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Hypocalcemia induced by immune activation is a conserved response across mammalian species; however, administration of Ca is discouraged in other species as it is associated with increased morbidity and mortality. Early postpartum cows experience a decrease in circulating Ca concentration following acute inflammation. Corrective Ca therapy during the transition period, particularly in dairy cows experiencing acute disease, is common practice. However, the effect of Ca administration on the inflammatory response during acute immune activation is unknown. Our objective was to compare the clinical, inflammatory, and metabolic response to an intravenous (IV) lipopolysaccharide (LPS) challenge between postpartum cows infused, or not, with IV Ca to maintain eucalcemia. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) or sterile 0.9% NaCl (CTRL) during an IV LPS challenge (0.040 or 0.045 µg of LPS/kg of body weight over 1 h). Ionized Ca (iCa) was monitored cow-side, and IV Ca infusion was adjusted in a eucalcemic clamp for 12 h following the start of LPS infusion. Cows were monitored during the 24 h following challenge and serial blood samples were collected to quantify concentrations of glucose, β-hydroxybutyrate, nonesterified fatty acids, urea nitrogen, cytokines, acute-phase proteins, and cortisol. Blood iCa concentration decreased to 0.87 ± 0.03 mM in CTRL during challenge, and by design, iCa concentration was maintained within 3% of baseline in IVCa. Body temperature, heart rate, and respiratory rate were monitored for 24 h following the start of challenge and did not differ between groups. A treatment × time interaction was identified such that serum cortisol concentrations increased in both groups at 2 h but decreased to a greater extent at 6 h in IVCa compared with CTRL. Rumination time (min/h) over the first 12 h following challenge was greater in IVCa, but total rumination time in the 24 h following challenge did not differ from CTRL. Serum glucose and nonesterified fatty acid concentrations decreased, and β-hydroxybutyrate and urea nitrogen concentrations increased over time, but did not differ between groups. Acute leukopenia occurred in both groups at 4 h before leukocytosis was observed at 24 h with total white blood cell counts returning to baseline within 72 h. Plasma concentrations of tumor necrosis factor (TNF) and interleukin-10 (IL-10) increased within 1 h following the start of challenge and did not differ between groups. Serum haptoglobin and serum amyloid A concentrations increased within the 24 h following challenge and were elevated through 72 h but did not differ between groups. Eucalcemia during the acute systemic inflammatory response did not alter the TNF or IL-10 cytokine response, or the acute-phase protein SAA and haptoglobin response in this LPS challenge model; however, eucalcemia was associated with a more rapid decline in cortisol response and greater rumination time in the first 12 h following challenge. We did not find evidence that eucalcemia exacerbated the inflammatory response in early postpartum cows, but Ca administration may alter the clinical response to acute systemic inflammation.
Nearly all cows will experience a decrease in circulating Ca concentrations around parturition as mammary demand for Ca to support lactation is greatly increased (
). Lactation-induced or metabolic hypocalcemia is due to inadequate intake and mobilization of bone Ca stores to support lactation demands. Circulating Ca concentrations also decrease following immune activation, and intravenous (IV) infusion of LPS to induce a systemic inflammatory response will rapidly decrease blood Ca concentrations in mid- and late-lactation cows (
). We have recently extended these findings in early postpartum cows and noted that an acute and transient period of hypocalcemia followed IV LPS challenge in cows during the first week of lactation (
), corrective Ca therapy may improve outcomes of infectious disease; however, when this was investigated in a retrospective cohort study, Ca therapy was associated with decreased organ function and increased mortality in septic human patients (
). Corrective Ca therapy appears to exacerbate the inflammatory response, and current practice in human emergency and critical care discourages the blanket use of Ca to treat hypocalcemia during sepsis (
Therapeutic Ca administration in the face of inflammatory disease is common practice in dairy cows. In particular, it is recommended to administer Ca intravenously, subcutaneously, or orally to cows experiencing toxic mastitis in clinical and on-farm settings (
). Relative to the discouraged use in other species, there is widespread use of therapeutic Ca to treat hypocalcemia during infectious disease and this is common in dairy cows during the early postpartum period when cows are at increased risk for infectious disease (
). Despite the widespread use of Ca in postpartum cows during infectious disease, it is unknown if supplemental Ca exacerbates the inflammatory response and potentially worsens outcomes as seen in humans and rodents (
). We were specifically interested in the possible beneficial or detrimental effects of Ca during acute immune activation in postpartum dairy cows due to the recommended use of Ca during infectious disease in current industry practices. We hypothesized that maintaining eucalcemia during the acute systemic inflammatory response to LPS challenge would alter the clinical response and circulating concentrations of selected inflammatory mediators in early postpartum cows compared with matched control cows. Our objective was to compare the clinical, inflammatory, and metabolic response to an IV LPS challenge between postpartum cows infused or not with IV Ca to maintain eucalcemia during the acute systemic inflammatory response.
