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

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.