The physiological function of the reticulorumen plays an essential role in ruminant nutrition, and detailed knowledge of rumen motility can further advance understanding of ruminant nutrition and physiology. Rumen motility was simulated by setting different stirrer rotation speeds in a rumen simulation technique (RUSITEC) system. The aim of this study was to investigate the effects of rotation speeds on rumen fermentation, saturation factor of dissolved gases, hydrogen (H₂) and methane (CH₄) emissions, microbial protein synthesis, and selected microbial population using RUSITEC. The experiment was performed according to a balanced 3 × 3 Latin square design, and each period included 7 d for adaptation and 3 d for sampling. Three motility treatments included 5, 15, and 25 rpm rotation speeds. Daily total gas and H₂ and CH₄ emissions had quadratic responses to the increasing rotation speed and were highest at 15 rpm. Quadratic and linear responses (highest at 5 rpm) to increasing rotation speed were observed for saturation factors of H₂ and CH₄, liquid-dissolved H₂ and CH₄ concentrations, and headspace concentration of H₂ in the gas phase, whereas increasing rotation speed linearly decreased saturation factors of CO₂ and liquid-dissolved CO₂ concentration. Quadratic and linear responses to increasing rotation speed were observed for molar percentages of acetate, ammonia, and microbial protein concentration, whereas increasing rotation speed quadratically increased pH and decreased total volatile fatty acid concentration and acetate-to-propionate ratio. The 15-rpm rotation speed had the highest values of total volatile fatty acids, acetate molar percentage, and microbial protein concentration. Quadratic and linear responses to increasing rotation speed were observed for copy numbers of solid-associated fungi and fluid-associated bacteria, fungi, and protozoa, while increasing rotation speed linearly increased copy numbers of solid-associated protozoa. Rotation at 15 rpm increased populations of fungi and protozoa in the solid rumen contents and the population of bacteria and fungi in the liquid rumen contents. In summary, this study provides insights on the biofunction of proper rumen motility (i.e., at a rotation speed of 15 rpm), such as improving feed fermentation, increasing gas emissions with decreased dissolved gas concentrations and saturation factors, and promoting microbial colonization and microbial protein synthesis, although further increase in rotation speed (i.e., to 25 rpm) decreases feed fermentation and microbial protein synthesis.

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