**Background:** Creatinine is widely used as an internal marker to estimate urinary volume in ruminant research, enabling less laborious collection protocols. However, no published data existed on optimal storage conditions for bovine urine to preserve creatinine concentration. Prior work in sheep showed that acidified urine (pH 2.5–3.5) stored at 28–39°C for 150 days had increased creatinine, likely due to conversion of creatine to creatinine under acidic, warm conditions. Since urine must be acidified (pH < 3) to prevent purine derivative degradation, this creates a potential conflict: acidification may promote creatine-to-creatinine conversion, inflating creatinine values and causing experimental errors.
**Methods:** Urine was collected via spontaneous urination from 25 cattle (10 Nellore, 15 Holstein). Samples (40 mL) were diluted in 160 mL distilled water and acidified to pH < 3 with sulfuric acid. A baseline aliquot was analyzed immediately (day 0). The remaining urine was fractionated into 2-mL Eppendorf tubes and stored at four temperatures: room temperature (mean 20.6 ± 1.1°C, max 26.4 ± 1.1°C), refrigerated (4°C), frozen (-20°C), and frozen (-40°C). Twenty urine samples (10 Nellore, 10 Holstein) were used to assess creatinine recovery without added creatine, analyzed on days 1, 3, 7, 10, 15, 30, 45, 60, 90, 120, and 150. Five additional Holstein urine samples were spiked with creatine solutions (20, 40, and 60 mg/dL) to evaluate creatine-to-creatinine conversion, analyzed on days 1, 3, 7, 15, 30, and 45. Creatinine was measured using a colorimetric kinetic method (Bioclin K067 kit) on an automated biochemistry analyzer at 510 nm. Relative creatinine was calculated as the ratio of stored-sample concentration to day-0 reference. Statistical analysis used SAS 9.4 with MIXED and REG procedures; α = 0.05.
**Key Results:** For urine without added creatine, there was no significant difference in relative creatinine concentration from days 1 to 15 at any storage temperature (P > 0.05). From day 30 onward, a significant effect of time and/or temperature emerged (P < 0.001). At room temperature, relative creatinine increased progressively: 1.168 at day 30, 1.248 at day 45, 1.250 at day 60, 1.304 at day 90, 1.369 at day 120, and 1.329 at day 150 — representing up to approximately 33% increase over baseline. Refrigerated samples (4°C) also showed significant increases over time (P = 0.002), reaching 1.117 at day 120. Frozen samples at -20°C and -40°C showed no significant change over 150 days (P = 0.409 and P = 0.560, respectively). For creatine-spiked urine, significant creatine-to-creatinine conversion occurred at room temperature and 4°C after 30 days (P < 0.05). By day 7, approximately 5% of added creatine was converted to creatinine; by day 45, conversion reached approximately 35%. No conversion occurred in frozen samples (P > 0.05).
**Clinical Implications:** Bovine urine acidified to pH < 3 can be stored at any temperature (room, 4°C, -20°C, or -40°C) for up to 15 days without significant changes in creatinine concentration. For storage exceeding 15 days, freezing at -20°C or -40°C is essential to prevent artifactual increases in creatinine. Storage at room temperature or 4°C beyond 15 days leads to overestimation of creatinine (up to 33% at 150 days at room temperature), which would cause substantial errors when using creatinine as a urinary volume marker. The mechanism appears to be conversion of endogenous creatine to creatinine under acidic, non-freezing conditions. Researchers should plan sample handling accordingly: short-term storage (≤15 days) is flexible, but long-term storage requires freezing. These findings provide evidence-based guidelines for urine sample management in bovine metabolic and nutritional studies.