Variation Of Rumen Microbiome,Rumen Epithelial Morphology Function,and Metabolome Of Rumen,Plasma,and Milk In Dairy Cows With Different Levels Of Subacute Rumen Acidosis Tolerance

Subacute ruminal acidosis（SARA）is a common metabolic disease in ruminant.Previous studies have shown that a wide variation in tolerance to SARA exists among ruminant animals.However,most of the research conducted on the variation in SARA tolerance focused on the factors related to diet and feeding behavior,and the physiological factors which remain unclear.In this study,we performed an integrated analysis of rumen microbiota,ruminal epithelium in lactation cow with variation in SARA tolerance.Besides,we also compared the difference in plasma and milk metabolome between the two groups of cows.1.Comparision of the rumen fermentation parameters and ruminal epithelial morphology and function in dairy cows with variation in SARA toleranceThe objective of this study is to investigate the rumen fermentation parameters,rumen epithelial morphology,and function in dairy cows with variation in SARA tolerance.Twelve multiparous ruminally cannulated mid-lactating Holstein cows were housed in individual tie-stalls and fed a diet with a concentrate to forage ratio of 4:6throughout the experimental period.The experimental period was 35 days,and samples were collected on the 34^th and 35^th days.Based on the ruminal p H,cows with relatively lower and higher mean ruminal p H was assigned to the susceptible（SUS,n=4）and tolerant（TOL,n=4）groups.The ruminal p H and VFA concentrations of rumen fluid were measured,while the rumen papilla was collected through the rumen fistula and analyzed for morphology and function.Then,50 m L milk was collected for composition analysis.The results showed that the ruminal p H was lower in the SUS group（P<0.05）compared with the TOL group,while no significance difference in the DMI,milk yield and component were observed between two groups（P>0.05）.Rumen fermentation parameters analysis showed that the concentrations of TVFA,propionate,butyrate,and valerate were higher in the SUS group（P<0.05）than those in TOL group.The examination of ruminal epithelial morphology and function revealed that the thickness in the spinous process and basal layer of ruminal epithelium were higher in the SUS group.In the SUS group,the expression levels of PAT1,MCT4,and DRA genes involved in VFA absorption were significantly lower（P<0.05）compared with the TOL group,while the NHE1,NHE2,NHE3,v H⁺ATPase,and Na⁺/K⁺ATPase expression were higher in the SUS group than those in TOL group（P<0.05）.However,the SUS group had a significantly lower expression of HMGCL-2 than that in the TOL group.Compared with the TOL group,the expression levels of cell involved in cell proliferation and apoptosis such as the CDK2,CDK6,Cyclin D1,Bad and Caspase-9were significantly higher in the SUS group（P<0.05）.In conclusion,the dairy cow has a wide variation in SARA tolerance which is independent of dry matter intake and milk performance.In SUS cows,the ability to absorb free fatty acid by ruminal epithelial cell was relatively weak,while the ability of undisintegrated fatty acid absorption was higher.These results lead to accumulation of VFA in the rumen,thereby decreasing the ruminal p H and increasing the risk of SARA in the dairy cows.2.Study on the difference in rumen microbiota of dairy cows with variation in SARA toleranceThe purpose of this chapter was to explore the difference in rumen microbiota of dairy cows with variation in SARA tolerance.Rumen contents were collected through rumen fistulas before morning feeding at 35 d,and microbial DNA was extracted and used for 16S r RNA and 18S r RNA sequencing.The results of 16S r RNA gene sequencing showed that the bacterial communities were clearly separated in the two groups（PCo A,P<0.05）,while the alpha diversity indexes showed no difference（P>0.05）.Compared with the TOL group,at the genus level,the relative abundance of Papillibacter and unclassified Family＿XIII were significantly higher in the SUS group（P<0.05）.At the OTUs level,abundance of 15 OTUs were significantly higher in the SUS group（P<0.05）,and 9 OTUs among them belonged to Prevotella.Moreover,the abundance of OTU239,OTU156,and OTU146 were negatively correlated with ruminal p H（r<-0.8,P<0.05）,while OTU239 and OTU146 were positively correlated with propionate concentration（r>0.8,P<0.05）.In addition,compared with the TOL group,the relative abundance of 20 OTUs were significantly lower in the SUS group（P<0.05）.The result of 18S r RNA genus sequencing revealed that protozoal