Breed-specific microbiomes drive differential responses to 3-nitrooxypropanol and Acacia mearnsii in dairy cows

<h2>Abstract</h2> Methane (CH₄) inhibitor 3-nitrooxypropanol (3-NOP) shows variable efficacy in Brown Swiss (BS) and Holstein (HF) cows, but the underlying mechanisms remain unclear. Rumination bolus, a proxy of rumen microbiota, combined with exhalomics (analysis of exhaled volatiles, offered a non-invasive approach to differentiate breed responses to CH₄ mitigation strategies. We investigated whether differences in rumination bolus microbiota and exhaled VFA (eVFA) between BS and HF cows contribute to their responses to 3-NOP. Sixteen multiparous cows (8 BS, 8 HF) were studied in a replicated 4 × 4 Latin Square design with a 2 × 2 factorial arrangement of 3-NOP (0 or 60 mg/kg DM) and <i>Acacia mearnsii</i> tannin extract (TAN; 0 or 3% DM). Rumination bolus samples collected 6 h post-feeding during the fourth period of the experiment were sequenced for full-length 16S rRNA sequences (PacBio) to assess bacterial diversity, while exhaled samples from all 4 periods were analyzed for VFA using secondary electrospray ionization-MS. Microbial community shifts were analyzed using Bray–Curtis, unweighted, and weighted UniFrac distances. Spearman correlations were performed between genus-level information with eVFA, CH₄, hydrogen (H₂), carbon dioxide (CO₂), DMI, and milk yield between breeds. Supplementation of 3-NOP induced a modest but consistent shift in microbial composition, while TAN effects were minimal and inconsistent. Breed-specific differences were evident: BS harbored more <i>Prevotella</i> and <i>Rikenellaceae</i>, while HF were enriched in <i>Succinivibrionaceae</i> and <i>Acetitomaculum</i>. Under 3-NOP, HF genera aligned more strongly with propionate-producing pathways and showed stronger negative correlations with CH₄, while BS genera remained more associated with acetate and butyrate proportions. Correlations with feed intake and milk yield were generally weak and inconsistent across genera. Overall, our results indicate that HF cows are more responsive to 3-NOP, likely redirecting spared H₂ toward propionate-utilizing bacteria, while BS appear more resilient to H₂ fluxes and consequently less responsive to 3-NOP. Findings from this pilot study highlight the importance of host-microbiome interactions in evaluating and implementing enteric CH₄ mitigation strategies in dairy cattle.