# Comparative analysis of rumen metagenomes with dietary supplementation of 3-nitrooxypropanol revealed divergent modes of action in hydrogen metabolism and reductant pathways between beef and dairy cattle

## TL;DR

Adding 3-NOP to cattle diets changes rumen microbes and hydrogen metabolism differently in beef and dairy cattle, reducing methane.

## Contribution

This study reveals divergent microbial and functional responses to 3-NOP in beef and dairy cattle rumen, offering new mechanistic insights.

## Key findings

- 3-NOP supplementation altered rumen bacteria and methanogen abundances in beef and dairy cattle.
- Hydrogenotrophic methanogenesis decreased, with distinct gene expression changes in beef and dairy cattle.
- Protozoal species like Isotricha prostoma decreased, while others like Polyplastron multivesiculatum increased in beef cattle.

## Abstract

The compound 3-nitrooxypropanol (3-NOP), an inhibitor of methyl-coenzyme M reductase (MCR), reduces enteric methane production in both beef and dairy cattle. Although the proposed mechanisms of 3-NOP involve on inhibiting the activity of MCR in vivo, it is unknown how this process could affect rumen microbiome as a whole and if it differs between beef and dairy cattle. This study conducted a comparative analysis of the rumen microbiome and its functional shifts in four different cattle studies (two beef and two dairy cattle studies) that evaluated 3-NOP supplementation using metataxonomics and metagenomics.

Comparative analysis of 281 rumen metataxonomic datasets (143 beef and 138 dairy cattle) revealed that dietary supplementation with 3-NOP affected rumen bacteria and methanogens. Further, comparative analysis of 54 metagenomic datasets (24 beef and 30 dairy cattle) revealed that 3-NOP inhibited mcrA, decreased the abundances of Methanobrevibacter gottschalkii and the protozoal species Isotricha prostoma, while increased the abundances of Methanobrevibacter ruminantium and Methanosphaera sp., Prevotella sp. was a significant bacterial taxon in both beef and dairy cattle, contributing to various pathways such as propionate and butyrate production. Its increased abundance after 3-NOP supplementation may also be linked to the decrease in Isotricha prostoma. Hydrogenotrophic methanogenesis decreased after 3-NOP supplementation with the abundance of genes involved in methylenetetrahydromethanopterin dehydrogenase decreased in beef cattle, while that of 4Fe-4S ferredoxin gene decreased in dairy cattle. The abundance of protozoal Polyplastron multivesiculatum increased after long-term 3-NOP supplementation in beef cattle, potentially due to changes in hydrogen (H2) partial pressure. During 3-NOP-mediated methanogenesis reduction, abundance of genes encoding methanogenic hydrogenase and H2 producing hydrogenase were decreased, while those encoding H2 sensory hydrogenase increased. Acyl-CoA dehydrogenase gene involved in propionate and butyrate production pathways increased in both beef and dairy cattle, while nitrite reductase increased specifically in beef cattle, indicating a rise in alternative H2 sinks.

Video Abstract

Video Abstract

Our findings revealed broad effects of 3-NOP on rumen microbiome and functions in vivo, with varied effects in beef and dairy cattle, which provide mechanistic insights into the supplementation of 3-NOP in both beef and dairy cattle, supporting its more sustainable and effective use in the future.

The online version contains supplementary material available at 10.1186/s40168-025-02201-y.