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Simple Summary  The rumen is the main digestive and absorption organ of dairy cows. It contains abundant microorganisms and can effectively use human-indigestible plant mass. Therefore, we used metagenomics to explore the role of rumen microbes in the regulation of milk protein and fat in dairy cows. This study showed that  Prevotella  species and  Neocallimastix californiae  in the rumen of cows are related to the synthesis of milk components due to their important functions in carbohydrate, amino acid, pyruvate, insulin, and lipid metabolism and transportation metabolic pathways.    Abstract  The rumen contains abundant microorganisms that aid in the digestion of lignocellulosic feed and are associated with host phenotype traits. Cows with extremely high milk protein and fat percentages (HPF;  n  = 3) and low milk protein and fat percentages (LPF;  n  = 3) were selected from 4000 lactating Holstein cows under the same nutritional and management conditions. We found that the total concentration of volatile fatty acids, acetate, butyrate, and propionate in the rumen fluid was significantly higher in the HPF group than in the LPF group. Moreover, we identified 38 most abundant species displaying differential richness between the two groups, in which  Prevotella  accounted for 68.8% of the species, with the highest abundance in the HPF group. Functional annotation based on the Kyoto Encyclopedia of Gene and Genome (KEGG), evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), and Carbohydrate-Active enzymes (CAZy) databases showed that the significantly more abundant species in the HPF group are enriched in carbohydrate, amino acid, pyruvate, insulin, and lipid metabolism and transportation. Furthermore, Spearman’s rank correlation analysis revealed that specific microbial taxa (mainly the  Prevotella  species and  Neocallimastix californiae ) are positively correlated with total volatile fatty acids (VFA). Collectively, we found that the HPF group was enriched with several  Prevotella  species related to the total VFA, acetate, and amino acid synthesis. Thereby, these fulfilled the host’s needs for energy, fat, and rumen microbial protein, which can be used for increased biosynthesis of milk fat and milk protein. Our findings provide novel information for elucidation of the regulatory mechanism of the rumen in the formation of milk composition.

Identification of the Potential Role of the Rumen Microbiome in Milk Protein and Fat Synthesis in Dairy Cows Using Metagenomic Sequencing