From Composition to Function: Lime Essential Oil (Citrus aurantifolia) and (R)-(+)-Limonene and Their Impact on Rumen Microbiota, Fermentation, Methane Emission and Blood Metabolic Parameters in Dairy Cows.

In this study, we characterized the chemical composition of lime essential oil (Citrus aurantifolia) by GC-MS and evaluated the effects of lime essential oil and its major constituent, (R)-(+)-limonene, on rumen fermentation, methane production, microbial community structure and metabolic responses in dairy cows. In vitro assays were used to establish effective doses for in vivo application. In vivo supplementation with lime essential oil on day 14 significantly increased ruminal pH (6.96 vs. 6.66; p < 0.05), total VFA concentration (4.84 vs. 4.11 mg mL-1; p < 0.05), serum glucose (3.92 vs. 3.63 mmol L-1; p < 0.05) and bicarbonate concentration (26.75 vs. 22.35 mmol L-1; p < 0.05), indicating improved fermentation efficiency and buffering capacity. Metabolic profiling revealed elevated glucose and reduced non-esterified fatty acids, suggesting enhanced energy utilization and decreased lipid mobilization, without adverse effects on liver enzymes (AST, ALT, GGT), lipid profile or acid-base balance. Rumen microbiota remained stable and was dominated by Bacteroidota and Bacillota; no significant changes occurred in alpha or beta diversity. (R)-(+)-limonene selectively reduced the abundance of Methanobacteriota (p = 0.04). Methane concentrations were not significantly affected. Both additives exerted beneficial, dose-dependent effects on rumen function and host metabolism and show promise as natural feed additives in dairy cows.