In vitro evaluation of selected compounds affecting ruminal fermentation and methane production

Rumen fermentation yields VFA for host metabolism but also generates methane (CH₄), a gaseous byproduct associated with dietary energy losses. Feed additives such as nitro compounds and phytochemicals can reduce CH₄ emissions; however, they may also impair VFA synthesis, necessitating careful evaluation before in vivo application. To address this, we conducted 3 sequential in vitro assays to (1) screen 11 compounds for their methane-reducing potential, including nitro compounds, plant secondary compounds, and phytochemicals; (2) identify minimal effective doses (0, 1.25, 2.5, 5, and 10 mM) that promote CH₄ reductions without compromising VFA production; and (3) evaluate the combined effects of carvacrol (CAR) and 2-nitroethanol (2NEOH) at low (LC; 1.25 mM each) and high (HC; 2.5 mM each) doses on rumen fermentation parameters, including pH, in vitro dry matter digestibility, VFA profiles, gas production (CH₄, CO₂, H₂), and fermentation kinetics. Of the 11 compounds screened, 4 treatments, including CAR and 2NEOH, significantly reduced CH₄ production by at least 20% and were selected for assessment. Increasing dose levels, particularly for CAR, significantly reduced CH₄ but also negatively affected VFA production. In the combination phase, the HC dose combination nearly eliminated CH₄ production but also reduced total VFA by ~50% and increased H₂ accumulation, indicating an overall reduction in microbial fermentation rather than a targeted effect on methanogenesis. The LC dose combination decreased acetic acid concentrations, thus reducing the acetate-to-propionate ratio and H₂ accumulation. The decrease in CH₄ (18%) and modest increase in propionate did not differ from the control. Our findings indicate that CAR and 2NEOH exert distinct effects on rumen fermentation and methanogenesis. Notably, the high dose combination negatively affected fermentative parameters, whereas the low dose combination performed similarly to 2NEOH alone. Further investigation into how these compounds modulate the rumen microbiota will help to elucidate the mechanisms underlying their differential effects before they can be tested in vivo.