| Summary: | The objective of the research was to evaluate the different levels of <i>Mitragyna speciosa</i> Korth leaves powder (MSLP) added to rations with 60:40 or 40:60 roughage to a concentrate (R:C ratio) on in vitro nutrient digestibility, rumen fermentation characteristics, microbial population, and methane (CH<sub>4</sub>) production. The treatments were arranged according to a 2 × 8 factorial arrangement in a completely randomized design. The two factors contain the R:C ratio (60:40 and 40:60) and the levels of MSLP addition (0, 1, 2, 3, 4, 5, 6, and 7% of the total substrate). There was no interaction between the R:C ratio and MSLP supplementation on gas production kinetics, ammonia nitrogen (NH<sub>3</sub>-N), and microbial populations. The gas production rate constant for the insoluble fraction (c) was increased by the R:C ratio at (40:60), whilst there was no difference obtained among treatments for cumulative gas production, whilst the gas production rate constant for the insoluble fraction (c) was increased by the R:C ratio at 40:60. The concentration of NH<sub>3</sub>-N was influenced by the R:C ratio and MSLP addition both at 4 and 8 h after incubation. In vitro dry matter degradability (IVDMD) and organic matter degradability (IVOMD) were significantly improved by the R:C ratio and supplementation of MSLP at 12 h. Increasing the R:C ratio and MSLP concentrations increased total volatile fatty acid (VFA) and propionic acid (C<sub>3</sub>) concentrations while decreasing acetic acid (C<sub>2</sub>) and butyric acid (C<sub>4</sub>) concentrations; thus, the C<sub>2</sub>:C<sub>3</sub> ratio was reduced. MSLP addition reduced protozoa and methanogen populations (<i>p</i> < 0.05). The calculated CH<sub>4</sub> production was decreased (<i>p</i> < 0.05) by the R:C ratios at 40:60 and supplementation of MSLP. Finally, the addition of MSLP as a phytonutrient may improve nutrient degradability and rumen fermentation properties while decreasing protozoa, methanogen population, and CH<sub>4</sub> production.
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