Abstract: Changes in venous blood gas composition and acid-base equilibrium in Holstein cattle blood samples kept under different temperatures at various time points were investigated for 24 h. The blood samples were collected from 3 healthy multiparous Holstein cows. A total of 9 blood samples obtained from the animals were allocated into three groups and kept at +4°C for group 1 (n = 3), at room temperature, 22°C, for group 2 (n = 3) and in an incubator at 37°C for group 3 (n = 3). Blood gases were analyzed at 0, 1, 3, 6, 12 and 24 h after the storage. The analyses of the blood samples indicated no change at pH and pCO₂ at 4 and 22°C for the 1st h. Similarly, in addition to O₂SAT at 4°C for 3 h and at 22°C for 6 h; O₂CT at 4°C and at 22°C for 24 h, HCO₃ and BB values at 4, 22 and 37°C for 24 h remained unchanged. By contrast, PO₂, BE and glucose levels in the samples were markedly altered within the 1st h.

INTRODUCTION

Blood gas composition and acid-base equilibrium in cattle alter during many of the respiratory system and metabolic diseases (Carlson, 1996). The precise measurement of the blood parameters is essential in the diagnosis of the diseases and the determination of their prognosis for veterinarians and researches. However, several factors including sampling method, measurement time and storage conditions are reported to affect blood gas composition and acid-base balance in cattle. The time passed between the blood sampling and its analyses is revealed to affect blood acid-base concentrations due to continuing anaerobic and aerobic activities and byproducts in humans, cattle and dogs (Haskins, 1977; Krokavec et al., 1987; Szenci and Besser, 1990; Boink et al., 1991; Liss and Payne, 1993; Gokce et al., 2004).

CO₂ is generated during aerobic metabolism, while lactic acid is produced all through aerobic activity in blood in vitro. The use of O₂ is altered depending on leukocytes, cytochrom in reticulocytes and citric acid enzymatic activities, all of which are directly regulated by the number of the cells. Moreover, glucolysis is a predominant function of the mature erythrocytes. The temperature is shown to be key-factor in the regulation of these metabolic changes in blood (Liss and Payne, 1993). Leucocytosis and anemia are also reported to yield changes in acid-base balance in blood (Haskins, 1977; Liss and Payne, 1993).

At the present study, we aimed to investigate the effect of discrete temperature points and different measurement times on venous blood gas composition and acid-base equilibrium in cattle blood samples.

MATERIALS AND METHODS

Animals and blood sampling: In this study, 3 healthy 4 years old multiparous Holstein cows were used for blood sampling and the analyses of several parameters in blood. Three blood samples obtained from the jugular vein and pulled into 10 mL injectors containing 0.08 mL heparin were collected from each animal. Before tightly capping with plastic air tight covers, air bubbles were carefully removed from the syringes. The blood samples were divided into 3 groups and were kept at refrigerator (4°C) for group 1, at room temperature (22°C) for group 2 and at an incubator (37°C) for group 3. In all of the blood samples, pH, pCO₂, pO₂, TCO₂, O₂SAT, O₂CT, HCO₃, SBC, BE, BB, Hb, Hct and glucose levels were measured at 0, 1, 3, 6, 12 and 24 h after the storage.

Blood analyses: In the samples, using portative blood gas system measuring several blood parameters automatically and with temperature option adjustable to subject’s body temperature (GASTAT-mini, Techno Medica, Japan), blood pH, partial CO₂ pressure (pCO₂), partial O₂ pressure (pO₂), total CO₂ (TCO₂), oxyhaemoglobin saturation (O₂SAT), O₂ saturation (O₂CT), actual bicarbonate (HCO₃), Standard Bicarbonate (SBC), Base Excess levels (BE), the sum of the total negative ion Buffer in Blood (BB), Hemoglobin (Hb), Heamatocrite (Hct) and glucose levels were measured. After adjusting the temperature of GASTAT-mini to the animal’s body temperature, pH, pCO₂ and pO₂ were analyzed.

Statistical analyses: The effect of temperature and time on blood parameters as described above was determined using SPSS 13.0 (Windows) and Anova-Tukey test.

RESULTS

The mean hemoglobin and heamatocrite values of the sampled animals were measured to be 7.97±1.83 g dL^-1 and 27.63±0.17%, respectively. At the time of sampling, the mean body temperature for the animals was found to be 38.4±0.1°C.

Blood borne acid-base, gas and glucose concentrations measured at different time points as designated and pertaining to all 3 groups are illustrated on Table 1-11.

The parameters determined for the groups at different hours were compared to the 0 h measurement results. The results showed that pH (Fig. 1) and pCO₂ remained stable for the 1st h in group 1 and 2. Whereas, there was a statistically significant sudden decrease in pH values and swift marked increase in PCO₂ in group 1.

Table 1:	Effect of storage time on pH of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)

Table 2:	Effect of storage time on pCO2 of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)

Table 3:	Effect of storage time on p02 of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)

Table 4:	Effect of storage time on TCO2 of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)

Table 5:	Effect of storage time on O2 SAT of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)

Table 6:	Effect of storage time on O2 CT of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)

Table 7:	Effect of storage time on HCO3 of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)

Table 8:	Effect of storage time on SBC of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)
S.D.: HCO3=SBC

Table 9:	Effect of storage time on BE of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)

Table 10:	Effect of storage time on BB of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n=3) (mean±S.E.)

