Differential alteration of rumen microbial composition in response to 3-nitrooxypropanol supplementation in dairy cattle fed high-grain and high-forage diets

While 3-nitrooxypropanol (3-NOP) is known to effectively reduce methane (CH₄) emissions in ruminants, its impact on rumen microbial communities under different dietary composition remains less understood. This study investigated how different diet compositions with 3-NOP supplementation affected rumen microbial communities in dairy cows. Rumen samples were obtained from two previous studies: a crossover design study with two 3-NOP levels [0 and 130 mg/kg of dry matter (DM)] under a high-grain diet (HG; grain:forage = 60:40, n = 12), and a 3×3 Latin square design study with three 3-NOP levels (0, 68, and 132 mg/kg of DM) under a high-forage diet (HF; grain:forage = 40:60, n = 15). A total of 138 rumen samples (HG: 48 and HF: 90 samples) were subjected to metataxonomic analysis to identify the compositional shifts of rumen microbiota (bacteria, archaea, and protozoa) in response to 3-NOP supplementation. The ruminal bacterial response to 3-NOP was more pronounced under HG diet (11 genera affected) than under HF diet (2 genera affected), with <i>Lachnospiraceae</i> NK3A20 group consistently increased under both diets. This bacterial group showed diet-specific fermentation patterns, correlating with increased molar proportion of butyrate under HF diet and potentially contributing to increased molar proportion of propionate under HG diet through succinate production. Methanogen responses to 3-NOP supplementation were also diet-dependent. Although <i>Methanosphaera</i> <i>sp</i>. increased under both diets, while distinctive changes including contrasting responses between <i>Methanobrevibacter</i> (<i>Mbb</i>). <i>gottschalkii</i> and <i>Mbb</i>. <i>ruminantium </i>under HF diet, reflecting their different metabolic capabilities in substrate utilization for methanogenesis in response to 3-NOP. Notably, ruminal <i>Mbb</i>. <i>gottschalkii</i> (H₂-dependent) decreased while <i>Mbb</i>. <i>ruminantium</i> (H₂/formate-dependent) increased under HF diet, suggesting potential adaptation mechanisms to 3-NOP-induced changes in substrate availability. Ruminal protozoal communities remained unaffected under both diets. Further analysis of combined two studies with a batch effect correction approach revealed increases in <i>Lachnospiraceae</i> NK3A20 group, <i>Saccharofermentans</i>, <i>Mbb</i>. <i>ruminantium</i>, and <i>Methanosphaera</i> <i>sp</i>. Group5 and ISO3-F5, and decreases in <i>Mbb</i>. <i>gottschalkii</i> and <i>Methanomassiliicoccaceae</i> Group4 sp. MpT1 under 3-NOP supplementation. These findings demonstrate that 3-NOP has consistent effects on specific microbial taxa regardless of diet composition, and it also has diet-dependent impacts on other members of the rumen microbiota. This knowledge provides valuable insights for optimizing CH₄ mitigation strategies in dairy production systems under different dietary compositions.