Abstract
Environmentally sustainable feed additives that mitigate enteric methane (CH4) emissions are a priority for climate-smart livestock production. The present study investigated the effects of laurel (Laurus nobilis) ethanolic extract applied as a normal-sized bulk extract (B) or an oil-in-water nanoemulsion (N) at inclusion levels of 0, 15, 30, and 45 µL/g dry matter (DM) on in vitro gas production (GP) kinetics, CH4 and carbon dioxide (CO2) emissions, nutrient degradability, and rumen fermentation end-products. A 2 × 3 + 1 factorial arrangement within a completely randomized design yielded seven treatment groups: Control, B15, B30, B45, N15, N30, and N45, with three incubation runs serving as replicates. The asymptotic GP (b) and gas yield at 24 h (GY24) exhibited linear and quadratic responses within both laurel extract forms, peaking at 15 µL/g DM for bulk (b: 332.9 mL/g DM; GY24: 377.8 mL/g DM) and nano (b: 342.2 mL/g DM; GY24: 385.5 mL/g DM) treatments before declining at higher doses, with N15 producing the highest values overall (p < 0.05). Metabolizable energy followed the same linear and quadratic trends (p < 0.001), reaching 9.01 MJ/kg DM (B15) and 9.40 MJ/kg DM (N15) versus 8.01 MJ/kg DM in the control. Both laurel extract forms linearly (p < 0.001) reduced CH4 as a percentage of total gas; however, on an absolute mL/g DM basis, CH4 output declined only at 30 and 45 µL/g DM, whereas B15 and N15 showed a slightly higher absolute CH4 output than the control alongside greater total gas production; N45 achieved the greatest absolute reduction (45.9 mL/g DM versus 75.6 mL/g DM in the control, representing a 39.3% reduction). The nanoemulsion form was consistently superior to the bulk form in reducing CH4 concentration (p < 0.001). Dry matter degradability peaked at N15 (61.0% versus 57.5% in the control) and declined at higher nano doses to 49.2% at N45 (p < 0.001). Total volatile fatty acid concentrations and individual acetate, propionate, and butyrate values were highest at 15 µL/g DM for the nanoemulsion and declined with increasing dose (p < 0.01). Rumen pH increased linearly (p < 0.01), and ammonia-N decreased as dose escalated (p < 0.001). The superior fermentation response of the nanoemulsion at 15 µL/g DM, together with the greater and dose-dependent CH4 reduction achieved at 30–45 µL/g DM, underscores the capacity of nanoemulsification to enhance the bioavailability and dose-efficiency of laurel extract bioactives in the rumen, while indicating that no single dose tested here simultaneously maximized fermentation and CH4 mitigation. These in vitro findings support laurel extract nanoemulsion as a promising environmentally sustainable feed additive candidate for enteric CH4 mitigation, warranting an in vivo dose–response evaluation to identify a practical inclusion level that balances CH4 mitigation, fermentation efficiency, and animal acceptability.