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

N. Martinez, L. D.P. Sinedino, R. S. Bisinotto, E. S. Ribeiro, G. C. Gomes, F. S. Lima, L. F. Greco, C. A. Risco, K. N. Galvão, D. Taylor-Rodriguez, J. P. Driver, W. W. Thatcher, J. E.P. Santos

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179 Scopus citations


The objectives were to study the effects of induced subclinical hypocalcemia [SCH, blood ionized Ca (iCa2+) <1.0mM, 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 43g of oral Ca, as Ca sulfate and Ca chloride, at -1 and 11h) or an induced SCH [SCHI, 5% ethylene glycol tetraacetic acid (EGTA), a selective iCa2+ chelator, intravenous infusion] treatment for 24h, 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 72h, to monitor concentrations and adjust the rate of infusion, maintaining blood iCa2+ <1.0mM 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, β-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic iCa2+ 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 iCa2+ concentrations throughout the 24-h treatment period (0.77±0.01 vs. 1.26±0.01mM iCa2+). Induction of SCH reduced dry matter intake on the day of infusion (5.3±0.8 vs. 9.1±0.8kg/d) and rumen contractions (1.9±0.2 vs. 2.7±0.2 contractions/2min) for the last 12h of infusion. Cows in SCHI had decreased plasma insulin concentration (1.44±0.23 vs. 2.32±0.23ng/mL) evident between 6 and 18h after the beginning of the infusion, accompanied by increased concentrations of glucose (4.40±0.04 vs. 4.17±0.04mM). Plasma nonesterified fatty acids concentration was greater for SCHI than NC cows (0.110±0.019 vs. 0.061±0.014mM). Neutrophils of cows in SCHI had a faster decrease in cytosolic iCa2+ 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.

Original languageEnglish (US)
Pages (from-to)874-887
Number of pages14
JournalJournal of Dairy Science
Issue number2
StatePublished - Feb 2014
Externally publishedYes

Bibliographical note

Funding Information:
The authors thank Gergeli Toldi (Semmelweis University, Budapest, Hungary) for helping us with the analysis of the intracellular calcium using FacsKin software and Eric Diepersloot (Dairy Unit, University of Florida, Gainesville) for assistance with the study. Appreciation is extended to Doug Ensley (Boehringer Ingelheim Vetmedica, St. Joseph, MO). Financial support for this study was provided by a student scholarship from Boehringer Ingelheim Vetmedica and by a grant from the Southeast Milk Check-Off Dairy Research and Education Projects .


  • Dairy cow
  • Immune function
  • Metabolism
  • Subclinical hypocalcemia


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