(Angiopoietin-like protein 4) gene expression in ileum,PPARγ(peroxisome proliferator-activated receptorsγ) and H-FABP(heart-type fatty acid binding proteins) gene expression in fat and muscle tissues. The results indicated that:HCHO diet made the fat deposit increased(P＜0.05),and especially increased the intramuscular fat content(P＜0.01).The gene expression of PPARγ(P=0.093) and H-FABP(P=0.095) was higher in HCHO than in LCHO treatment in DLY pigs.Meishan pigs had more Firmicutes proportion(P＜0.05) and higher ratios of FD/BD (P=0.127) and FD/BS(P=0.129) comparing with DLY pigs at the start,and Firmicutes proportion(P=0.106) and FD/BS(P＜0.05) in Meishan were still higher after the 2 months. Feeding HCHO diet for 2 months increased the abundance of Firmicutes(P=0.076) and the ratio of FD/BS(P＜0.01),and reduced the percentages of Bacteroides based on all Bacteria (P=0.094) and Bacteroidetes(P＜0.05) in the feces.But there was no difference in the abundance of bacterial groups between HCHO and LCHO treatments after the 3 months.It is concluded that the diets with different carbohydrate/protein had a significantly effect on fat storage,relative gene expression and gut microbial communities,and the changes in gut microbiota were consistent with the dynamic changes of body fat.In summary,data reported here demonstrated that:1) The Real-Time PCR assays for detecting the 16S rRNA gene of Firmicutes and Bacteroidetes in the gut microbiota were specific for their target strains;2) It is similar to in mice and humans that the fat storage is relative to the abundance of Firmicutes and Bacteroidetes of the gut microbiota in pigs.In general,there is a higher ratio of Firmicutes/Bacteroidetes in obese individual than in lean individual;3) The Bacteroides spp.may represent the Bacteroidetes division,and had a negative correlation with the body fat in pigs; 4) With the age increasing,the copy numbers and abundance of Bacteroides were reduced, the abundance of Firmicutes and the ratios of Firmicutes/Bacteroidetes and Firmicutes/Bacteroides increased;5) Feeding diet with higher carbohydrate increased the deposit of body fat and the ratio of Firmicutes/Bacteroides of the gut microbiota in pigs.