Mechanism Of Concentrate Starter/Alfalfa Hay Introduction Regulating Rumen Wall Developmental Plasticity In Suckling Lambs

The rumen is a unique organ of digestion,absorption and metabolism in ruminants,and it also plays an important role in barrier and immunity.Therefore,in young ruminants,it is of great significance to promote the establishment of rumen wall morphology and function through nutritional interventions.Various of previous studies showed that concentrate starter introduction tend to promote the development of ruminal epithelial papillae,while hay introduction tends to promote the development of rumen muscle layer.However,the mechanism of rumen developmental plasticity with different solid diet remains unclear.Therefore,based on the 16S r RNA,metagenomics,metabolites,transcriptome and in vitro primary cell culture techniques,this study explored the microbial-host interaction mechanism of rumen wall development with concentrate starter or hay feeding in sucking lambs.1.Effects of concentrate starter/alfalfa hay introduction on animal performance and rumen morphological development of suckling lambsTwenty-four 11-day-old Hu lambs were selected.At 14-day-old,the lambs were randomly assigned by sex to three groups:milk powder only（M,n=8）,milk powder plus concentrate starter（MC,n=8）,and milk powder plus alfalfa hay（MH,n=8）.The feed intake was recorded every day.The body weights were weighed weekly.At 42-day-old,lambs were slaughtered after jugular blood collection.Rumen contents and fluid were collected for analysis of VFA concentration.The sample of the rumen wall from the ventral sac was collected for analysis of rumen epithelium and muscle development.The results showed that,the average daily crude protein intake（P<0.001）,metabolic energy（P<0.001）and average daily body weight gain（P=0.002）were higher in MC and M group lambs than those in M group lambs.The average daily neutral detergent fiber intake（P<0.001）was highest in MH group lambs,followed by MC,and then M group lambs.The average daily starch intake was significantly（P<0.001）higher in MC group than that in MH and M group lambs.There was no significant difference in the body weight of lambs among the three groups during the experiment.The results of rumen fermentation parameters showed that the concentrations of total VFA,acetate,propionate,butyrate,valerate and microbial protein were highest in the rumen of MC group lambs,followed by MH,and then M group lambs（P<0.001）.The results of rumen morphological development of lambs showed that,compared with the M group,concentrate starter or alfalfa hay introduction significantly increased the empty rumen weight（P<0.001）and relative rumen weight（P<0.001）,alfalfa hay introduction increased the rumen volume of lambs（P=0.005）.The rumen epithelial absorption area,papillae length,papillae width,total epithelium thickness,stratum corneum,and stratum granulosum thickness were thickest in the MC group lambs,followed by MH,and then M group lambs（P<0.001）.The rumen muscle layer thickness was thickest in the MH group lambs,followed by MC,and then M group lambs（P<0.001）.The above results indicated that concentrate starter or alfalfa hay introduction both promoted the development of rumen morphology.Concentrate introduction especially promoted the development of rumen epithelium,while alfalfa hay introduction especially promoted the development of rumen muscle layer.2.Differentiated characteristics of gene expression profiles and functions of rumen wall after concentrate starter/alfalfa hay introduction in suckling lambsThe experimental design is the same as in Chapter 1.The lambs were slaughtered and sampled at 42 days of age,the sample of the rumen wall from the ventral sac was collected for analysis of transcriptomics.The principal components analysis of gene expression in rumen tissue samples showed the marked clustering among three groups.Compared with M group,1108 genes were significantly differentially expressed in the MC group（MC vs.M）,544 genes were significantly differentially expressed in the MH group（MH vs.M）.Moreover,there were 223 differentially expressed genes between MC and MH group（MC vs.MH）.Venn diagram showed there were 312 shared DEGs between the“MH vs M”and“MC vs M”,and 232 and 796 unique DEGs observed in the“MH vs M”and“MC vs M”,respectively.Functional enrichment of unique 796 DEGs observed in the“MC vs M”showed concentrate introduction mainly altered nutrient transport and metabolism,especially ion transport,amino acid,and fatty acid metabolism.The results of function analysis of 232 unique DEGs observed in the“MH vs M”showed alfalfa hay introduction specifically downregulated the expression of genes associated with cytokine production,immunocyte activation,and immune response,while upregulated the expression of genes related to epithelial resistance to bacterial invasion.The functional enrichment of 312shared DEGs observed in the“MH vs M”and“MC vs M”was mainly in VFA absorption and metabolism.In addition,the results of weighted gene co-expression network analysis identified that M16 and M18 modules were positively correlated with rumen epithelium morphology.The M16 module was mainly related to metabolism pathway,while the M18module was mainly associated with Wnt and cell cycle signaling pathways.The M10module was most correlated with the thickness of rumen muscle layer,and its genes were mainly enriched in smooth muscle contraction signaling pathways and calcium ion signaling pathways.The above results indicated that concentrate starter introduction enhanced nutrient transport and metabolism,especially amino acid and fatty acid metabolism;alfalfa hay introduction facilitated the ability of rumen epithelium to resist bacterial invasion,and reduced inflammatory response.3.Microbial mechanism of concentrate starter/alfalfa hay regulating rumen wall developmental plasticity in lambsThis study aims to reveal the rumen microbial