Dietary supplementation with linseed, saponins, and nitrate is a promising methane mitigation strategy in ruminant production. Here, we aimed to assess the effects of these additives on the rumen microbiota in order to understand underlying microbial mechanisms of methane abatement. Two 2-by-2 factorial design studies were conducted simultaneously, which also allowed us to make a broad-based assessment of microbial responses. Eight nonlactating cows were fed diets supplemented with linseed or saponin in order to decrease hydrogen production and nitrate to affect hydrogen consumption; also, combinations of linseed plus nitrate or saponin plus nitrate were used to explore the interaction between dietary treatments. Previous work assessed effects on methane and fermentation patterns. Rumen microbes were studied by sequencing 18S and 16S rRNA genes and ITS1 amplicons. Methanogen activity was monitored by following changes in mcrA transcript abundance. Nitrate fed alone or in combination in both studies dramatically affected the composition and structure of rumen microbiota, although impacts were more evident in one of the studies. Linseed moderately modified only bacterial community structure. Indicator operational taxonomic unit (OTU) analysis revealed that both linseed and nitrate reduced the relative abundance of hydrogen-producing Ruminococcaceae Linseed increased the proportion of bacteria known to reduce succinate to propionate, whereas nitrate supplementation increased nitrate-reducing bacteria and decreased the metabolic activity of rumen methanogens. Saponins had no effect on the microbiota. Inconsistency found between the two studies with nitrate supplementation could be explained by changes in microbial ecosystem functioning rather than changes in microbial community structure.IMPORTANCE This study aimed at identifying the microbial mechanisms of enteric methane mitigation when linseed, nitrate, and saponins were fed to nonlactating cows alone or in a combination. Hydrogen is a limiting factor in rumen methanogenesis. We hypothesized that linseed and saponins would affect hydrogen producers and nitrate would affect hydrogen consumption, leading to reduced methane production in the rumen. Contrary to what was predicted, both linseed and nitrate had a deleterious effect on hydrogen producers; linseed also redirected hydrogen consumption toward propionate production, whereas nitrate stimulated the growth of nitrate-reducing and, hence, hydrogen-consuming bacterial taxa. This novel knowledge of microbial mechanisms involved in rumen methanogenesis provides insights for the development and optimization of methane mitigation strategies.
Keywords: linseed; methane; microbiota; nitrate; rumen; saponin.
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