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Simple Summary  There is a great interest among the scientific community in breeding animals that emit less methane (CH 4 ) as a strategy to mitigate the environmental impact of ruminants. The present study ranked individual dairy cows as low and high CH 4  emitters fed the same diet and evaluated whether the original in vivo ranking was maintained in the in vitro conditions using two contrasting substrates each without or with an antimethanogenic compound. Results do not support a strong effect of rumen microbiome on observed CH 4  production in vivo, and thus, ranking was not successfully replicated in in vitro conditions. Instead, it appears that animal-related factors such as digesta passage rate are more important drivers of enteric CH 4  production.    Abstract  This study evaluated if ranking dairy cows as low and high CH 4  emitters using the GreenFeed system (GF) can be replicated in in vitro conditions using an automated gas system and its possible implications in terms of fermentation balance. Seven pairs of low and high emitters fed the same diet were selected on the basis of residual CH 4  production, and rumen fluid taken from each pair incubated separately in the in vitro gas production system. In total, seven in vitro incubations were performed with inoculums taken from low and high CH 4  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 CH 4  production. Except for the aimed differences in CH 4  production, no statistical differences were detected among groups of low and high emitters either in in vivo animal performance or rumen fermentation profile prior to the in vitro incubations. The effect of in vivo ranking was poorly replicated in in vitro conditions after 48 h 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 CH 4  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 CH 4  emitter group, CNSL decreased predicted in vivo CH 4  (26.7 vs. 11.1 mL/ g of dry matter; DM) and stoichiometric CH 4  (CH 4 VFA; 304 vs. 235 moles/mol VFA), with these being also reflected in decreased total gas production per unit of volatile fatty acids (VFA). Microbial structure was assessed on rumen fluid sampled prior to in vitro incubation, by sequencing of the V4 region of 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.  Genus  Methanobrevibacter  represented 93.7 ± 3.33% of the archaeal sequences. There were no clear differences between groups in relative abundance of  Methanobrevibacter.

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