Effects Of The Different Dietary Fibers On Gut Mucosal Gene Expression,Microbiota Composition And Metabolites In Growing Pigs

Dietary fibers refer to plant-derived carbohydrates which can’t be degraded by digestive enzymes in the small intestine of monogastric animals.They are mainly fermented by the microorganisms in the hind intestine and then improve functions of them.Dietary fibers with different types have distinct physical and chemical properties,which exert different effects on the intestinal functions.Inulin,resistant starch,and pectin,these three fibers are commonly used to improve intestinal health.Many studies have shown that the three different fibers can cause different changes in the gut microbiota composition.However,these studies mainly focused on a single fiber and the effects of different fibers on the intestine in the same experiment were unclear.Therefore,this research mainly took modern technical means such as transcriptomics,16S rRNA gene high-throughput sequencing,and metabolomics,to investigate the effects of different dietary fibers on gut mucosal gene expression,microbiota composition,and metabolites in growing pigs and the links among dietary fibers,intestinal flora,and the host.The paper includes the following three parts:1. Effects of different dietary fibers on intestinal morphology and mucosal gene expression in pig colonThe purpose of this experiment was to compare the effects of different dietary fibers on colonic morphology and mucosal gene expression in growing pigs.28 healthy Duroc × Landrace × Large White growing pigs with similar body weight(8.79 ± 0.09 kg) were randomly divided into four groups:control group (CON),inulin group (INU),raw potato starch group (RPS) and pectin group (PEC).Each group had seven replicates with one pig for each replicate.The CON group was fed with a basal diet of corn-soybean meal,and 8% (w/w) corn starch in the basal diets were replaced with inulin,raw potato starch and apple pectin,respectively in the three fiber-treated groups.The trail lasted for 40 days and all pigs were slaughtered at the end of the experiment.Sections of the proximal colon were collected for intestinal histomorphology analysis,and colonic mucosal samples were collected for transcriptome analysis.The results showed that the thickness of the colonic mucosa in the PEC group was significantly lower than that in the INU group,and the surface of colonic epithelium in the PEC group was obviously damaged.According to the screening conditions (P <0.05,FC≥ 1.5 or <0.67),we selected out the differential expressed genes (DEGs) between each fiber-treated group and the CON group,and then carried out the Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on them.It was found that 161 genes were up-regulated and 79 genes were down-regulated in the INU group.The pathway enrichment analysis showed that DEGs were mainly related to pathways about starch and sucrose metabolism,steroid biosynthesis,retinol metabolism,ascorbic acid and aldehyde acid metabolism,etc.;61 genes were up-regulated and 56 genes were down-regulated in the RPS group.And these DEGs were mainly enriched in pathways about fat digestion and absorption,retinol metabolism,RNA degradation,etc.;the expressions of 64 genes were enhanced and 75 genes were lowered in the PEC group.More than half of the DEGs were enriched in GO items related to immune function,and the enriched pathways included retinol metabolism,cytokine-cytokine receptor interaction,ascorbic acid and aldehyde metabolism pathways.The results of the protein-protein interaction (PPI) network analysis showed that the supplementation of pectin up-regulated the expression of UGT2B31 involving the metabolism of important endogenous compounds such as bile acids and steroids.These results suggested that the supplementation of 8% pectin could cause damage to the colon mucosa of growing pigs,but the supplementation of inulin and raw potato starch had no significant effect on the morphology of the colon epithelium.In addition,dietary fibers significantly changed the gene expression related to the pathways of glucose metabolism and lipid metabolism in the colon mucosa.Moreover,the supplementation of pectin had significant effect on the gene expression related to the mucosal immunity.2.Effects of different dietary fibers on gut microbiota composition in growing pigsThe purpose of this experiment was to compare the effects of dietary fibers on intestinal microbiota composition in growing pigs.On the basis of the first experimental design,contents of ileum and colon were collected for 16S rRNA gene high-throughput sequencing analysis.The results showed that,compared with the CON group,the addition of pectin significantly increased the Chao index of colonic microbiota composition,while the addition of these fibers all had no significant effect on the a diversity of ileal microbiota composition.The results of PCoA analysis showed that pectin significantly changed the bacterial communities of colon contents in growing pigs.Compared with the CON group,the relative abundances of Firmicutes and Proteobacteria were increased significantly (P <0.05),but the abundance of Bacteroidetes decreased significantly (P <0.05) in the PEC group,and inulin and raw potato starch had no significant effect on the bacterial communities at the phylum level(P> 0.05).Compared with the CON group,pectin significantly increased the relative abundances of 17 genera (P <0.05),including [Eucharacterium]coprostanigenes group,Ruminocaceae NK4A214 group,Megasphaera and Leeia,while the abundance of Bacteroides was predominantly reduced (P <0.05).Besides,the addition of inulin predominantly increased the abundance of Rickenellaceae RC9 gut group (P <0.05),and the addition of raw potato starch significantly reduced the relative abundance of Bacteroides (P <0.05).With the further analysis of unique OTUs in pectin group,we found the pectin group had the greatest number of OTUs and Megasphaera took up the most.The results showed that pectin exerted the most noticeable effect on colonic microbiota composition,which may relate to the gelation of pectin,but the effects of all these fibers on the ileal flora was not significant.3.Effects of different dietary fibers on microbial metabolism in pig colonThe purpose of this experiment was to compare the effects of dietary fibers on the colonic microbial metabolism in growing pigs.On the basis of the first experimental design,the colonic content was collected for metabolomics analysis.The results showed that,compared with the CON group,pectin significantly increased the concentration of isobutyric acid in the colonic content (P <0.05),while the other two fibers had no significant effect on the concentrations of short chain fatty acids in pig the colon (P> 0.05).Basing on the screening conditions (VIP> 1 and P <0.05),compared with the CON group,the results showed that the concentration of pyroglutamic acid was decreased significantly (P <0.05),but concentrations of 2-methyl-butanedioic acid and eicosanoic acid,nicotinic acid and hypoxanthine were increased significantly in the RPS group,and at the same time,the supplementation of raw potato starch significantly altered pathways related to glutathione metabolism,nicotinate and nicotinamide metabolism,and purine metabolism.In the PEC group,lipids accounted for the largest proportion among totally 26 differential metabolites,and concentrations of 10 lipid compounds such as nonanoic acid and arachidonic acid were significantly increased,but creatinine decreased significantly.At the same time,the addition of pectin could affect pathways related to fatty acid synthesis,arginine synthesis,β-alanine metabolism and carbon metabolism;the addition of inulin had no significant effect on microbial metabolism.The results suggested that the supplementation of pectin significantly changed the microbial metabolites related to lipid metabolism,carbon metabolism and amino acid metabolism,and the supplementation of raw potato starch affected several microbial metabolites,while the supplementation of inulin had no significant effect on colon microbial metabolism.In conclusion,this study showed that different fibers could exert different effects on gut mucosal gene expression,bacterial structure and metabolites in growing pigs.In addition,pectin had the most distinct impact on the pig gut with the same addition levels as others.Nevertheless,the supplementation of pectin with the high addition level was adverse to gut health in growing pigs.