Harnessing bioactive compounds from Cannabis sativa residue to improve rumen fermentation and reduce methane production: in silico, in vitro, and in situ nylon bag studies

The inhibition of methyl-coenzyme M reductase (MCR) effectively suppresses ruminal methanogen activity, thereby mitigating enteric methane emissions in ruminants. However, the development of highly specific and environmentally sustainable inhibitors remains a significant challenge. This study aimed to identify key bioactive compounds from Cannabis sativa L. residue (CSR) that function as novel methane inhibitors that specifically target MCR by computational molecular screening. Cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) were identified as potential candidate compounds using the molecular docking technique. CBD and THC were found to be compounds that can navigate through a narrow channel and bind to the active sites of MCR, with calculated binding free energies of -8.8 and -5.5 kcal/mol, respectively. Furthermore, the effects of CSR supplementation levels on rumen fermentation, nutrient digestibility, methane production, and the ruminal microbial population were investigated through in vitro rumen fermentation simulations, as well as in situ digestibility analysis according to a completed randomized design (CRD). Compared with the control, supplementation with 2% total DM substrate in a total mixed ration (TMR) diet resulted in a 34% decrease in methane production. In addition, CSR increased the molar proportion of propionic acid and the concentration of ammonia-nitrogen (NH₃-N), whereas the molar proportion of acetic acid and the acetic acid to propionic acid ratio decreased. At the bacterial level, the population of R. flavefaciens decreased with CSR supplementation. At the archaeal level, the population of Methanobacteriales decreased with CSR supplementation. These findings suggest that CSR has the potential to be used as a novel natural additive for inhibiting ruminal methane production. Further in vivo studies are recommended to validate these findings.