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 identity and inclusion level. This study evaluated phenylpropanoid-derived phenolics identified in Brassica rapa subsp. chinensis and selected commercial feed additives for their effects on methane production and ruminal fermentation. Lucerne ( Medicago sativa ) pellets (0.5 g dry matter) were incubated for 24 h in three independent runs, each using rumen fluid collected from a pasture-fed, ruminally cannulated cow. Each treatment was represented by three technical bottles per run, run × treatment was the experimental unit, and variation among bottles was retained as residual error. Data were analysed separately by category using mixed models. Compound or product, active-treatment dose, and their interaction were fixed effects, whereas incubation run and run × treatment were random effects. Control-inclusive overall dose-response tests, compound- or product-specific orthogonal-polynomial contrasts, and Dunnett-adjusted comparisons with the control were conducted separately. Treatments included p-coumaric acid (5, 10, and 15 mM), trans-cinnamic acid (5, 10, and 15 mM), their 1:1 combination (2.5 + 2.5, 5 + 5, and 7.5 + 7.5 mM), gallic acid (5, 10, and 15 mM), tannic acid (actual concentrations of 0.41, 0.81, 1.22, and 4.05 mM after correction for 81% purity), N-carbamyl-L-glutamic acid (5, 10, and 15 mM), and two proprietary formulations (4.5% and 9% of substrate dry matter). The proprietary products comprised a medium-chain fatty-acid blend and an organic-acid blend with plant essential oils; individual ingredient concentrations were not disclosed by the manufacturer. 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 CH₄ concentration and yield in a dose-dependent manner, with CH₄ yield reduced by up to 43.3% at 15 mM; however, IVDMD was reduced at ≥ 10 mM. CH₄ yield decreased linearly with trans-cinnamic acid, although only the 27.4% reduction at 15 mM differed from the control. The combination reduced CH₄ yield by up to 41.4% and maintained IVDMD at the two lower inclusion levels. Gallic acid did not affect CH₄. Tannic acid reduced CH₄ yield by 19.4%–85.0%; the lowest dose caused no detectable reductions in gas production, IVDMD, or total VFA concentration, whereas higher doses increasingly inhibited fermentation. N-Carbamyl-L-glutamic acid and the two proprietary formulations did not reduce CH₄. The hypothesis was therefore partially supported: the lowest tested doses of p-coumaric acid and its combination with trans-cinnamic acid produced the clearest selective reductions in CH₄, while low-dose tannic acid was also effective but had a narrow margin before adverse fermentation effects emerged. These responses warrant in vivo validation and dose optimisation.