Ruminal Microbiome-host Crosstalk Stimulates The Development Of The Ruminal Epithelium Under Early Starter Feeding In A Lamb Model

The development of the rumen is an important physiological challenge for ruminants,which was related to the host metabolism,immunity and health.Previous studies have shown that starter feeding can effectively facilitate the growth and development of the rumen in ruminants.However,the mechanism through which starter feeding stimulates the development of the rumen is not clear.Here,we performed an integrated analysis in the structure and function of ruminal microbiota,metabolite pool,and host transcriptomic profiles in a lamb model with the intervention of starter feeding to understand the ruminal microbiome-host crosstalk in stimulating the development of the ruminal epithelium.The present study comprehensively described the symbiotic relationship between the rumen microbiota and the host in lambs to provide a theoretical basis for subsequent research.1.Effects of early starter feeding on rumen fermentation parameters,epithelial morphology and microbial community of lambsTo explore fermentation parameters,rumen epithelial morphology,and rumen microbiome structure response of lambs to starter feeding was the objective of this study.We used 20 healthy 10-day-old Hu sheep with similar body conditions,which were randomly assigned to groups,namely the control group(CON,n=10)and the starter treatment group(ST,n=10).Slaughter at 56 days of age,rumen fluid was used to measure pH and VFA concentrations.Then,a small rumen abdominal sac(2*2cm)was used for determination of rumen papillae surface area.The rumen contents were used for bacterial and protozoal sequencing analysis.The results showed that starter feeding significantly reduced rumen pH(P<0.001),meanwhile increased total VFA concentration(P=0.034),acetate concentration(P=0.028),butyrate concentration(P=0.028),and the proportion of butyrate(P=0.019).Rumen epithelial morphology showed that,compared with the CON group,the length(P<0.001),width(P<0.001),and surface area(P=0.002)of rumen papillae all increased in the ST group,implying starter feeding promoted the development of rumen epithelium.16S rRNA gene sequencing analysis showed bacterial communities were significantly separated in the two groups,and starter feeding significantly reduced the richness(P<0.001)and evenness of bacteria(P=0.001).Compared with the CON group,at the phylum level,starter feeding significantly increased the relative abundance of Actinobacteria and Proteobacteria,whereas decreased the relative abundance of Tenericutes(P<0.05).At the genus level,starter feeding significantly increased the relative abundance of five genera(P<0.05)and decreased the relative percentage of six genera(P<0.05).At the OTU level,starter feeding also caused the missing of 640 OTUs and the emergence of 333 OTUs.In addition,we observed that the relative abundance of many bacteria had significant change after starter feeding(P<0.05).18S rRNA sequencing results showed that protozoal communities were significantly separated in the two groups.Starter feeding significantly reduced the species evenness(P<0.05),but the protozoal richness had no significant shift.Meanwhile,Entodinium had the highest abundance and was also the only genus with increased relatively abundance(P=0.010).In conclusion,starter feeding can accelerate rumen fermentation of lambs,affect the structure and composition of rumen bacterial and protozoan communities,and promote rumen epithelium development.2.Effects of early starter feeding on carbohydrate enzyme gene abundances and fermentation pathways in rumen microbiome in lambsThe purpose of this chapter was to study the effect of starter feeding on the rumen microbial function of lambs.This experiment used the lamb as an animal model and adopt the interventional method of early starter feeding.The rumen contents were collected at the 56-day slaughter for shotgun metagenome sequencing.Metagenomic results showed that starter feeding reduced the total carbohydrate enzyme gene abundance(P=0.021)and four carbohydrate enzyme genes,including carbohydrate esterases(CEs,P=0.043),glycoside hydrolases(GHs,P=0.021),glycosyl transferases(GTs,P=0.021),and polysaccharide lyases(PLs,P=0.021).To explore the degradation process of starch in starter food,we focused on the amylase genes.We found that only GH13,belonging to α-amylase,had a significantly shift that increased in the ST group(P=0.021),and this enzyme can efficiently degrade starch.In parallel,Prevolella and Butyrivibrio were the most assigned genera for the majority of genes enriching in the ST group.To in-depth dissect the process of increased acetate and butyrate concentrations,we found that 21 enzyme genes were involved in the acetate and butyrate fermentation pathway,but the relative abundances of these 21 enzyme genes had no significant change(P>0.05).Therefore,starter feeding significantly increased the relative abundance of GH13 genes family belonging to α-amylases to promote the degradation of starch in starter food,but had not affected the relative abundance of all enzyme genes involved in the fermentation process.In conclusion,starter feeding increased the production of acetate and butyrate by accelerating the degradation rate of substrate starch in starter food.3.Effects of early starter feeding on rumen epithelial growth-related gene expressions and signalling pathways in lambsTo hunt for the mechanism of VFA promoting epithelial development,we explored the growth associating gene expression and signalling pathways of rumen epithelium in lambs.In this study,we used lambs as animal model to adopt early intervention methods of starter food Rumen epithelial tissue was collected at 56 days of slaughter for transcriptome sequencing analysis.Transcriptome results showed that total 604 differentially expressed genes(DEGs)were identified(FDR<0.05;Fold Change>2).Among them,compared with the CON group,358 genes had higher expressions,whereas 246 genes were down-regulated by starter feeding.The GO enrichment analysis of 604 DEGs revealed that 73 significantly changed GO terms were annotated,mainly including three modules of regulation,namely protein activity processes(modification and degradation),substance transport,and cell growth(apoptosis and proliferation).Then,we focused on cell growth module enriching with 51 DEGs,from which eight genes(MAPK1,PIK3CB,SAVl,SNA12,DLG1,ITGA6,TNFSF10,and BAD)located in growth-related signalling pathways.The expressions of these eight genes had strong correlations with acetate and butyrate concentration.Finally,we established a network of rumen epithelial growth-promoting gene-signalling pathwayfunction co-expression model.Therefore,starter feeding increased the concentration of acetate and butyrate in the rumen of lambs.Then,the increased acetate and butyrate,which were generated by microbiota,had a strong association with the growth-related genes locating in growth-associated signaling pathways in the ruminal epithelium.These co-development networks promoted rumen epithelial growth.