Effect of induced subclinical hypocalcemia on physiological responses and neutrophil function in dairy cows

The objectives were to study the effects of induced subclinical hypocalcemia [SCH, blood ionized Ca (iCa(2+)) <1.0 mM, without recumbency] on physiological responses and function of immune cells in dairy cows. Ten nonpregnant, nonlactating Holstein cows were blocked by lactation and assigned randomly to a normocalcemic (NC; intravenous infusion of 0.9% NaCl i.v. plus 43 g of oral Ca, as Ca sulfate and Ca chloride, at -1 and 11 h) or an induced SCH [SCHI, 5% ethylene glycol tetraacetic acid (EGTA), a selective iCa(2+) chelator, intravenous infusion] treatment for 24 h, using a crossover design. The sequence of treatments was either NC-SCHI or SCHI-NC, with a 6-d washout period. Ionized Ca was evaluated before, hourly during the infusion period, and at 48 and 72 h, to monitor concentrations and adjust the rate of infusion, maintaining blood iCa(2+) <1.0 mM in SCHI throughout the 24-h infusion period. Additional measurements included heart and respiratory rates, rectal temperature, dry matter intake, rumen contractions, whole-blood pH, concentrations of glucose and K in whole blood, concentrations of total Ca, Mg, nonesterified fatty acids, P-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic iCa(2+) in neutrophils and lymphocytes was quantified and neutrophil function was assayed in vitro. Infusion of a 5% EGTA solution successfully induced SCH in all SCHI cows, resulting in decreased blood iCa(2+) concentrations throughout the 24-h treatment period (0.77 +/- 0.01 vs. 1.26 0.01 mM iCa(2+) ). Induction of SCH reduced dry matter intake on the day of infusion (5.3 +/- 0.8 vs. 9.1 +/- 0.8 kg/d) and rumen contractions (1.9 +/- 0.2 vs. 2.7 +/- 0.2 contractions/2 min) for the last 12 h of infusion. Cows in SCHI had decreased plasma insulin concentration (1.44 +/- 0.23 vs. 2.32 +/- 0.23 ng/mL) evident between 6 and 18 h after the beginning of the infusion, accompanied by increased concentrations of glucose (4.40 +/- 0.04 vs. 4.17 +/- 0.04 mM). Plasma nonesterified fatty acids concentration was greater for SCHI than NC cows (0.110 +/- 0.019 vs. 0.061 +/- 0.014 mM). Neutrophils of cows in SCHI had a faster decrease in cytosolic iCa(2+) after stimulation with ionomycin (9.9 +/- 1.0 vs. 13.6 +/- 1.4 Fluo-4:Fura Red post-end ratio) in vitro. Furthermore, induction of SCH reduced the percentage of neutrophils undergoing phagocytosis (22.1 +/- 2.1 vs. 29.3 +/- 2.1%) and reduced the oxidative burst response after incubation of pathogenic bacteria (16.1 +/- 1.7 vs. 24.2 +/- 1.7%). Subclinical hypocalcemia compromised appetite, altered metabolism, and impaired function of immune cells in dairy cows.