mechanism of concentrate starter/alfalfa hay introduction regulating rumen wall developmental plasticity in lambs.The experimental design is the same as in Chapter 1.The rumen fluid of lambs in the milk powder plus concentrate starter group（MC,n=8）and milk powder plus alfalfa hay group（MH,n=8）were used for metabolomic analysis.The rumen content was used for16S r RNA and metagenomics analysis.PCA analysis of ruminal fluid metabolites showed that the composition of ruminal fluid metabolites was significantly different between the two groups of lambs（P=0.001）.Compared with MH group,the level of 65 metabolites were significantly higher in the MC group,while the level of 41 metabolites were significantly lower in the MC group.The result of KEGG enrichment showed that the different metabolites were mainly involved in butyrate metabolism,tryptophan metabolism and linoleic acid metabolism.The level of tryptophan metabolites（3-methyldioxindole,2-indolecarboxylic acid and IAld）in the rumen of MC group was significantly higher than those of MH group,and were positive correlation with rumen epithelial development indicators.The level of linoleic acid metabolite（PGD2）in the rumen of lambs in the MH group was significantly higher than that in the MC group,and was positively correlated with the thickness of the rumen myometrium.Based on the results of random forest analysis and correlation analysis,IAld and PGD2 were potential microbial characteristic metabolites in MC and MH group,respectively.Metagenomics KEGG-L2 pathway analysis showed that the relative abundance of metabolic pathways in the MC group was significantly higher than that in the MH group,including carbohydrate metabolism,amino acid metabolism,lipid metabolism,nucleic acid metabolism and energy metabolism.Based on metagenomics,the microbial producer and metabolic pathways of IAld and PGD2 were explored.The results showed that concentrate introduction increased the relative abundance of IAld producer（Bifidobacterium longum）and aromatic amino acid aminotransferase（Ar AT）genes in rumen microbiota,and enhanced the metabolism of tryptophan to indolepyruvic acid,and then increased the synthesis of IAld.Alfalfa hay introduction increased the relative abundance of PGD2 producucer（Candida albicans）,TGL4 and COX1 genes in rumen microbiota,and enhanced the biosynthesis of PGD2 in the microbial linoleic acid metabolic pathway.The above results show that concentrate starter and alfalfa hay introduction established different rumen microbial environments.Concentrate introduction elevated production of microbial IAld through enhancing rumen microbiota indole pyruvate pathway.Alfalfa hay introduction enhanced microbial biosynthesis of PGD2 via elevating linoleic acid metabolism of rumen microbiota.IAld and PGD2 may play an important role in promoting the development of the rumen epithelia and muscle layer,respectively.4.Molecular mechanism of key microbial metabolites IAld and PGD2 regulating development of rumen epithelium and muscle layer in vitroThe key microbial metabolites IAld and PGD2,screened in Chapter 3,were selected to further validate their roles in regulation of rumen epithelia and muscle development in vitro.Primary ruminal epithelia cells and smooth muscle cells were isolated from the rumen wall of Hu lambs（42 day of age）.Primary ruminal epithelia cells were divided into 4groups after adherence:control group（Con,n=5）,IAld group（IAld,n=5）,IAld+AHR inhibitor group（IAld+Ai,n=5）and IAld+β-catenin inhibitor group（IAld+βi,n=5）.Cell proliferation,cell cycle progression and relative expression of genes and proteins in AHR-Wnt/β-catenin signaling pathway were detected after 24 hours of cell treatment.The results showed that IAld significantly accelerated the cell cycle process and increased the relative m RNA and protein expressions of AHR,CYP1A1 and CTNNB1.Pretreatment with Ah R inhibitor orβ-catenin inhibitor remarkably attenuated the function of IAld on promoting the proliferation of primary rumen epithelial cells.Primary smooth muscle cells were divided into 3 groups after adherence:control group（Con,n=5）,PGD2 group（PGD2,n=5）,and PGD2+CAMK2 inhibitor group（PGD2+Ci,n=5）.Cell proliferation,cell cycle progression,intracellular Ca²⁺content and relative expression of genes and proteins in Ca²⁺signaling pathway were detected after 24h of cell treatment.The results showed that PGD2 promoted the proliferation of primary smooth muscle cells,increased the intracellular Ca²⁺level of primary smooth muscle cells,and upregulated the relative expression of CAMK2 protein.The effect of PGD2 on promoting the proliferation of primary rumen smooth muscle was remarkably attenuated by the addition of a CAMK2protein inhibitor.The above results show that IAld promoted the proliferation of rumen epithelial cells through AHR-WNT/β-catenin signaling pathway.PGD2 promoted the proliferation of primary smooth muscle cells via Ca²⁺signaling pathway.In conclusion,this study found that the differential development of the rumen wall morphology was accompanied by the differential development of the rumen wall function.Concentrate starter introduction was likely to promote the nutrient metabolism function in the rumen wall,while alfalfa hay introduction primarily enhanced the ability of rumen epithelium to resist bacterial invasion and reduced immune response in the rumen wall.This study revealed the microbial-host interaction of concentrate starter/alfalfa hay introduction in regulating differential development of rumen wall in preweand lambs.Concentrate starter introduction elevated production of microbial IAld through enhancing rumen microbiota tryptophan metabolism,and then IAld promoted ruminal epithelial development by Ah R-Wnt/β-catenin pathway.Alfalfa hay introduction enhanced rumen microbial linoleic acid metabolism and increased PGD2 level.PGD2 promoted the development of rumen muscle layer through the Ca²⁺signaling pathway.