PSV-11 - In vitro screening of selected compounds to reduce ruminal methane production

Abstract: Methane is a natural product of rumen fermentation and emissions from ruminant livestock represent a major environmental concern. Methane is not only a potent greenhouse gas but also represents a loss of energy efficiency for the host. The objective of this study was to perform in vitro screening of a range of compounds with potential anti-methanogenic activity, including polyphenols, flavonoids, fatty acids, amino acids, and salts. We aimed to select compounds conferring up to a 20% reduction in CH₄ for further investigations. The substances tested included carvacrol, coumaric acid, coumarin, flavanone, gallic acid, glycerol monolaurate (GML), glycine, methylene blue, naringin, quercetin, quinic acid, resorcinol, rutin, tannic acid, vanillic acid, and zinc acetate. These substances varied in their mechanisms of action and were selected based on known antimicrobial or anti-methanogenic properties. The experiment was designed as a randomized block experiment, with treatments tested in triplicates across four batches (n=228 samples) using the same treatments and experimental conditions. Fresh rumen contents were manually collected from two rumen-fistulated lactating Holstein cows, 3 hours after their morning feeding. Rumen contents were blended at a 2:1 ratio, filtered through a four-layered cheesecloth, and added to McDougall's solution at a 2:1 ratio. Nine milliliters of the final media were added to a 20 ml Bellco flask containing 150 mg of total mixed ration (TMR) ground to 1 mm. Each batch also had three flasks without TMR and no treatment (blank flasks), three flasks containing only TMR without treatment (negative control), and three flasks containing TMR and treated with chloroform (positive control), resulting in a total of 19 treatments. The end-products of in vitro batch fermentations were assessed after 24 h of incubation, and samples from each flask were collected for gas composition analysis using gas chromatography. Volatile fatty acids were determined using high-performance liquid chromatography (HPLC). The data was analyzed using R Studio, and the following model was applied: yij = µ + treatment_i + batch_j + ℇ_ij. Methane production (measured in parts per million, ppm) increased when GML, naringin, and quercetin were added compared to the negative control. In contrast, the addition of carvacrol, glycine, tannic acid, and methylene blue resulted in a decrease of up to 25% in methane production. These compounds show potential to alter ruminal fermentation and mitigate methane production and will be selected for evaluation of synergistic interactions and effects on the rumen microbial communities.