Effect Of Severe Feed Restriction On Rumen Fermentation,epithelium-associated Microbiota And Epithelial Transcriptional Profile Of Hu Sheep During Late Pregnancy

During the late pregnancy,a severe decrease in feed intake in ewes carrying multiple fetuses because rumen volume decreases due to the fetuses growth in the abdominal cavity.It will lead to negative energy balance and result in metabolic disorder such as pregnancy toxemia.As a special digestive organ of ruminants,rumen has been shown to be responsive to changes in dietary protein and energy level,but little is known about the mechanism for the rumen rumen epithelial morphology,rumen epithelium-associated microbiota and epithelium transcriptional profile in response to the feed restriction.The objective of this study was to investigate the effect of severe feed restriction on rumen fermentation,epithelium-associated microbiota and epithelial transcriptional profile of Hu sheep during late pregnancy and provide theoretical basis for the nutritional regulation of late pregnancy1.The effect of severe feed restriction on rumen fermentation,rumen epithelial morphology and function of Hu sheep in the late pregnancy.The objective of this study was to evaluate the changes in the ruminal fermentation,epithelial morphology and function of Hu sheep in response to severe feed restriction(SFR)during the late pregnancy.Sixteen pregnant ewes(108 days of gestation)were randomly blocked and assigned to one of two treatments:control(CON,n=8)and SFR(n=8).Ewes were fed a common diet with a 60:40 forage to concentrate ratio for 7 days baseline period followed by a SFR challenge period.Ewes of the SFR treatment group were restricted to 30%of the base for 15 days.At the end of the experimental period,the body weight of Hu sheep was measured before slaughter.Then all animals were slaughtered and ruminal contents and ruminal epithelial tissue were collected.Results showed that,the body weight gain was significantly greater(P=0.017)in the CON group than that in SFR group.The body weight of fetuses from the CON group is greater(P=0.016)compared with the SFR group.Ruminal pH was greater in SFR group(P=0.040)compared with CON group,while SFR decreased the concentrations of ruminal acetate(P<0.001),propionate(P<0.001),butyrate(P=0.025),valerate(P=0.039)and total VFA(P<0.001).The molar proportion of propionate was greater(P=0.022)in the CON group than that in the SFR group,and the ratio of acetate to propionate tended to increase(P=0.059).Plasma IGF-1(P=0.007)concentration of ewes in SFR group was lower than that of the CON group.Compared with the CON group,SFR decreased the empty rumen tissue weight(P=0.003),the length(P<0.001),width(P=0.001),and surface area(P<0.001)of rumen papillae.The real-time PCR results showed that SFR decreased the mRNA expression of Claudin-1(P=0.001)and ZO-1(P=0.009).SFR down-regulated the mRNA expression of genes involved in cell cycle and cell apoptosis including CDK1(P=0.001),CDK2(P=0.028),CDK6(P=0.035),Cyclin A2(P=0.009),Cyclin B1(P=0.004),Cyclin D1(P=0.001),Cyclin El(P<0.001)and Bad(P=0.017).The results of mRNA expression of VFA absorption and metabolism showed that the mRNA expression of MCT-1(P=0.022),NHE-2(P=0.009),NHE-3(P=0.050),PAT-1(P=0.032),BDH-1(P=0.028),and HMGCL(P=0.047)in SFR group decreased compared with the CON group.Generally,our data suggest that SFR decreased rumen VFA concentration,altered the rumen fermentation pattern during the late pregnancy.Severe feed restriction led to a decrease in the surface area of the rumen epithelial papillae,inhibited cell cycle progress,and altered the rumen barrier function.2.The effect of severe feed restriction on the composition and function of epitheliumassociated microbiota of Hu sheep in the late pregnancy.The objective of this study was to evaluate the changes in the composition and function of epithelium-associated microbiota of Hu sheep in response to severe feed restriction(SFR)during the late pregnancy.At the end of the experimental period,all animals were slaughtered and ruminal epithelial tissue were collected for microbial DNA extraction.A plot of principal coordinate analysis and analysis of molecular variance revealed that the composition of ruminal epithelial bacterial communities in the CON group was distinct from that of the ruminal epithelial microbiome in the SFR animals.At the phylum level,20 phyla were found across all samples.The abundances of Elusimicrobia(P=0.036)and Lentisphaerae(P=0.012)were greater in the SFR group than the CON group.At the genus level,172 genera were detected across all samples.Compared with CON group,SFR increased the abundance of unclassified Neisseriaceae(P=0.046),Comamonas(P=0.002)and Papillibacter(P=0.005),and decreased the proportion of Howardella(P=0.027),Desulfobulbus(P=0.036)and Suttonella(P=0.036).The metagenome of ruminal epithelium-associated microbiota predicted by PICRUSt revealed that the SFR group had a greater relative abundance of genes related to amino acid metabolism(P=0.003),cellular processes and signaling(P=0.021),lipid metabolism(P=0.001),xenobiotics biodegradation and metabolism(P=0.009),metabolism of terpenoids and polyketides(P=0.002),metabolism of other amino acids(P=0.002),endocrine system(P=0.001),transport and catabolism(P=0.016),excretory system(P=0.006),and a lower percentage of gene families associated with genetic information processing(P=0.046),transcription(P=0.021),metabolism(P=0.046),enzyme families(P=0.046),and environmental adaptation(P=0.003).In conclusion,these results demonstrated that the feed restriction altered the composition and function of epithelium-associated microbiota.3.Effect of severe feed restriction on rumen epithelium gene expressions of Hu sheep in the late pregnancy based on the RNA-SeqThe objective of this study was to evaluate the effect of severe feed restriction on rumen epithelium gene expressions of Hu sheep in the late pregnancy based on the RNASeq.At the end of the experimental period,all animals were slaughtered and ruminal epithelial tissue were collected for RNA extraction.Transcriptome analysis revealed that with the criterion of FDR<0.05,FC>1.5 or<0.67,106 genes were differed significantly between two groups,of which 46 significantly up-regulated genes,and 60 significantly down-regulated genes in SFR group.KEGG pathway analysis indicated that the differentially expressed genes(DEGs)were associated with ECM-receptor interaction,PPAR signaling pathway,steroid biosynthesis,focal adhesion,PI3K-Akt signaling pathway,protein digestion and absorption,DNA replication,cell cycle,fatty acid metabolism,biosynthesis of unsaturated fatty acids,AMPK signaling pathway,platelet activation,fatty acid elongation,fructose and mannose metabolism and fatty acid degradation.DEGs related to growth and development were screened out.Compared with the CON group,the DEGs related to cell cycle and DNA replication(MCM2,MCM4,MCM5,BUB1B,and ORC1)were down-regulated in the SFR group,the DEGs involved in PI3K-Akt signaling pathway(THBS4,CSF1,COL1A1,COL1A2,COL3A1,COL5A1,and JAK3)were down-regulated in the SFR group.These results indicated that severe feed restriction regulated the growth and development of rumen epithelium by PI3K-Akt signaling pathway,cell cycle,and DNA replication,and then the rumen epithelial structure and function had an adaptive response to the feed restriction.