Short communication: do milk purine derivatives reflect the urinary PDC index in dairy cows? Insights from integrative milk–urine targeted and untargeted screening

Urinary purine derivatives (PD) are widely used as non-invasive indicators of microbial proteinsynthesis in dairy cows, whereas their potential use in milk as an alternative biofluid remainspoorly understood. This study investigated whether milk PD reflect urinary purine metabolism,assessed the effects of dietary protein degradability on PD profiles, and explored coordinatedmetabolic responses in milk and urine using an integrative targeted and untargeted approach.We hypothesised that differences in dietary protein degradability would induce coordinatedmetabolic changes detectable across both biofluids. Milk and urine samples from nine lactatingHolstein cows fed diets differing in rumen-degradable (RDP) to rumen-undegradable (RUP) pro-tein ratio, namely CTR (64:36 ratio), SOL (67:33 ratio), and BY (57:43 ratio), were analysed by tar-geted and untargeted liquid chromatography coupled with high-resolution mass spectrometry.Targeted quantification of allantoin, uric acid, and creatinine revealed no association betweenmilk PD concentrations and the urinary purine derivatives-to-creatinine (PDC) index, indicatingthat milk PD do not directly reflect urinary purine metabolism. However, untargeted profilingidentified diet-related metabolic differences in both biofluids, with changes involving pathwaysrelated to amino acid metabolism, particularly in cows receiving the high-rumen-undegradable-protein diet. Integration of milk and urine datasets using DIABLO further revealed coordinatedmetabolite patterns across biofluids and dietary groups. Overall, these findings indicate thatmilk PD alone may not be a suitable proxy for urinary PD-based assessment of microbial proteinsynthesis. Integrated profiling of multiple biofluids can reveal systemic metabolic responses todietary protein degradability and provide complementary insights into nitrogen metabolism indairy cows.