Comparison Of Growth Performance,Ruminal And Colonic Microbiome,and Ruminal Epithelium Transcriptome Of Hu Sheep Fed Low-Grain,Non-Pelleted Or Pelleted High-Grain Total Mixed Ration

Diet composition and animal feed processing significantly affects feedlot management,feed intake as well as animal performance.Among various processing methods,feed pelleting is widely used in animal production to reduce the dustiness of feed and feed sorting.However,little information is available on the effect of pelleted high grain total mixed ration on the health of gastrointestinal tract in ruminants.In addition,few studies have been carried out to explore the effects of high grain feeding on the health of rumen and hindgut in meat sheep.In the present study,we tested the effects of the pelleted or nonpelleted high-grain total mixed ration,and mashed low grain total mixed ration on the growth performance,ruminal and colonic microbiome and transcriptome of ruminal epithelium in sheep.1-Comparison of low grain,non-pelleted or pelleted high-grain TMR on the growth performance,rumen fermentation,rumen morphology and gene expression of proinflammatory cytokines and tight junction proteins in rumen epithelium of Hu sheepThis study aimed to compare the rumen fermentation,growth performance,rumen morphology and gene expression of proinflammatory cytokines and tight junction proteins of Hu sheep fed non-pelleted low grain,non-pelleted high grain or pelleted high-grain total mixed ration(TMR).Fifteen male lambs were allocated into 3 groups(n=5 for each).One group(LG)received a low-grain non pelleted TMR(19.2%corn),the second group(HG)received a high-grain non-pelleted TMR(40.6%corn),while the third group(HP)received the same high-grain TMR in a pelleted form.In the comparison between the LG and HG groups,results showed that the animals of the HG group showed a significantly higher average daily gain(P=0.032)and body weight gain compared with the LG group(P=0.032).The disruption of the stratum corneum that appeared in the histological examination in the HG group as compared with the LG,and the upregulation of TNF-? and IL-6 in HG group,indicate the presence of an inflammation that caused this disruption and loss of integrity.As compared with the HG group,the rumen pH of the HP group was lower(P=0.012),while lactate concentration was higher(P=0.024).While there was no significant difference between HG and HP in any of the growth performance parameters.In summary,this study showed that the high grain mashed TMR enhanced the growth performance as compared with the low grain.On the other hand,pelleting the high-grain TMR altered the rumen fermentation,in addition to a negative effect on the rumen epithelium health.2-Comparison of the rumen digesta and epithelium-associated microbiota of Hu sheep fed non-pelleted low grain,non-pelleted high grain or pelleted high-grain total mixed rationThis study aimed to compare the rumen digesta and epithelium-associated microbiota of fattening lambs fed non-pelleted low grain,non-pelleted high grain or pelleted high-grain TMR in Hu sheep.The experimental animals,diet preparation and experimental design are shown in chapter 2.The composition and CowPI predicted function of ruminal digesta and epithelium-associated microbiome were examined.During the comparison between the LG and HG groups,the unweighted unifractional principle coordinates analysis and the analysis of the molecular variance(AMOVA)revealed that both groups clustered separately from each other in both rumen digesta and epithelium-associated microbiota.All the alpha diversity estimators were observed to decrease in the HG compared with the LG.On the other hand,Bacteroidetes and Firmicutes were the predominant phyla of the rumen digesta and epithelium-associated microbiota in both the LG and HG groups.At genus level,Prevotella showed a significant increase in the HG group compareepithelial d with the LG group(P=0.009).In contrast,the unclassified Lachnospiraceae showed a significant decrease in the HG group compared with the LG group(P=0.009).The analysis of predominant OTUs is consistent with the analysis on genus level of Prevotella and unclassified Prevotellaceae,where they increased in the HG group as compared with the LG group.In the comparison between the HG and HP groups,the unweighted unifractional principle coordinates analysis and the analysis of the molecular variance(AMOVA)revealed that two groups clustered separately from each other in both rumen digesta and epithelium-associated microbiota.All the alpha diversity estimators were decreased in the HG compared with HP.In addition to that.the abundance of Fibrobacteres was significantly decreased in the HP group compared with the HG group(P=0.047).Also,at the genus level,pelleting resulted in a decrease in the percentage of unclassified BS11 gut group and Succiniclasticum,while it led to an increase in the abundance of some simple sugar fermenters like Megasphaera(P=0.008),Olsenella(P=0.007),Dialister(P=0.009)and Sharpea(P=0.019)as compared to the HG group.Among the predominant OTUs,the proportion of some OTUs belonging to the Prevotella species increased in the HG group with a severe decrease in the HP group.In contrast,Megasphaera(P=0.008),Rumincoccus(P=0.016),Olsenella(P=0.007)and Sharpea(P=0.005)showed a significant increase in the HP group compared with the HG group.The CowPI predicted functional profile of the rumen digesta and epitheliumassociated microbiota revealed the enrichment of the pentose phosphate pathway in the HP group only.In summary,this study showed that high grain diet altered the rumen microbiota diversity compared with low grain diet.In addition,pelleting of high-grain TMR altered the rumen microbial diversity and amplified the ability of ruminal microbiome to utilise simple sugar compared to the non-pelleted diets.3-Transcriptome analysis of the rumen epithelium in Hu sheep fed non-pelleted low grain,non-pelleted high grain or