A data fusion approach unveils the effects of 3-nitrooxypropanol on the rumen fluid and milk metabolomes of lactating Holstein dairy cows

: This study used a data fusion approach, combining rumen and milk metabolomic profiling, to investigate the biochemical effects of 3-nitrooxypropanol (3-NOP), an effective methanogenesis inhibitor. A controlled feeding trial was conducted in 2 phases (i.e., summer and fall) with Holstein dairy cows (n = 30/phase) fed a diet supplemented for 9 wk with 60 mg/kg DM of 3-NOP or a placebo. Rumen fluid and milk samples were collected at the end of the experimental phases and analyzed using ultra-HPLC coupled with high-resolution mass spectrometry. Metabolomic profiles from the cows supplemented with 3-NOP and the control group were compared to assess the influence of 3-NOP on rumen fermentation and milk composition. Both 3-NOP and the experimental phase significantly influenced rumen metabolites, whereas no significant differences were observed in the milk metabolome with 3-NOP supplementation. Untargeted metabolomic analysis, supported by supervised and unsupervised multivariate statistics, identified significant alterations in rumen metabolites. Results confirmed that 3-NOP significantly alters rumen fermentation by reducing methanogenesis-related compounds (e.g., methyl-coenzyme M and coenzyme B) and influencing nitrogen and nucleic acid metabolism, indicative of changes in microbial fermentation and protein synthesis. Regarding the milk metabolome, changes in certain discriminant metabolites, such as glycerophospholipids, small peptides, and nucleic acids, reflect alterations in rumen fermentation. Although 3-NOP supplementation induced marked changes in the rumen metabolome, including pathways related to methanogenesis and nitrogen metabolism, these alterations were not reflected in major changes in milk quality or its overall metabolomic profile.