Mechanism Of Rumen Microbiome Involved In Milk Protein Vield In Lactating Dairy Cows

Milk protein content is the important indicator to evaluate milk quality,and the key component to determine the price of raw milk in China.Milk protein synthesis is affected by various factors,including biological and environmental factors.The biological factors include animal genetics,digestion,metabolism and nutrients partitioning,and the environmental factors include diets,management practices,etc.Synthesis of milk protein is a complex biological process that can be regulated by various molecular mechanisms.As the rumen serves as a large bioreactor that enables dairy cows to obtain nutrients from the fermentation of plant mass that is undigestible by humans,we speculated that the rumen microbiome and its function may directly and indirectly affect host milk protein yield.In the current study,we applied system biology-based approaches,including 16 S rRNA sequencing,metagenomics sequencing,metatranscriptomics sequencing and metabolomics,to characterize the biological systematic mechanism for regulation of the milk protein production in the dairy cows that selected from a large commercial dairy herd with same feeding and management regime.The outcomes are expected to provide implication to improve feed efficiency,increase milk protein yield,and contribute to scientific knowledge for China dairy industry.1.Assessment of rumen microbiota from a large cattle cohort reveals the pan and core bacteriome contributing to varied phenotypes(Expt.1)The present study was conducted to characterize the rumen microbiota of a large cohort of mid-lactating Holstein dairy cows(n=334)that were fed the same diet and raised under the same environment.Milk yield was recorded and samples of milk and rumen fluid were collected for determination of milk composition and rumen fermentation characteristics.The amplicon sequencing of 16 S rRNA sequencing was used to determine the rumen microbial composition.The aim of this study was to uncover the inter-animal variations in rumen microbial composition and the linkages between rumen microbiomes and host phenotypes.Amplicon sequencing of the partial 16 S rRNA gene identified 391 bacterial genera as the pan bacteriome and 33 genera as the core bacteriome.The core genera belonged to six phyla,icluding Firmicutes(21.67 ± 0.18%,23 genera),Bacteroidetes(20.68 ±0.49%,four genera),Proteobacteria(0.52±0.01%,three genera),Spirochaetes(1.35± 0.04%,one genus),Fibrobacteres(0.86±0.02%,one genus),and Tenericutes(0.44±0.01%,one genus).Large inter-animal variations in the abundances of these core and pan taxa were observed,with the CVs ranged from 14.1 to 64.8%.Spearman correlation network analysis revealed significant correlations among rumen bacteria,volatile fatty acids(VFA),and lactation performance,with the core and non-core genera accounting for 53.9 and 46.2% of the network,respectively.These results suggest that even when cows were under the same management and were the same species,the rumen microbiome could be largely inter-animal varied.The pan rumen bacteriome together with the core bacteriome potentially contributes to variations of milk production traits.2 Assessment of rumen bacteria in dairy cows with varied milk protein yield(Expt.2)The present study was conducted to assess rumen bacteria in lactating cows with different milk protein yield(MPY),aiming to understand the role of rumen bacteria in this trait and the relationship between rumen bacteria and fermentation characteristics variables.Cows with high milk protein yield(high milk yield × high milk protein content [HH],n=20)and low milk protein yield(low milk yield × low milk protein content [LL],n=20)were selected from 334 mid-lactation Holstein dairy cows fed thesame high grain diet.The concentrations of ruminal total VFA,propionate,butyrate,valerate,and the proportion of isobutyrate were higher in HH cows than in LL cows(P <0.05).The rumen bacterial community identified by amplicon sequencing revealed that the richness(Chao 1 index)of rumen microbiota was higher in LL cows(P <0.05).The relative abundance of Succinivibrio(P <0.05)was significantly higher and that of Clostridium tended to be higher in the rumen of LL cows(P <0.10).Sharpea was 2.28-fold enriched in HH cows compared with LL cows(P <0.05).Different relationships between the relative abundances of rumen microbial taxa and VFA concentrations were observed in HH and LL animals,respectively,revealing that Succinivibrio and Prevotella were positively correlated with acetate,propionate and valerate in LL cows;and Sharpea was positively correlated with propionate and valerate concentrations in HH cows.Collectively,our results reveal that rumen bacterial richness and the relative abundances of several bacterial taxa significantly differed between dairy cows with high and low milk protein yield,suggesting the potential roles of rumen microbiota contributing to milk protein yield in dairy cows.Moreover,our results reveal the relationships between the bacterial taxa and VFA as well as milk protein yield,providing an evidence that the rumen microbiota could be one of factors that contribute to variations in milking performance of dairy cows.3 The rumen microbiome and its metabolome together with the host metabolome contribute to individualized dairy cow performance(Expt.3)In this part,some of HH and LL cows were used to analysze rumen metagenomics and metabolomics,together with serum metabolomics to identify potential mechanisms for regulation of MPY at both the rumen microbiome and host levels.Metage nomics analysis revealed that several Prevotella species were significantly abundant in the rumens of high-MPY cows(P <0.01),contributing to improved functions related to branched-chain amino acid biosynthesis.In addition,the rumen microbiome of high-MPY cows had lower relative abundances of organisms with methanogen and methanogenesis functions(P <0.01),suggesting that these cows may produce less methane.Metabolomics analysis revealed that the relative concentrations of rumen microbial metabolites(mainly amino acids,carboxylic acids,and fatty acids)and the absolute concentrations of VFAs were higher in the high-MPY cows(P <0.05).By associating the rumen microbiome with the rumen metabolome,we found that specific microbial taxa(mainly Prevotella species)were positively correlated with ruminal microbial metabolites,including the amino acids and carbohydrates involved in glutathione,phenylalanine,starch,sucrose,and galactose metabolism.To detect the interactions between the rumen microbiome and host metabolism,we associated the rumen microbiome with the host serum metabolome and found that Prevotella species may affect the host's metabolism of amino acids(including glycine,serine,threonine,alanine,aspartate,glutamate,cysteine,and methionine)(R> 0.5,P <0.05).Further analysis using the linear mixed effect model estimated contributions to the variation in MPY based on different omics and revealed that the rumen microbial composition,functions,and metabolites,and the serum metabolites contributed 17.81,21.56,29.76,and 26.78% to the host MPY,respectively.These findings provide a fundamental understanding of how the microbiome-dependent and host-dependent mechanisms contribute to varied individual performance in the milk production quality of dairy cows under the same management conditions.In summary,the current study investigated the rumen microecology of dairy cows by using system biology tools including microbial 16 S rRNA sequencing,metagenomics,metatranscriptomics,and metabolomics,aiming to systematically analyze mechanisms with which the microbial species,functions,metabolites and host metabolism exert their effects on host mik protein yield.The results from the current study will provide implication to improve host metabolism,increase milk protein yield,contributing scientific knowledge to the cow's feeding and breeding in China dairy industry.