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

Authors: PERES ASSUMPÇÃO, ALICE - University Of Wisconsin; Young, Juliana; FERREIRA, IGOR - University Of Wisconsin; RODRIGUES, ARLAN - University Of Wisconsin; Anderson, Robin; SUEN, GARRET - University Of Wisconsin; MANTOVANI, HILARIO - University Of Wisconsin

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/8/2026
Publication Date: 6/1/2026
Citation: Peres Assumpção, A., Young, J.D., Ferreira, I., Rodrigues, A., Anderson, R.C., Suen, G., Mantovani, H.C. 2026. In vitro evaluation of selected compounds affecting ruminal fermentation and methane production. Journal of Dairy Science. https://doi.org/10.3168/jds.2026-28405.
DOI: https://doi.org/10.3168/jds.2026-28405

Interpretive Summary: When cows digest feed, microbes in their rumen (i.e., a specialized stomach chamber), break down carbohydrates and produce volatile fatty acids (VFAs), which are the main source of energy for the animal. However, this process also generates gases like carbon dioxide and hydrogen. These gases are converted into methane, a natural byproduct of digestion that unfortunately leads to a loss of usable dietary energy. Here, we tested 11 different compounds in the lab for reducing methane production, but only four of them [carvacrol (CAR), nitroethanol (NE), 2-nitroethanol (2NEOH), nitropropanol (NEPOH)] stood out by reducing methane production by at least 20%. These four were selected for further testing to determine the lowest effective doses that would reduce dietary energy losses from methane production without compromising fermentation/digestion (VFA production) in the cow’s rumen. CAR was particularly effective at reducing methane, but at higher doses, it also reduced the production of VFAs, which defeats the purpose of improving energy efficiency through suppression of methane production. In contrast, 2NEOH lowered methane losses while maintaining normal VFA levels, showing a better balance between methane reduction and energy preservation. Overall, the results suggest that nitro compounds like 2NEOH are promising tools for reducing dietary energy losses in cows. They offer the added benefit of being easy to incorporate into existing feeding routines without major changes to management practices.

Technical Abstract: Carbohydrate fermentation in the rumen produces volatile fatty acids (VFAs), which serve as a key energy source for ruminants. This process also generates carbon dioxide (CO2) and hydrogen (H2), substrates for methane (CH4) production—a biologically necessary but energetically inefficient pathway that results in dietary energy losses. Feed additives such as nitro compounds and phytochemicals can reduce CH4 emissions through distinct mechanisms; however, they may also impair VFA synthesis, necessitating careful evaluation prior to in vivo application. To address this, we conducted three sequential in vitro assays to: i) screen eleven compounds for their methane-reducing potential (e.g., carvacrol [CAR], nitroethanol [NE]); ii) identify minimal effective doses (0, 1.25, 2.5, 5, and 10 mM) that preserve fermentation; and iii) evaluate the combined effects of CAR and 2NEOH (2-nitroethanol) at LOW (1.25 mM each) and HIGH (2.5 mM each) doses on rumen fermentation parameters, including pH, dry matter digestibility (IVDMD), VFA profiles (acetic and propionic acids), gas production (CH4, CO2, H2), and fermentation kinetics. Of the eleven compounds screened, CAR, NE, 2NEOH, and NEPOH (nitropropanol) reduced CH4 production by at least 20% and were selected for dose-response assessment. Increasing dose levels, particularly of CAR, significantly reduced methane but also negatively affected VFA production. In the final phase, the HIGH-dose combination of CAR and 2NEOH nearly eliminated CH4 production but reduced total VFAs by ~50%. In contrast, the LOW-dose combination increased propionate, decreased the acetate-to-propionate ratio, and reduced CH4 by 18%. Altogether, these findings highlight the contrasting mechanisms of CAR and 2NEOH and suggest that low-dose combinations may offer a balanced strategy to reduce dietary energy losses via methanogenesis while maintaining rumen fermentation efficiency.