Short- and long-term dietary supplementation as well as withdrawal of the enteric methane inhibitor 3-nitrooxypropanol reveal distinct effects on the rumen microbial community

Abstract Background The enteric methane inhibitor 3-nitrooxypropanol (3-NOP) inhibits the key enzyme in ruminal methanogenesis, but whether short-term (ST) and long-term (LT) dietary supplementation has similar effects on rumen microbiota in beef cattle and how microbes change after 3-NOP withdrawal have not been studied. This study investigated changes in rumen bacteria, archaea, and protozoa after ST and LT dietary supplementation and removal of 3-NOP using metataxonomic analysis. Results A total of 143 rumen samples were collected from two beef cattle studies with 3-NOP supplementation. The ST study (95 samples) used eight ruminally cannulated beef cattle in a 4 × 4 Latin square design with four 28-d of 3-NOP treatments [mg/kg of dry matter (DM)]: control: 0, low: 53, med: 161, and high: 345. The LT study (48 samples) was a completely randomized design with two 3-NOP treatments [control: 0, and high: 280 mg/kg of DM) fed for 112-d followed by a 16-d withdrawal (without 3-NOP). Bacterial and archaeal communities were significantly affected by 3-NOP supplementation but limited effects on protozoal communities were observed. Under ST supplementation, the relative abundances of Prevotella, Methanobrevibacter (Mbb.) ruminantium, Methanosphaera sp. ISO3-F5, and Entodinium were increased (Q < 0.05), whereas those of Mbb. gottschalkii and Epidinium were decreased (Q < 0.05) with 3-NOP supplementation. In LT study, relative abundances of Mbb. ruminantium, and Methanosphaera sp. Group5 were increased (Q < 0.05), while those of Saccharofermentans and Mbb. gottschalkii were decreased (Q < 0.05) with 3-NOP supplementation. Comparison between 3-NOP supplementation and the withdrawal revealed increased relative abundances of Clostridia UCG-014 and Oscillospiraceae NK4A214 group and decreased those of Eubacterium nodatum group and Methanosphaera sp. Group5 (P < 0.05) after 3-NOP withdrawal. Further comparison of rumen microbiota between control and 3-NOP withdrawal showed significantly higher (P = 0.029) relative abundances of Eggerthellaceae DNF00809, p-1088-a5 gut group, and Family XII UCG-001 in control group while no significant differences were detected for archaea and protozoa. Microbial network analysis revealed that microbial interactions differed by both 3-NOP dose and durations. Conclusions Both ST and LT supplementation affected overall rumen microbial profile, with individual microbial groups responded to 3-NOP supplementation differently. After 3-NOP withdrawal, not all microbes showed recovery, indicating that the 3-NOP driven shifts were only partially reversible. These findings provide an understanding of the effects of 3-NOP on rumen microbial communities and their adaptability to methane mitigation strategies.