The Adaptive Response Of The Microbiota And Epithelial Morphology And Function To High Grain Diets In The Colon Of Hu Sheep

The hindgut is an important organ in the digestive tract of ruminants,which mainly participates in the digestion and absorption of nutrients from the outside.Under the high grain（HG）diet,the structure of the microbial flora and the epithelial morphology and function of the rumen were changed in order to adapt to the feeding pattern,but it is not very clear at present what mechanism for the hindgut to adapt to the HG diet.In this paper,an animal experiment was conducted to study the adaptive response process of the colonic microbial fermentation,colonic epithelial morphology and function,and colonic microbiota to an HG diet under the condition of abruptly increasing the proportion of grains.The full text includes the following three parts.1.Dynamic shifts in the microbial fermentation and epithelial morphology and function in the colon of sheep fed a high grain dietThis study investigated the dynamic shifts in microbial fermentation and epithelial morphology and function in the colon of sheep fed a high grain（HG）diet.A total of 20 male sheep were randomly assigned to 4 groups（n=5 for each）.The sheep in the first group received hay diet（CON）.The animals in the other 3 groups were fed an HG diet for 7（HG7）,14（HG14）or 28（HG28）days,respectively.The colonic digesta and mucosa samples were collected for further analysis.Following the days of the sheep being fed an HG diet,results showed that the colonic pH linearly reduced（P=0.007）,and the concentrations of acetate,propionate,butyrate,total VFA（TVFA）,and lactate linearly increased（P<0.05）.A linear decrease was detected in the molar proportions of the isobutyrate,isovalerate,and valerate with the days being fed an HG diet（P<0.05）,while the proportion of butyrate（P<0.001）increased linearly with the number of days fed an HG diet.The microscopic examinatiorn showed that the mucosal epithelial injury was observed in HG7 group while the trend of restoration of the mucosal epithelium was observed in HG14 and HG28 groups.The results of gene expression of tight junction revealed that the mRNA expression of claudin-4 in HG7 group decreased from the CON group then increased in the HG14 group,after that decreased to the CON level in HG28 group（P<0.001）.The results of genes expression related to proliferation and apoptosis revealed that an HG diet decreased the expression of CDK-1,CDK-6 and cyclin-E（P<0.05）.The expression of caspase-8 in HG7 group increased from the CON group then decreased to the CON level in HG14 and HG28 group（P=0.046）;No significant difference was observed in the expression of caspase-3 between the CON and HG7 groups until a decrease was observed in HG14 and HG28 groups（P=0.025）.The results of mRNA expression of genes assocated with VFA absorption and metabolism showed that the expression of MCT-1 in HG7 group decreased from the CON group then gradually increased to the CON level in HG14 and HG28 group（P=0.013）.The HG diet decreased the mRNA expression of NHE-3 and BDH-1（P<0.05）.No significant shift was observed in the expression of PAT-1 and HMGCS-1 in the CON,HG7,and HG14 groups until a decrease was observed in HG28 group（P<0.05）.Generally,HG feeding altered the microbial fermentation and colonic structure and function,and these changes occurred gradually over at least 4 weeks.2.Dynamic shifts in the composition of digesta-associated microbiota in the colon of sheep fed a high grain dietThis study investigated the dynamic shifts in digesta-associated microbiota composition in the colon of sheep fed a high grain（HG）diet.The experimental design is samed to the first part.The colonic digesta was sampled to characterize the bacterial communities using Illumina MiSeq sequencing and to determine the 16S rRNA gene copies of total bacteria,Firmicutes,and Bacteroidetes using quantitative real-time PCR.As time advanced,microbial analysis showed that an HG diet linearly reduced（P<0.05）the diversity and richness of the colonic microbiota.The principal coordinate analysis results showed that the colonic digesta-associated bacterial communities of the four groups significantly shifted with number of days fed an HG diet.During the experimental process,7 phyla significantly changed within the 18 detected phyla.At the genus level,HG feeding significantly decreased the relative abundance of Akkermansia,unclassified Clostridiales,unclassified BS11_gut_group,Desulfovibrio,and unclassified Bacteroidales（FDR<0.05）,and increased the relative abundance of some taxa including unclassified Lachnospiraceae,Coprococcus,unclassified Erysipelotrichaceae,Prevotella,Anaerostipes,and Clostridium_sensu_stricto₁（FDR<0.05）,and the percentage of these taxa was not affected by days fed an HG diet.However,with the increasing number of days fed an HG diet,the proportion of Ruminococcus,Roseburia,and Bifidobacterium dynamically changed（FDR<0.05）.Generally,HG feeding altered colonic digesta-associated bacterial communities,and these changes occurred gradually over at least 4 weeks.3.Dynamic shifts in the composition of mucosa-associated microbiota and mucosal inflammatoty cytokines in the colon of sheep fed a high grain dietThis study investigated the dynamic shifts in the mucosa-associated microbiota composition in the colon of sheep fed a high grain（HG）diet.The experimental design is samed to the first part.The colonic mucosa was sampled to characterize the bacterial communities using Illumina MiSeq sequencing and to determine the mRNA expression levels of cytokines using quantitative real-time PCR.As time advanced,microbial analysis showed that an HG diet linearly reduced（P<0.05）the diversity and richness of the colonic microbiota.The principal coordinate analysis results showed that the colonic mucosa-associated bacterial communities of the four groups significantly shifted with number of days fed an HG diet.During the experimental process,7 phyla significantly changed within the 21 detected phyla.At the genus level,HG feeding significantly decreased（FDR=0.016）the relative abundance of Treponema,and increased（FDR<0.05）the relative abundance of some taxa including Prevotella,Oscillibacter,Acetitomaculum,Anaerostipes,and Clostridium_sensu_stricto₁,and the percentage of these taxa were not affected by days fed an HG diet.With the increasing number of days fed an HG diet,the proportion of Roseburia,unclassified Lachnospiraceae,Butyrivibrio,Bifidobacterium,and Helicobacter dynamically changed（FDR<0.05）.The realtime-PCR results showed that the 16S rRNA gene copies of total bacteria（P=0.024）,Firmicutes（P=0.002）,and Bacteroidetes（P=0.002）in HG28 group were significantly higher than those in CON,HG7,and HG14 groups,and no significantly difference was observed among these three groups.Results of real-time PCR found that the HG diet increased the mRNA expression of IL-1β,TNF-α,and TLR-3（P<0.05）in colonic mucosa.The correlation analysis revealed a negative association between the mRNA expression of TNF-α and colonic pH（r=-0.517,P=0.020）.The mRNA expression of IL-1β（r=0.672,P=0.001）and TNF-α（r=0.617,P＝0.004）was positively related to the abundance of Closrridium—sensu_stricto₁.Generally,these findings revealed that HG feeding altered colonic mucosal bacterial communities and induced the epithelial inflammation,and these changes occurred gradually over at least 4 weeks.