Humic Substances Supplementation Reduces Ruminal Methane Production and Increases the Efficiency of Microbial Protein Synthesis in Vitro

The effects of humic substances (HS) on rumen fermentation and methane (CH₄) production were investigated using an in vitro rumen batch culture technique. HS was included in cultures with a 60% barley silage:40% concentrate diet (dry matter (DM) basis) at 0, 108, 216, 324 or 432 mg HS/g of substrate incubated. Rumen in vitro batch cultures were conducted as a completely randomized design with three runs. Gas (GP) and CH₄ production were measured after 3, 6, 9, 12, 24, and 48 h of incubation. Total volatile fatty acid (TVFA), microbial nitrogen (MN) and ammonia nitrogen (NH₃-N), as well as disappearences of true dry matter (TDMD), neutral detergent fiber (NDFD), starch (SD) and nitrogen (ND) were determined after 12 and 48 h. Total GP was linearly increased (P < 0.05) with increasing HS inclusion during the first 12 h of incubation, but it was not affected (P > 0.05) after 24 and 48 h. Increasing HS linearly decreased (P < 0.05) CH₄ production (expressed as ml/g DM) at all time points evaluated. After 48 h of incubation, CH₄ production was reduced by 12.83% at the highest HS dose compared to control. Increasing HS linearly decreased NH₃-N concentration, and the molar proportion of acetate (P < 0.05) at 12 h of incubation, while MN, TDMD and the molar proportion of butyrate linearly increased (P < 0.05), and SD responded quadratically (P < 0.05). After 12 h, ND, NDFD, TVFA, the molar proportion of branched-chain VFA and A:P ratio were not affected (P > 0.05) by increasing HS. After 48 h, HS linearly increased (P < 0.01) MN (35.15%), TDMD (7.31%) and the molar proportion of butyrate, but did not affect (P > 0.05) ND, SD, the molar proportion of acetate, propionate and branched-chain VFA, A:P ratio or NH₃-N concentration. Increasing HS resulted in quadratic response (P < 0.05) in NDFD after 48 h. Overall, inclusion of HS effectively reduced CH₄ production and increased in vitro substrate disappearance and the efficiency of microbial protein synthesis. Further studies are needed to evaluate the effects of HS on in vivo ruminal fermentation, CH₄ production, the microbiome, and its consequent effect on growth performance.