Table 11:	Effect of storage time on glucose of bovine venous blood samples at +4°C (Group 3), +22°C (Group 2) and +37°C (Group 3) (n = 3) (mean±SE)
NM = Not measured

Fig. 1:	Alterations of pH levels dependent on storage time

Moreover, pO₂ in the entire groups was also markedly and promptly increased.

The decrease in TCO₂ was meaningful and fast in group 1 but it stayed constant for 12 h in group 2 and for 3 h in group 3. We also determined that O₂SAT showed sudden change in group 1 but remained steady for 6 h in group 2 and for 3 h in group 3. O₂ CT maintained its level for 3 h in group 1 and for 24 h in group 2 and 3; by contrast, HCO₃ concentration had a rapid change in group 1 but stayed constant for 24 h in group 2 and for 3 h for group 4. Furthermore, SBC and BE amounts showed sudden alteration in the groups; in contrast, BB remained stable in all groups for 24 h. Similar to SBC and BE, augmentation in glucose levels was swift and statistically consequential.

DISCUSSION

In living beings, the maintenance of blood buffering system at pH between 7.35-7.45 is required for normal metabolic activities. Nearly, all of the enzyme systems in the body affect the H⁺ ion concentration and therefore, the sustenance of H⁺ ion concentration at physio- logical levels is a very delicate balance in the body (Di Bartola et al., 1994).

Temperature is reported to be one of the leading factors affecting most of the biological reactions in blood. At their study on the measurement of the blood gases in dogs, Di Bartola et al. (1994) report that the determination of blood gases should be done within in 15-30 min at the room temperature or within 2 h at 4°C after withdrawing. In contrast to dogs, controversies exist among the studies performed to elucidate the effect of temperature on acid-base balance in cow blood samples stored at different temperatures. According to the work of Szenci and Basser (1990), blood samples can be used within 24 h for diagnostic purposes. Other researches indicate that the pH determined in blood samples kept at 4°C for 5-6 h (Paulsen and Surynek, 1977) or stored at 4°C for 48 h (Gokce et al., 2004) can be used in clinical applications; in addition, they show that storage time and temperature can change pH and bicarbonate values. At the present study, in contrast to earlier studies, pH values clearly decreased after 1 h in blood samples kept at 4°C but BE values, similar to other reports, were lower 1 h after storage. It appears that ongoing anaerobic activities and lactic acid formation during storage can alter BE and SBC via reducing pH. Rapid changes in pH values 1 h after storage at 22°C or immediately at 37°C that we measured in this study, further support our observation.

Anaerobic, aerobic metabolisms and their byproducts are the most prevailing factor in the increase of pCO₂ in dog and cow blood owing to storage (Haskins, 1977; Poulsen and Surynek, 1977). In addition, oxygen infiltration through plastic syringe wall is shown to cause increase in pO₂ in blood sample (Gokce et al., 2004). A recent study reported by Knowles et al. (2006) suggests that measurements of blood gases in the samples kept in plastic syringes should be done as soon as possible. In the present study, we found sudden noticeable increase in PO₂ at all temperatures studied, in PCO₂ when compared to 0 h measurements, at 37°C and 1 h after the storage in group 2 and 3. Rapid and marked increases in PCO₂ levels measured in this study, seem to be owing to anaerobic and aerobic activities of blood cells (Szenci and Beser, 1990; Beaulieu et al., 1999). Moreover, in addition to anaerobic and aerobic activities of blood cells, oxygen infiltration through plastic syringe wall appeared to be responsible for the increase in PO₂ (Gokce et al., 2004; Knowles et al., 2006).

Glucolysis is a predominant metabolism of mature erythrocytes and it is strictly regulated by temperature (Liss and Payne, 1993). In the present study, initiate of sudden decrease in glucose levels at different temperatures tested and the presence of little or no glucose at 12th h at 37°C and 24th h at 22°C indicate the consumption of glucose during erythrocyte metabolism.

Most of the circulating oxygen is bound to hemoglobin and only a small portion of it exists in dissolved form. The amount of circulating O₂ bound to hemoglobin is the oxygen saturation (O₂SAT) (Carlson, 1996). Our observation of sudden decrease in O₂SAT at +37°C is in line with the study of Gokce et al. (2004). In their interpretation, they reason that low pH induces separation of O₂ from hemoglobin thereby reducing O₂SAT. Our present data are similar and further support their view.

CONCLUSION

Moreover, the present study results suggest that changes in blood parameters are time and temperature dependent therefore, all parameter should be measured and combined carefully prior to interpretation for precise diagnosis. For instance, we observed no change in blood pH and PCO₂ values at 4 and +22°C’ for the 1st h; O₂SAT at 4°C for 3 h and 22°C 6 h; O₂CT at 4 and 22°C for 24 h in addition to HCO₃ and BB values at 4, 22 and 37°C 24 h, while PO_2, BE and glucose levels showed marked changes within first hour. Therefore, acid-base and blood gases should not be evaluated separately but rather combined for proper diagnosis. Moreover, current study also indicates that the storage of samples at 4°C or 22°C extends sample stability for the measurement of blood gas composition and acid-base balance in the diagnoses of diseases. Measurements should be preferably done right after or within 1 h after sampling; for this purpose, portative blood gas system can be used for the measurement at the site.