In Vitro Incubations do not Reflect In Vivo Differences Based on Ranking of Low and High Methane Emitters in Dairy Cows

This study evaluated if ranking dairy cows as low and high CH4 emitters using the GreenFeed system (GF) can be replicated in vitro conditions using an automated gas system and their possible implications in terms of fermentation balance and microbial communities in rumen conditions. Pairs of low and high emitters fed the same diet were selected on the basis of residual CH4 production, and rumen fluid taken from each pair was incubated separately in the in vitro gas production system. In total, seven in vitro incubations were performed with inoculums taken from low and high CH4 emitting cows incubated in two substrates differing in forage to concentrate proportion, each without or with the addition of cashew nutshell liquid (CNSL) as an inhibitor of CH4 production. Except for the aimed differences in CH4 production, no statistical differences were detected among groups of low and high emitters either on in vivo animal performance or rumen fermentation profile at the beginning of the in vitro incubations. The effect of in vivo ranking was poorly replicated in vitro conditions after 48 hours of anaerobic fermentation. Instead, the effects of diet and CNSL were more consistent. The inclusion of 50% barley in the diet (SB) increased both asymptotic gas production by 17.3%, and predicted in vivo CH4 by 26.2 %, when compared to 100% grass silage (S) substrate, respectively. The SB diet produced on average more propionate (+ 28 mmol/mol) and consequently less acetate compared to the S diet. Irrespective of CH4 emitter group, CNSL decreased predicted in vivo CH4 (26.7 vs 11.1 ml/ g of dry matter; DM) and stoichiometric CH4 (CH4VFA; 304 vs 235 moles/mol VFA), with these being also reflected in decreased total gas production per unit of volatile fatty acids (VFA). The microbial structure was assessed, on rumen fluid sampled prior to in vitro incubation, by sequencing of the V4 region of the 16S rRNA gene. Principal coordinate analysis (PCoA) on operational taxonomic unit (OTU) did not show any differences between groups.Some differences appeared of relative abundance between groups in some specific OTUs mainly related to Prevotella Archaeal sequences were filtered out separately in a second step and relative abundance was calculated in relation to total archaeal counts. Genus Methanobrevibacter represented 93.7 ± 3.33 % of the archaeal sequences. There were no clear differences between groups in the relative abundance of Methanobrevibacter.