Multiomics analysis revealed that the metabolite profile of raw milk is associated with the lactation stage of dairy cows and could be affected by variations in the ruminal microbiota.

The nutritional components and quality of milk are influenced by the rumen microbiota and its metabolites at different lactation stages. Hence, rumen fluid and milk samples from 6 dairy cows fed the same diet were collected during peak lactation, early mid-lactation, and later mid-lactation. Untargeted metabolomics and 16S rRNA sequencing were applied for analyzing milk and rumen metabolites, as well as rumen microbial composition, respectively. The levels of lipid-related metabolites, l-glutamate, glucose-1-phosphate, and acetylphosphate in milk exhibited lactation-dependent attenuation. Maltol, N-acetyl-d-glucosamine, and choline, which are associated with milk flavor or coagulation properties, as well as l-valine, lansioside A, clitocine, and ginsenoside La, increased significantly in early mid-lactation and later mid-lactation, especially in later mid-lactation. The obvious increase in rumen microbial diversities (ACE and Shannon indices) were observed in early mid-lactation compared with peak lactation. Twenty-one differential bacterial genera of the rumen were identified, with Succinivibrionaceae_UCG-001, Candidatus Saccharimonas, Fibrobacter, and SP3-e08 being significantly enriched in peak lactation. Rikenellaceae_RC9_gut_group, Eubacterium_ruminantium_group, Lachnospira, Butyrivibrio, Eubacterium_hallii_group, and Schwartzia were most significantly enriched in early mid-lactation. In comparison, only 2 bacteria (unclassified_f__Prevotellaceae and Prevotellaceae_UCG-001) were enriched in later mid-lactation. For rumen metabolites, LysoPE(16:0), l-glutamate, and l-tyrosine had higher levels in peak lactation, whereas PE(17:0/0:0), PE(16:0/0:0), PS(18:1(9Z)/0:0), l-phenylalanine, dulcitol, 2-(methoxymethyl)furan, and 3-phenylpropyl acetate showed higher levels in early mid-lactation and later mid-lactation. Multiomics-integrated analysis revealed that a greater abundance of Fibrobacter contributed to phospholipid content in milk by increasing ruminal acetate, l-glutamate, and LysoPE(16:0). Prevotellaceae_UCG-001 and unclassified_f_Prevotellaceae provide substrates for milk metabolites of the same category by increasing ruminal l-phenylalanine and dulcitol contents. These results demonstrated that milk metabolomic fingerprints and critical functional metabolites during lactation, and the key bacteria in rumen related to them. These findings provide new insights into the development of functional dairy products.