Multi-omics analysis reveals the mechanism of rosemary extract supplementation in increasing milk production in Sanhe dairy cows via the "rumen-serum-milk" metabolic pathway.

Rosemary extract (RE) has shown potential as a plant-derived feed additive, but its effects on Sanhe dairy cows are still unknown. In this study, 30 multiparous Sanhe dairy cows (days in milk 171 ± 17 days) with similar body condition were randomly divided into two groups: the RE group (n = 15) was fed the basal diet plus 20 g RE/d, and the CON group (n = 15) was fed only the basal diet. The experiment lasted for 57 days, including a one-week adaptation period. Compared with the CON group, milk yield (P = 0.022) increased significantly with RE supplementation, while milk fat (P = 0.071) also tended to increase. Milk urea nitrogen (P = 0.003) and serum urea nitrogen (P = 0.013) contents were significantly reduced in the RE group compared with the CON group. In rumen fermentation, the content of butyric acid (P = 0.035) in RE group was significantly increased, while valeric acid (P = 0.080) content had an increasing trend. In addition, RE supplementation improved the antioxidant capacity of Sanhe dairy cows by significantly increasing the serum total antioxidant capacity (P < 0.001), superoxide dismutase activity (P = 0.001), immunoglobulin A content (P < 0.001), and immunoglobulin G content (P = 0.005), while decreasing serum malondialdehyde content (P < 0.001), to improve immunity and also affect the composition of serum free amino acids. Metabolomic results showed that a total of 13 co-differential metabolites were identified in rumen and serum, including ursolic acid, a major component of RE, which was higher in both rumen and serum. The milk metabolome analysis identified glycerides, glycerophospholipids, and sphingolipids as the three lipid types that exhibited higher identification intensity in RE. Rumen metagenomic results showed that RE supplementation affected the composition of rumen microorganisms, and differential microbial Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses revealed that the RE group was significantly enriched in the fatty acid biosynthesis pathway and the glycerophospholipid metabolism pathway; two pathways related to lipid synthesis. By associating the genus-level differential microorganisms in the rumen with the "rumen-serum-milk" metabolome and mapping the correlation network, it was found that g_Sharpea, g_Tistlia, and g_Acetobacter, which were more abundant in RE, correlated with more differential metabolites and clustered in the same module. Among the 10 microbial biomarkers screened in the rumen, g_Acetobacter and g_Prevotella were more abundant in the RE, and Mantel's analysis showed that they correlated with rumen fermentation parameters and oxidative and immunological indicators in serum. These results reveal the regulatory mechanism of RE supplementation feeding to enhance milk production and improve milk quality by improving oxidative stress capacity and immunity and reducing nitrogen loss in Sanhe dairy cows, suggesting that RE has the potential as a feed additive for dairy cows.