Rumen-Plasma-Milk Metabolomics Profiling Revealed Metabolic Alterations Associated with Milk Fat Synthesis in Chinese...

Milk fat synthesis in dairy cows is a complex process affected by ruminal fermentation, systemic metabolism, and mammary gland activity. To explore the metabolic interplay across these systems, a multi-tissue metabolomics approach (rumen fluid, plasma, and milk) using ultra-high-performance liquid chromatography-mass spectrometry was used to identify metabolic differences between Chinese Holstein cows with high (H-MF, 5.82 ± 0.41%) and low (L-MF, 3.60 ± 0.12%) milk fat content under the same diet. The bovine mammary epithelial cells (BMECs) were also cultured to evaluate the impact of a key metabolite, malic acid (MA), on lipid metabolism. Our findings reveal distinct metabolic profiles across rumen fluid, plasma, and milk, with 96, 109, and 79 differential metabolites, respectively, between the L-MF and H-MF groups. In rumen fluid, H-MF cows showed higher levels of lauric acid and succinic acid, linked to fatty acid biosynthesis, while the L-MF cows had elevated citraconic and orotic acids, associated with amino acid metabolism and liver stress. Plasma from the H-MF cows contained higher β-hydroxybutyric acid, methionine sulfoxide, and phosphatidylcholine, supporting lipogenesis, whereas L-MF plasma showed increased 3-hydroxy-L-proline, indicating tissue catabolism. In milk, the L-MF cows had higher MA, while the H-MF cows exhibited elevated L-carnitine, linked to fatty acid β-oxidation. Metabolite trend analysis during rumen fluid-plasma-milk showed that 211 metabolites were classified into 8 profiles. Profile 1 had the largest number of metabolites whose levels were down-regulated from rumen to plasma and enriched in lipid metabolism. Profile 3 (mainly related to amino acid metabolism) and profile 4 (mainly related to energy metabolism) exhibited opposite trends from plasma to milk. In vitro, 200 μM of MA reduced the triglyceride content in BMECs and down-regulated lipogenic genes and their protein expression levels (fatty acid synthase, stearoyl-CoA desaturase and sterol regulatory element binding protein 1). These results highlight how rumen fluid, plasma, and milk metabolites collectively influence milk fat synthesis, with MA acting as a key regulator of lipid metabolism in mammary epithelial cells.