Thyme essential oil (Thymus vulgaris) modulates methane mitigation and rumen fermentation in vitro: a dose-response study.

This study evaluated the effects of thyme (Thymus vulgaris) essential oil (EO), applied as a conventional bulk extract or a nanosized nanoemulsion at inclusion levels of 0, 15, 30, and 45 µL/g dry matter (DM), on in vitro ruminal gas production kinetics, methane and carbon dioxide emissions, nutrient degradability, and fermentation end-products. Gas chromatography-mass spectrometry identified thymol (33.15%), 1,3,8-p-menthatriene (22.65%), and γ-terpinene (12.8%) as the dominant volatile constituents, classifying the extract within the thymol chemotype. The nanoemulsion showed a unimodal droplet size distribution peaking near 244.3 nm by dynamic light scattering. Inclusion level significantly affected all measured fermentation variables except pH (P < 0.05), and droplet size form significantly influenced several gas production and greenhouse gas parameters. Gas production and nutrient degradability were greatest at 15 µL/g DM for both forms. Asymptotic gas production (b) was maximized at 15 µL/g DM for both forms (+ 15.6 and + 24.1% over the control for the bulk and nanoemulsion forms, respectively) before declining at higher doses. Methane (CH4) as a percentage of total gas decreased linearly with increasing inclusion level (P < 0.001), reaching a 37.9% reduction at the highest nanoemulsion dose (N45). Dry matter degradability and total volatile fatty acid concentrations followed a similar pattern, peaking at 15 µL/g DM (55.9 and 57.1% for the bulk and nanoemulsion forms, respectively) before declining at higher doses, while ammonia nitrogen decreased linearly with EO inclusion for both forms (P < 0.001). These findings indicate that 15 µL/g DM was the best-performing inclusion level for balancing fermentation efficiency with CH4 mitigation, and that nanoformulation conferred modest rather than consistently superior benefits over the bulk form. Further in vivo validation is warranted before field application.