The aim of the present study was to evaluate the changes of ionized calcium (iCa) and glucose in refrigerated heparinized blood samples from dairy cows. Blood samples were collected from the coccygeal vessels of 16 adult cows into vacuum tubes coated with heparin. Measurements of iCa and glucose were performed using a biochemical analyzer (Stat Profile PRIME CCS Analyzer, Nova Biomedical, Waltham, MA). After collection, samples were immediately refrigerated, stored at 4°C and analyzed at 0, 5, 20 and 35 min, 1, 2, 4, 26, 50 and 74 h. Before each analysis, samples were homogenized for proximately 30 s using a tube rocker. After homogenization a specimen was extracted from the vacuum tube using a syringe to minimize gas exchange. The iCa and glucose changes were evaluated with the MIXED procedure of SAS with cow as a random measure and time as a repeated measure. Initial iCa ranged from 1.27 to 0.92 mmol/L and glucose from 58 to 87 mg/dL. Least squares means of iCa and glucose concentrations over time are shown in Table 1. There was a significant effect of storage time for glucose (P < 0.001) and a tendency for iCa concentration (P < 0.10). The observed changes in iCa over time were minimal (<1%). Glucose concentration remained stable from 0 to 26 h but decreased 2.37% at 50 h and 5.24% at 74 h. Our results suggest that iCa concentration remains stable for 74 h and glucose concentration for 26 h when heparinized samples are kept refrigerated in vacuum tubes. Table 1. Variations in the concentrations of iCa and glucose in refrigerated blood samples from 16 dairy cows

Duration of storage	iCa (mmol/L)	Glucose (mg/dL)
0 min	1.146 ± 0.026	64.64 ± 3.30a
5 min	1.148 ± 0.026	65.13 ± 3.30a
20 min	1.151 ± 0.026	64.13 ± 3.30a
35 min	1.154 ± 0.026	64.47 ± 3.30a
1h	1.157 ± 0.026	64.48 ± 3.27a
2 h	1.158 ± 0.026	64.81 ± 3.24a
4 h	1.156 ± 0.026	64.44 ± 3.24a
26 h	1.154 ± 0.026	63.38 ± 3.24a
50 h	1.152 ± 0.026	61.88 ± 3.24b
74 h	1.152 ± 0.026	60.06 ± 3.24c

^a–cDifferent letters show significant difference within column (P < 0.05).