Variability of urinary traits in lactating Holstein cows and their associations with milk quality traits

Monitoring urine composition is a valuable option for assessing animal health and physiological status, including the detection of nutritional imbalances and renal disorders. Although 24-h total urine collection is considered the gold standard, its application under commercial farming conditions is unfeasible due to housing constraints, collection procedures, and animal stress. Spot urine sampling offers a practical alternative; however, adjustment for urine concentration—through specific gravity—is required to account for individual variability. Building on a large-scale sample set, this study aimed to (i) evaluate the effects of stage of lactation and parity on urinary traits and (ii) investigate associations between urinary and milk traits to demonstrate which urine traits provide independent information. Spot urine and milk samples were collected from 1,271 Holstein cows across 17 farms in northern Italy. Farms were visited repeatedly, with different cows randomly sampled at each visit. Urinary pH, specific gravity, urea, protein, creatinine, allantoin, uric acid, glucose, and the protein-to-creatinine ratio were determined using reference methods. Milk composition was analysed using infrared spectroscopy and flow cytometry. Generalized linear mixed models were applied to assess sources of variation in urinary traits, including urinary specific gravity (as a covariate), stage of lactation and parity as fixed effects, and herd-test-date as a random effect. Pearson correlations were calculated within urinary traits and between urinary and milk traits. Urinary traits showed wide variability, with the protein-to-creatinine ratio exhibiting the greatest variability, whereas pH and specific gravity were the least variable. Specific gravity was significantly affected by stage of lactation and parity and explained a substantial proportion of the variability in all urinary traits, confirming its strength in adjusting for urine concentration. Stage of lactation influenced all specific-gravity adjusted urinary traits except pH and uric acid, whereas parity significantly affected only creatinine concentration. Most nitrogenous compounds were most concentrated in urine in early lactation and followed distinct temporal patterns across lactation. Herdtest- date accounted for a considerable proportion of variance, particularly for urinary pH and urea concentration. Strong correlations were observed within urinary nitrogenous compounds, whereas associations between urinary and milk traits were generally weak, confirming their independence in diagnostic value. Only milk urea concentration was closely correlated with urinary total nitrogen and urea concentrations. Milk urea concentration showed the strongest positive correlations with urinary total nitrogen and urea. These findings provide a robust baseline for interpreting urinary traits in lactating cows and identify key sources of physiological variability, thereby laying the foundations for the use of spot urine samples in diagnostic and herd management applications. This study received support from the project Cool-Cow 2023 of the Breeders Association of Veneto Region (ARAV, Vicenza, Italy).