After carbon dioxide, methane is a greenhouse gas that has the greatest impact on global warming. Methane produced through ruminal fermentation in ruminants not only contributes substantially to greenhouse gas emissions from the livestock sector, but also represents a loss of dietary energy, thereby reducing feed energy utilization efficiency and animal productivity. Therefore, elucidating the regulatory mechanisms underlying ruminal methane production and emission and establishing scientific, standardized mitigation assessment methods applicable to different production systems are of considerable importance for promoting the green and low-carbon development of the livestock industry and achieving the “Dual Carbon” goals. This paper systematically reviewed recent advances in the mechanisms and detection methods associated with methane mitigation in ruminants. First, according to the processes involved in ruminal methanogenesis and their regulatory points, methane inhibitors were classified into feed additives that regulated volatile fatty acid production and reduced hydrogen accumulation, inhibitors targeting methanogens and their key enzymes, and substances that promoted methane oxidation. The methane mitigation effects of 3-nitrooxypropanol (3-NOP), seaweed, nitrate, essential oils, tannins, lipids and combined additives were summarized, with particular emphasis on their effects on feed intake, average daily gain, milk yield and methane emission intensity in ruminants. Secondly, the effects of different physiological stages, dietary compositions, production systems and experimental durations on the assessment of mitigation efficacy were discussed in relation to Latin-square, crossover and continuous experimental designs. The importance of combining short-term trials with long-term monitoring was also emphasized. Thirdly, research approaches for elucidating the mechanisms of action of methane inhibitors were systematically reviewed from the perspectives of microbiology, cellular and molecular biology, ruminal fermentation and ecosystem functioning, animal metabolism, and integrated multidisciplinary research. These mechanisms included the regulation of methanogen communities, inhibition of methyl-coenzyme M reductase and coenzyme M methyltransferase activities, modification of hydrogen metabolism and volatile fatty acid production, alteration of electron acceptor utilization, and promotion of methane oxidation. In addition, this paper compared empirical screening with target-based design strategies for discovering bioactive substances, and introduced technologies including high-throughput screening, isolation and purification, structural identification using mass spectrometry and nuclear magnetic resonance spectroscopy, molecular docking and computational modelling. With regard to methane emission measurements, the principles, application conditions, advantages and limitations of respiration chambers, the sulphur hexafluoride tracer technique, the GreenFeed system, face masks, portable accumulation chambers, house-flux methods, open-path laser detection, unmanned aerial vehicles and satellite remote sensing were reviewed. The selection of an appropriate detection method should be based on the research objectives, animal production system, available equipment and the required measurement accuracy. Finally, the applications of empirical, mechanistic and machine-learning models in predicting methane emissions and mitigation potential were summarized. The need to strengthen the integration of high-quality data, standardize additive dosage and delivery methods, assess both short-term effects and long-term adaptation, and conduct integrated evaluations of mitigation efficacy, animal performance, product safety and life-cycle environmental impacts was highlighted. This review provided a theoretical basis and technical reference for the screening of methane inhibitors in ruminants, elucidation of their mechanisms of action, experimental design, methane emission measurement and prediction of mitigation potential.