communities were not distinct separation between the two groups（P>0.05）,and there was also no difference in Alpha diversity indexes（P>0.05）.The results of statistical analysis showed that no significant difference was observed in the composition between the two group（P>0.05）.In summary,the increased abundance of OTUs belong to the genus Prevotella,the dominant starch-degrading bacteria in the rumen of SUS dairy cows,promotes the degradation of starch polysaccharides in the diet,which may be an important factor leading to the increase of rumen VFA concentration and the decrease of ruminal p H.3.Study on the difference in rumen,plasma,and milk metabolome of dairy cows with variation in SARA toleranceThis chapter aims to explore the differences in the rumen,plasma,and milk metabolites in dairy cows with different levels of SARA tolerance,and to explain the potential association among them.Ruminal fluid was collected before morning feeding.Milk samples were collected in the morning and evening,while the blood samples were collected at 6 h after morning feeding.The rumen fluid,milk,and blood samples were further used for metabolomics analysis.Based on the results of ruminal LC-MC metabolomics,a total of 195 metabolites were identified in the two groups,and 135metabolites were significantly different among of them（P<0.05）.Compared with the TOL group,126 differential compounds were high in the SUS group（P<0.05）,which mainly including amino acids,lipids,organic acids,sugars,biogenic amines and other substances.Meanwhile,9 metabolites were low in the SUS group,such as stearic acid,inosine,and 9-HETE.According to the pathway topology analysis,10 metabolic pathways were significantly enriched by differential metabolites（Impact>0.1,P<0.05）,including amino acid metabolism and biosynthesis,purine metabolism,tricarboxylic acid cycle and butyrate metabolism,etc.Analysis of plasma metabolome showed that 208 metabolites were identified in two groups.In SUS group,the levels of MG（18:0/0:0/0:0）,9-HODE,12（13）Ep-9-KODE,niacinamide,Isovalerylcarnitine,phosphocreatine,and L-glutamic acid were increased（P<0.05）,while the L-Phenylalanine level was lower（P<0.05）.The result of pathway topology analysis indicated that 6 metabolic pathways related to phenylalanine,tyrosine and tryptophan biosynthesis,D-glutamine and D-glutamate metabolism,phenylalanine metabolism,nicotinic and nicotinamide metabolism,and arginine biosynthesis were significantly enriched（Impact>0.1,P<0.05）.Milk metabolome analysis revealed that 188metabolites were identified,and 16 metabolites were significantly different among them（P<0.05）.Compared with the TOL group,excepted the levels of 1-Stearoylglycerophosphoserine and Inosine 2’,3’-cyclic phosphate were higher,14metabolites were significantly lower in the SUS group.The pathway topology analysis showed that glycerolipid metabolism was enriched（P<0.05）.In conclusion,the rumen physiological metabolism was significantly stronger in the SUS group,such as propionate metabolism and amino acid metabolism.This will promote the accumulation of VFA and increase the pro-inflammatory factors released in the rumen,such as biogenic amines.Consequently,it could lead to a higher risk of SARA.In addition,the plasma and milk metabolome of cows had a less affected by ruminal metabolites changes.In summary,we found that there was widely individual difference in the SARA tolerance of lacating cows,and it was independent of dry matter intake.In the present study,the main reasons for the variation in tolerance of SARA was that:The increased abundance of some OTUs in the SUS group which belong to Prevotella dpromote the degradation of starch polysaccharides in the rumen,which may accelerate the accumulation of organic acids in the rumen,such as propionate,butyrate,and lactate,and lead to a low ruminal p H.Moreover,the expression levels of PAT1,MCT4,and DRA genes involved in non-free VFA absorption were significantly lower in the SUS group,while the expression of H⁺transporters NHE1,NHE2,NHE3,v H⁺ATPase,and Na⁺/K⁺ATPase were higher.These results indicate tha the absorption of free VFA by SUS cows is relatively lower,and the absorption of non-free fatty acids was higher,which indirectly leads to the accumulation of VFA in the rumen.In addition,the amino acids synthesis and metabolism of rumen microbes were stronger in the SUS group,and these resulted in a higher level of biogenic anmines.A high concentration of biogenic amines may lead to the injury of the barrier function of rumen epithelium and potentially increase the risk of SARA in lactation cows.