Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted me -tab olomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multi-variate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most dis-criminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The dis-criminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vi-tamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, deter-mining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degrada-tion product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial ni- trogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.