A reliable in vitro rumen culture system and workflow for screening anti-methanogenic compounds

Arguably the biggest man-made challenge of the century is to halt the progression of the climate change. Livestock’s methane (CH ₄ ) emissions, a greenhouse gas with a higher global warming potential than carbon dioxide (CO ₂ ), represent a prime target for reducing anthropogenic impact. While the reduction of enteric methane emissions through feed additives has been demonstrated, potent and affordable compounds inhibiting methanogenesis in ruminants are hardly established. Reliable methods for reproducible cultivation of the rumen microbiome in the laboratory are an essential tool for the study of methanogenesis.

We have developed a versatile setup that allows for the cultivation of ruminal fluid in a benchtop configuration. This setup combines, miniaturises and improves existing systems. We use everyday labware to create a setup capable of the long term cultivation of primary cultures extracted from the rumen of slaughtered cows. We describe the detailed preparation and cultivation procedure and demonstrate the expected function of the setup. The efficacy of the system is demonstrated through the administration of various concentrations of state-of-the-art methanogenesis inhibitors, including lyophilised Asparagopsis taxiformis (AT) powder, bromoform (BF), iodoform (IF), 3-nitrooxypropanol (3-NOP), rapeseed oil and BF dissolved in rapeseed oil. The parameters of the system exhibit a change in accordance with the literature. In comparison with conventional methodologies, our system offers enhanced versatility and ease of use. Furthermore, a novel approach for the quantification of the exhaled gases, CH ₄ and CO ₂ , is presented.