pelleted high-grain total mixed rationThis study investigated the effects of non-pelleted low grain,non-pelleted high grain or pelleted high-grain TMR on the transcriptome analysis of the rumen epithelium in Hu lamb.The experimental animals,diet preparation and experimental design are shown in chapter 2.Among the encoded genes,1817 DEGs were identified from the comparison between the LG and HG groups(P value<0.05;fold change>1.5).Among these DEGs,there were 847 upregulated genes and 969 downregulated genes.GO(Gene Ontology)enrichment analysis indicated that the DEGs were classified into three main categories:biological process,cellular component,and molecular function.In addition to that,the KEGG enrichment analysis of differentially expressed genes indicated that the most enriched categories were "Ribosome"(P<0.001,including 75 DEGs),"Alzheimer's disease"(P<0.001,including 50 DEGs),"parkinson's disease"(P= 3.28E-13,including 50 DEGs),“Huntington's disease"(P=8.74E-13,including 50 DEGs),"Oxidative phosphorylation"(P=1.26E-12,including 50 DEGs),"Non-alcoholic fatty liver disease"(NAFLD)(P=1.10E-09,including 50 DEGs)and "Cardiac muscle contraction"(P=2.37E-06,including 25 DEGs).The downregulated in the DEGs related to the Oxidative phosphorylation pathway may be due to the upregulation of inflammatory cytokines(TNF-?)in the HG as compared with the LG group.The enrichment in"regulation of innate immune response" GO suggested that innate immune response could be one of the mechanisms involved in epithelial adaptation to the high grain diet.A total of 52 DEGs were identified from the comparison between the HG and HP groups(P value<0.05;fold change>1.5)out of which 32 genes were upregulated whereas 19 genes were downregulated.The transcriptome profiling analysis between HG and HP groups showed no significant difference in the KEGG pathways.4-Comparison of the fermentation,composition of digesta and mucosa-associated microbiota,and epithelium health in the colon of Hu sheep fed non-pelleted low grain,non-pelleted high grain or pelleted high-grain total mixed rationThis study investigated the effects of non-pelleted low grain,pelleted or non-pelleted high grain TMR on the fermentation,composition of digesta and mucosa-associated microbiota,and epithelium health in the colon of Hu Sheep.The experimental animals,diet preparation and experimental design are shown in chapter 2.The pH of the colon was significantly lower in the HG group as compared with the LG group(P=0.001).According to the(3-diversity result,the composition of the colonic digesta and mucosa-microbial community of the LG and HG groups were distinctly separated from each other.Also,the unweighted distance-based analysis of AMOVA result showed a statistically significant difference.At the phylum level,Firmicutes,Bacteroidetes and Spirochaetae were the most predominant phyla in both the colonic mucosal and digesta microbial community of the LG and HG groups.At the genus level,the colonic mucosa samples in the HG group showed a significant increase in Prevotella(P=0.009),Subdoligranulum(P=0.041),unclassified Lachnospiraceae(P=0.009),Succinivibrio(P=0.016),unclassified S24-7(P=0.009),Roseburia(P=0.016),Coprococcus(P=0.009)and Acetitomaculum(P=0.028)as compared with the LG group whereas Treponema(P=0.028),and Unclassified Gastranaerophilales(P=0.009)were significantly lower.In addition,the colonic digesta samples in the HG group showed a significant increase in population of Prevotella(P=0.009),Subdoligranulum(P=0.016),unclassified Lachnospiraceae(P=0.009)and unclassified S24-7(P=0.009)while the RC9_gut_group significantly decreased(P = 0.009)in comparison with the LG group.On the other hand,the HG group showed higher concentrations of acetate and total VFAs in comparison with the HP group(P=0.001,0.012,respectively).According to the ?-diversity result,the composition of the colonic mucosa-microbial community of the HG and HP groups were distinctly separated from each other.At the genus level,Alloprevotella(P=0.02)and Olsenella(P=0.009)were significantly higher in the HP group than the HG group.Roseburia(P=0.047)showed a significant decrease in the HP group compared to the HG group.The analysis of the predominant OTUs is consistent with the analysis of Prevotella at genus level.PICRUSt predicted the metagenomic contribution of the communities observed based on KEGG.There was an increase in the immune system diseases pathway of the colonic mucosaassociated microbiota in the HG group.The colonic epithelium revealed sloughing of the epithelial surface in both HG and HP groups.In conclusion,the study indicates the alternation of colonic fermentation characteristics by feeding pelleted or non-pelleted highgrain TMR,which caused a colonic epithelial injury compared with low grain TMR.Moreover,the colonic microbiota shows an alteration in the composition and diversity as a result of mashed or pelleted high grain diet.Finally,we can conclude that high grain(HG)TMR improved the ADG and BWG of feedlot sheep compared with the low grain(LG)TMR.While pelleting the high-grain TMR(HP)did not enhance the animal performance compared with its mashed-form(HG).Furthermore,High grain(HG)TMR increased the isobutyrate and valerate concentrations compared with LG.Also,pelleted TMR increased the ruminal lactate level and decreased the rumen pH compared with HG.On the other hand,the presence of some taxa specialized in complex carbohydrates fermentation was observed in the rumen and colonic digesta and epithelium-associated microbiome of the HG.Pelleted high grain TMR caused a shift in the rumen and colonic digesta and epithelium-associated microbiome towards improved simple sugar fermentation compared with HG group.In addition to that,the whole transcriptome of the rumen epithelial tissues revealed that the high grain diet(HG)has positively altered the gene expression in the rumen while no effect was observed in the HG group compared with the HP group.