Screening compounds of the phenylpropanoid pathway and commercial products to mitigate methane emissions using batch culture in vitro incubations

Enteric methane (CH₄) produced during ruminal fermentation is a major contributor to agricultural greenhouse gas emissions and represents an energy loss to the animal. Plant-derived phenolic compounds have been proposed as dietary strategies to mitigate methane formation; however, their efficacy depends on compound structure and inclusion level. This study evaluated phenylpropanoid-derived phenolics identified in Brassica rapa subsp. chinensis and selected commercial additives for their ability to reduce methane without impairing ruminal fermentation. Lucerne pellets (500 mg dry matter) were incubated for 24h in an anaerobic in vitro batch culture system using rumen fluid from three pasture-fed fistulated cows (n = 3). Treatments included p-coumaric acid (5, 10, 15 mM), trans-cinnamic acid (5, 10, 15 mM), their combination (2.5, 5, 7.5 mM each), gallic acid (5, 10, 15 mM), tannic acid (0.41, 0.81, 1.22, 4.05 mM), N-carbamyl-L-glutamic acid (5, 10, 15 mM), and two commercial formulations (4.5 and 9% of substrate dry matter). Total gas production, CH₄ concentration and yield, in vitro dry matter disappearance (IVDMD), fermentation pH, and volatile fatty acid (VFA) profiles were measured. p-Coumaric acid reduced (P < 0.01) CH₄ concentration and yield in a dose-dependent manner, with reductions up to 43% at 15 mM; however, IVDMD was reduced (P < 0.01) at ≥ 10 mM. Trans-Cinnamic acid reduced (P < 0.01) CH₄ yield only at 15 mM. The combination of p-coumaric acid and trans-cinnamic acid reduced (P < 0.01) CH₄ production by up to 41% while maintaining IVDMD at 2.5–5 mM. Gallic acid did not affect CH₄ (P ≥ 0.16). Tannic acid produced the greatest CH₄ suppression (up to 85%; P < 0.01) but reduced gas production, IVDMD, and total VFA concentration (P < 0.01) at higher doses. N-carbamyl-L-glutamic acid and the two commercial formulations did not reduce CH₄ under these conditions (P ≥ 0.05). Moderate inclusion of p-coumaric acid (5 mM) alone or combined with trans-cinnamic acid (2.5 mM each) reduced methane without measurable adverse effects on fermentation, supporting further in vivo validation and dose optimisation for practical methane mitigation strategies.