AimsTo investigate the effect of garlic oil (G), nitrate (N), saponin (S) and their combinations supplemented to different forage to concentrate substrates on methanogenesis, fermentation, diversity and abundances of bacteria and Archaea invitro. Methods and ResultsThe study was conducted in an 8x2 factorial design with eight treatments and two substrates using mixed ruminal batch cultures obtained. Quillaja S (06gl(-1)), N (5mmoll(-1)) and G (027gl(-1)) were used separately or in binary and tertiary combinations. The two substrates contained grass hay and a dairy concentrate mixture at a 70:30 (high-forage substrate) ratio or a 30:70 (high-concentrate substrate) ratio. Ruminal fermentation and cellulolytic bacterial populations were affected by interaction between substrate and anti-methanogenic compounds. The inhibitor combinations decreased the methane production additively regardless of substrate. For the high-concentrate substrate, S decreased methane production to a greater extent, so did G and N individually for the high-forage substrate. Feed degradability and total volatile fatty acid (VFA) concentrations were not decreased by any of the treatments. Fibre degradability was actually improved by N+S for the high-forage substrate. VFA concentrations and profiles were affected differently by different anti-methanogenic inhibitors and their combinations. All treatments inhibited the growth of Archaea, but the effect on Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens varied. ConclusionsThe results suggest that substrate influences the efficacy of these inhibitors when they are used separately, but in combinations, they can lower methanogenesis additively without much influence from the substrate. Significance and Impact of the StudyThe presented research provided evidence that binary and tertiary combination of garlic oil, nitrate and saponin can lower the methane production additively without adversely impacting rumen fermentation and degradability, and forage to concentrate ratio does not change the above effects. These anti-methanogenic inhibitors in combination may have practical application to mitigate methane emission from ruminants.