Diurnal Dynamics of Gaseous and Dissolved Metabolites and Microbiota Composition in the Bovine Rumen

Abstract

Diurnal patterns of ruminal fermentation metabolites and microbial communities are not commonly assessed when investigating variation in ruminal CH4 production. The aims of this study were to monitor diurnal patterns of: (i) gaseous and dissolved metabolite concentrations in the bovine rumen, (ii) H2 and CH4 emitted, and (iii) the rumen microbiota. Furthermore, the effect of dietary inclusion of linseed oil on these patterns was assessed. Four rumen cannulated multiparous cows were used in a cross-over design with two 17 days periods and two dietary treatments: a control diet and a linseed oil supplemented diet [40% maize silage, 30% grass silage, 30% concentrate on dry matter (DM) basis for both diets; fat contents of 33 vs. 56 g/kg of DM]. On day 11, rumen contents were sampled for 10 h after morning feeding to profile gaseous and dissolved metabolite concentrations and microbiota composition. H2 and CH4 emission (mass per unit of time) was measured in respiration chambers from day 13 to 17. A 100-fold increase in ruminal H2 partial pressure (contribution to the total pressure of rumen headspace gases) was observed at 0.5 h after feeding. This peak was followed by a decline to basal level. Qualitatively similar patterns after feeding were also observed for H2 and CH4 emission, ethanol and lactate concentrations, and propionate molar proportion, although the opposite pattern was seen for acetate molar proportion. Associated with these patterns, a temporal biphasic change in the microbial composition was observed as based on 16S ribosomal RNA with certain taxa specifically associated with each phase. Bacterial concentrations (log10 16S ribosomal RNA gene copies based) were affected by time, and were increased by linseed oil supplementation. Archaeal concentrations (log10 16S ribosomal RNA gene copies based) tended to be affected by time and were not affected by diet, despite linseed oil supplementation decreasing CH4 emission, tending to decrease the partial pressure of CH4, and tending to increase propionate molar proportion. Linseed oil supplementation affected microbiota composition, and was most associated with an uncultivated Bacteroidales taxon. In summary, our findings support the importance of diurnal dynamics for the understanding of VFA, H2, and CH4 production.

NG-Tax, a highly accurate and validated pipeline for analysis of 16S rRNA amplicons from complex biomes

“…Commonly employed quality filtering parameters based on Phred score, such as minimum average Phred score, maximum number of ambiguous positions, maximum bad run length, trimming and minimum read length after quality trimming, are not utilized in NG-Tax because quality scores from the Illumina base caller have been shown to be of limited use for the identification of actual sequence errors for 16S rRNA gene amplicon studies 9,37 . Additionally, these quality scores only check for errors that occurred during sequencing, but do not account for other sources of error, such as PCR amplification, whereas quality filtering by abundance is sensitive to any source of error.…”

“…The reproduction of MC compositional profiles generated by amplicon sequencing on Illumina platforms commonly suffers from a high fraction of poorly classified and spurious OTUs 9 . Using QIIME, up to 20% of OTUs per sample could not be assigned beyond the class level ( Figure 3).…”

“…For deep sequencing based approaches, filtering strategies that remove low-abundance reads make it impossible to apply richness estimation metrics such as the Chao1 index and the ACE coverage estimator, because low-abundance read counts are included in their calculations. Conversely, richness estimates based on unfiltered datasets are unlikely to be accurate, if many of the reads actually represent PCR and/or sequencing errors 9 . In contrast to purely OTU-based methods, divergence-based methods account for the fact that not all species within a sample are equally related to each other, considering two communities to be similar if they harbour the same phylogenetic lineages, even if the species representing those lineages in each of the communities are different.…”

“…Reproducibility can be evaluated by comparing separate sequencing runs and different primer pairs that cover distinct 16S rRNA gene regions. Although replicability is often achieved, accuracy has been shown to be challenging especially at higher taxonomic resolution such as at genus level 8,9 .…”

“…However, in 16S rRNA gene amplicon sequencing every sequencing error could potentially lead to the false discovery of a new species. To avoid overestimation of microbial diversity, stringent quality filtering is therefore considered essential 9 .…”