Abstract

This publication aims to provide guidelines of the knowledge required and the potential research to be conducted in order to understand the mode of action of antimethanogenic feed additives (AMFA). In the first part of the paper, we classify AMFA into 4 categories according to their mode of action: (1) lowering dihydrogen (H2) production; (2) inhibiting methanogens; (3) promoting alternative H2-incorporating pathways; and (4) oxidizing methane (CH4). The second part of the paper presents questions that guide the research to identify the mode of action of an AMFA on the rumen CH4 production from 5 different perspectives: (1) microbiology; (2) cell and molecular biochemistry; (3) microbial ecology; (4) animal metabolism; and (5) cross-cutting aspects. Recommendations are provided to address various research questions within each perspective, along with examples of how aspects of the mode of action of AMFA have been elucidated before. In summary, this paper offers timely and comprehensive guidelines to better understand and reveal the mode of action of current and emerging AMFA. © 2025 American Dairy Science Association