Design, implementation and interpretation of in vitro batch culture experiments to assess enteric methane mitigation in ruminants—a review

•There are many factors that influence the results from in vitro fermentation technique (IVFT) to assess methane mitigation.•Factors include donor animals and diet, inoculum collection and processing, substrate and incubation buffer and procedures.•In vitro CH4 production is more closely correlated with in vivo CH4 production expressed per unit of degraded material.•IVFT is a valuable approach although CH4 inhibition potential is often overestimated compared to in vivo.•Guidelines are provided to interpret results obtained from in vitro methods before assessing mitigation strategies in vivo. In vitro fermentation techniques (IVFT) have been widely used to evaluate the nutritive value of feeds for ruminants and in the last decade to assess the effect of different nutritional strategies on methane (CH4) production. However, many technical factors may influence the results obtained. The present review has been prepared by the ‘Global Network’ FACCE-JPI international research consortium to provide a critical evaluation of the main factors that need to be considered when designing, conducting and interpreting IVFT experiments that investigate nutritional strategies to mitigate CH4 emission from ruminants. Given the increasing and wide-scale use of IVFT, there is a need to critically review reports in the literature and establish what criteria are essential to the establishment and implementation of in vitro techniques. Key aspects considered include: i) donor animal species and number of animal used, ii) diet fed to donor animals, iii) collection and processing of rumen fluid as inoculum, iv) choice of substrate and incubation buffer, v) incubation procedures and CH4 measurements, vi) headspace gas composition and vii) comparability of in vitro and in vivo measurements. Based on an evaluation of experimental evidence, a set of technical recommendations are presented to harmonize IVFT for feed evaluation, assessment of rumen function and CH4 production.