The objective was to assess the supplementation with microencapsulation of hemp leaf extract (mHLE) utilized as a rumen enhancer on in vitro rumen fermentation and to enhance the bioavailability of active compounds for antimicrobial action, particularly in protozoa and methanogen populations. The feed treatments were totally randomized in the experimental design, with different levels of mHLE diet supplemented at 0, 4, 6 and 8% of total DM substrate and added to an R:C ratio of 60:40. During fermentation, gas kinetics production, nutrient degradability, ammonia nitrogen concentration, volatile fatty acid (VFA) profiles, methane production, and the microbial population were measured. The supplemented treatment at 6% of total DM substrate affected reductions in gas kinetics, cumulative gas production, and volatile fatty acid profiles, especially the acetate and acetate to propionate ratio. Whereas propionate proportion and total volatile fatty acid concentration were enhanced depending on the increase of nutrients in vitro dry matter degradability (IVDMD) after 12 h of post-fermentation at a R:C ratio of 60:40 (P < 0.05). Consequently, mHLE addition resulted in optimal ruminal pH and increased nutrient degradability, followed by ammonia nitrogen concentrations (P < 0.05), which were enhanced by dominant cellulolytic bacteria, particularly Ruminococcus albus and Ruminococcus flavefaciens, which showed the highest growth rates in the rumen ecology. Therefore, mHLE, a rich phytonutrient feed additive, affected the methanogen population, reduced the calculated methane production and can be a potential supplement in the ruminant diet.
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