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

Abstract

a b s t r a c tIn 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 4 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 4 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 4 production.

Comparison of In Vitro Long Digestion Methods and Digestion Rates for Diverse Forages

“…Concerns regarding costs and labor requirements of feed digestibility determinations in vivo have furthered the utilization of alternative laboratory techniques. Although it is desirable to have consistent guidelines relating to host animals, sampling, inoculum preparation, and digestion system (Yáñez-Ruiz et al, 2016), it is unlikely that any particular system would be accepted as a standard procedure (Mould et al, 2005). In vitro digestion, however, is considered only a guide to the potential, rather than realizable, value of a feed (Tilley and Terry, 1963).…”

The effect of diet of the donor cows on in vitro measurements of methane production from wheat and corn incubated in various forage‐to‐grain ratios

“…Whereas previous studies have found that more total gas and methane were produced when substrates were incubated in ruminal fluid from cows adapted to the substrate, this did not occur in our study. It is well known that the rumen inoculum is one of the key factors that influence in vitro fermentations . However, most previous studies that investigated this issue compared rumen inocula from donor cows fed forage (low starch) versus concentrate (high starch) diets .…”

“…In fact, we have made the important novel finding that the source of the donor fluid had a greater effect on MP than did the substrate used in the incubation. This is an indication that the original recommendation that the diet fed to donor animals should be of similar composition to the substrate incubated in vitro is in some cases not enough to elucidate the different methane productions between two different feeds.…”

“…However, it should be considered that in vitro experiments are used to screen substrates for their potential methane mitigation effect; in that context, all rates used in experiment 2 detected the methane mitigating potential of wheat grain when compared with corn grain. It has been suggested that ruminal fluid for in vitro studies should come from donor animals fed a diet similar to the feed to be tested . This may not always be practical or desirable for the health of the donor animals.…”

“…However, some aspects within the methodology can affect the results, and hence the conclusions drawn. A previously published review made a series of recommendations to standardize the in vitro methodology . One recommendation was that in vitro incubations should use ruminal fluid from donor animals fed a diet similar to the substrates that are screened in vitro in order to have an inoculum containing microbial populations adapted to the substrate in question .…”