Animal as the Solution III: Dairy Cows Selected for Low Milk Urea Nitrogen Show Modest Differences in Milk Metabolomic Profiles with Potential Nutritional Benefits

Societal expectations for dairy production increasingly demand reductions in environmental impacts alongside improvements in milk nutritional quality. Animal-based strategies have therefore been explored, including the phenotyping and selection of A1 Protein Free (A1PF) cows that excrete less milk urea nitrogen (MUN) and less urinary nitrogen (low-MUN cows). This study characterizes the milk metabolome of Low-MUN cows, examining metabolomic patterns associated with reduced nitrogen excretion and potential implications for milk quality. Three herd tests were conducted on a population of 1600 Holstein–Friesian cows to identify a sample of 200 individuals representing the extremes of MUN concentrations (the lowest 100 and highest 100). Milk samples were collected from these cows during mid and late lactation for MUN determination. From this group, metabolomic profiles of milk and urinary N concentrations were analyzed in 20 distinct individuals (the lowest 10 and highest 10 MUN concentrations). Overall, only modest differences in milk metabolomic composition were observed between Low-MUN and High-MUN cows; however, the differences did not remain significant after Benjamini–Hochberg correction for multiple comparisons (FDR < 0.05). Caffeic acid 3-sulfate, isovanillic acid sulfate, catechol sulfate and 3-indoxyl sulfate were more abundant in the high-MUN group, whereas n-acetyl-L-citrulline and guanosine monophosphate were significantly elevated in low-MUN cows. These metabolites are associated with antioxidant activity and nitrogen metabolism, suggesting subtle differences in metabolic function between groups. However, since the overall metabolomic profiles were largely similar, the presence of specific compounds linked to nitrogen-use efficiency and potential animal-based strategies targeting reduced nitrogen excretion may contribute to environmental sustainability without compromising milk metabolic quality. Nevertheless, further research is required to confirm these findings and establish more conclusive evidence regarding the metabolic and functional implications of the Low-MUN phenotype.