Untargeted Metabolomics of Dairy Cows as Influenced by the Combinations of Essential Oil Blends and Fumaric Acid as Natural Feed Additives Using RUSITEC

Background and Objectives: The potential of essential oils (EOs) and fumaric acid (FA) to modulate ruminal fermentation and mitigate greenhouse gas emissions in dairy cows has attracted significant attention. However, little is known about the specific metabolites produced as a result of their interaction. This study investigated the combined effects of essential oil blends (EOBs) and FA on rumen metabolites using a rumen simulation technique (RUSITEC) system. Materials and Methods: Three rumen-cannulated, non-lactating Holstein Friesian cows served as inoculum donors. The total mixed ration (TMR; CON) comprised corn silage (60%), alfalfa hay (20%), and concentrate (20%). Three distinct EOBs were formulated: EOB1 [Garlic, Lemongrass, Cumin, Lavender, and Nutmeg; at 4:2:2:1:1, respectively], EOB2 [Anise, Clove, Oregano, Cedarwood, and Ginger; at 4:2:2:1:1, respectively], and EOB3 [Clove, Anise, Peppermint, and Oregano; at 4:3:2:1, respectively]. Four treatments evaluated were control (CON), EFA1 (EOB1 + FA), EFA2 (EOB2 + FA), and EFA3 (EOB3 + FA). EOBs and FA were included at 10 µL/g feed and 3% of TMR, respectively. Rumen effluents were collected over 5 days for metabolome analysis using liquid chromatography-mass spectrometry (LC–MS). Results: A total of 661 metabolites were detected and identified. Volcano plot analysis revealed 13 differentially abundant metabolites for EFA1, 41 for EFA2, and 19 for EFA3 compared to CON group. PLS-DA analysis showed clear separation of treatments, indicating modifications in the rumen fluid metabolome. Conclusions: The treatments led to the enrichment of pathways involved in amino acid, nucleotide, cofactor, and energy metabolism. These additives have the potential to optimize nutrient utilization and overall animal health. Therefore, in vivo studies should be conducted to validate their efficacy.