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Although garlic oil and nitrate can effectively suppress ruminal methane (CH 4 ) production in vitro, the application of these compounds is associated with suppressed total volatile fatty acid (VFA) concentration. On the other hand, the effectiveness of fumarate as a ruminal CH 4 mitigating agent is variable but its application increases total VFA concentration. We therefore hypothesized that the different characteristics of the compounds can compensate for the shortcomings of the other. The objective of this study was to develop an optimal blend of garlic oil, nitrate and fumarate that can suppress in vitro ruminal CH 4 without affecting total VFA concentration. Three ruminal in vitro fermentation experiments were carried out. The first one, a one factor at a time experiment was employed to investigate the effective concentration of each of the compounds on CH 4 and VFA production by ruminal bacteria. We then applied the fractional factorial design and response surface methodology in the second experiment to determine optimal concentrations of the compounds in the blend. The optimal blending of garlic oil, fumarate and nitrate was determined to be 50 mg/l, 15 mm and 20 mm, respectively. This simulated optimal blend was verified in a 48 h in vitro batch fermentation experiment. The blend achieved the intended goal of suppressing CH 4 whilst maintaining total VFA concentration. The blend and nitrate suppressed archaea populations (p < 0.001) but did not affect the total microbial population (p = 0.945). The observed results could be explained by additive effects of the agents making up the blend. Supplementing a high concentrate diet with the blend can significantly decrease ruminal CH 4 and maintain total VFAin vitro. These findings however, need to be verified in vivo using the optimized ratio of combining the three methane inhibitors as a guide., (Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.)