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 CH 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 CH 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 CH production.

This review is part of the FACCE-JPI ‘Global Network’ project and the ‘Feeding and Nutrition Network’ of Livestock Research Group, in the Global Research Alliance for Agricultural Greenhouse Gases (www.globalresearchalliance.org). Authors acknowledge national funding from INIA (Spain, project MIT01-GLOBALNET-EEZ), the Ministry of Economic Affairs (The Netherlands; project Global Research Alliance on Agricultural Greenhouses Gases, BO-20-007-006), USDA-NIFA (USA), French National Research Agency through the program FACCE-JPI program, the Irish contribution to this JPI-FACCE project was funded by the Agricultural GHG Research Initiative for Ireland (AGRI-I), Department for Environment Food & Rural Affairs (UK), BLW (Switzerland) and Academy of Finland, Helsinki, Finland (Project 281337).

Peer Reviewed