MATERIALS AND METHODS
Animals and Management
All procedures were evaluated and approved by the Cornell University Animal Care and Use Committee (protocol no. 2020–0030). Postpartum Holstein cows (n = 14) entering their second or third lactation from the herd at the Cornell University Ruminant Center (Harford, NY) were enrolled between September and October 2020 in a matched-pair randomized controlled design. Cows were monitored after calving for signs of clinical disease and were eligible for enrollment in the absence of dystocia, diagnosed health events, or fever of unknown origin. At approximately 4 DIM, cows were moved to individual sawdust-bedded tie-stalls equipped with individual feed bins and intake was monitored for 3 d. All cows received the same fresh cow TMR and were fed ad libitum once daily (0700 h) during the study period. Diet formulation and sampling are described in detail in the companion paper (
). Throughout the experiment cows were observed at least once daily for the presence of health disorders. Briefly, rectal temperature was recorded at each a.m. milking and cows were monitored daily for signs of displaced abomasum, respiratory disorders, mastitis, and metritis. Cows were monitored for hyperketonemia using a handheld point of care device (Precision Xtra meter, Abbott Diabetes Care Inc.) in whole blood immediately after obtaining the sample (
) to receive an IV infusion of either sterile 0.9% NaCl (CTRL, n = 7) or IV Ca in a eucalcemic clamp (IVCa, n = 7) for 12 h following an IV LPS challenge (0.040 or 0.045 μg of LPS/kg of BW over 1 h). Cows were eligible for LPS challenge when blood ionized Ca (iCa) concentration was ≥1.2 mM to avoid confounding effects of concurrent metabolic hypocalcemia (
). To determine if blood iCa was ≥1.2 mM the day before challenge, cows were screened for blood iCa using a handheld VetScan i-STAT analyzer (Abaxis Inc.) and disposable cartridges (CG8+, Abbott) using a blood sample collected from the coccygeal vessels. At enrollment, BW was measured on a commercial scale (Tru Test, Datamars Inc.) and BCS was recorded by a single investigator using a 5-point scale (
). The evening before LPS challenge, one jugular vein of CTRL cows and both jugular veins of IVCa cows were catheterized with an extended duration 14 gauge × 13.3 cm catheter (Milacath, Mila International) as described previously (
). All cows were subjected to an LPS challenge by IV infusion of LPS (Escherichia coli O111:B4, Millipore Sigma) that was started between 0700 and 0900 h. Lyophilized LPS was dissolved in sterile 0.9% NaCl (VetOne, MWI Animal Health), diluted to a stock solution of 1 mg/mL, and stored at −20°C. On the day of challenge, LPS stock solution was diluted 2,000-fold in sterile 0.9% NaCl to 0.5 µg/mL. This solution was infused through a jugular catheter at a rate to supply a dose of 0.040 or 0.045 µg/kg of BW over 1 h using a Plum XL infusion pump (Abbott Laboratories). Cows within the same pair received the same dose of LPS per kilogram of BW. On the day of challenge, cows were fed immediately after milking and were allowed at least 1 h of ad libitum feed intake before LPS infusion. At the start of LPS infusion, feed was removed and cows were fasted for the next 12 h.
In IVCa, blood iCa concentration was maintained in a eucalcemic clamp for 12 h following the start of LPS infusion, where a 23% Ca gluconate solution (VetOne, MWI Animal Health) was IV infused at a known and adjustable rate using a Plum XL infusion pump. To establish baseline iCa values and confirm that blood iCa was ≥1.2 mM on the day of challenge, whole blood was collected at −24, −2, and 0 h relative to the start of LPS infusion and immediately analyzed cow-side for iCa using a handheld VetScan i-STAT analyzer (Abaxis Inc.) and disposable cartridges (CG8+, Abbott). These iCa values were averaged, and Ca gluconate infusion was adjusted to maintain iCa concentrations within a target of ±5% of baseline values. To maintain eucalcemia, blood iCa concentration was measured cow-side every 15 min during the first hour, every 20 min during the second hour, then every 30 min until 7 h, and hourly until 12 h relative to the start of LPS infusion. Calcium infusion was started when blood iCa concentrations were decreasing in order to maintain blood iCa within 5% of baseline values. Whole blood was sampled and LPS was infused into the contralateral jugular catheter that was used for Ca infusion in IVCa animals. In paired CTRL cows, animals were infused with sterile 0.9% NaCl for 12 h at a rate to deliver the same volume dose of fluid using a nonpyrogenic IV administration set with universal drip chamber and roller clamp (IV Admin Set UniverSet, PractiVet). Blood iCa was measured and recorded at −24, −2, and 0 h, hourly until 12 h, and at 24 h relative to the start of LPS infusion in both IVCa and CTRL animals.
Data Collection, Sampling, and Analysis
Ambient temperature and relative humidity were monitored continuously during the experiment by a HOBO temperature data logger (HOBO MX2301A, Onset Computer Cooperation) that was mounted approximately 2.5 m from the ground directly above the front of the tie-stalls in the barn. Hourly temperature-humidity index (THI) was calculated using the equation: THI = (1.8 × T + 32) − [(0.55 − 0.0055 × RH) × (1.8 × T − 26)], where T = ambient temperature (°C) and RH = % relative humidity (
Body Temperature, Heart Rate, Respiration, Rumen Contractions, and Rumination
Body temperature was recorded continuously at 1-min intervals starting 1 h before LPS challenge until 24 h after the start of challenge using a HOBO external temperature data logger (HOBO MX2304, Onset Computer Cooperation) with an external sensor probe that was mounted to a blank intravaginal cattle insert (Eazi-Breed CIDR, Zoetis). At 0, 1, 2, 3, 4, 6, 8, 12, and 24 h relative to the start of LPS challenge, heart rate, respiratory rate, and rumen motility were recorded. Heart rate and respiratory rate were determined by auscultation of the heart using a stethoscope and by observation of flank movement, respectively. Rumen motility was determined by auscultation in the left paralumbar fossa and recorded as rumen contractions counted in a 2-min interval. Rumination data were collected from a subset of animals (CTRL, n = 4; IVCa, n = 6) equipped with ear tag monitors (SMARTBOW, Zoetis) in 1-h intervals.
Blood Sampling and Analysis
During LPS challenge, blood was sampled into evacuated blood collection tubes (Becton Dickinson Vacutainer Systems) with 158 United States Pharmacopeia (USP) units of sodium heparin for plasma separation, and into serum tubes for the separation of serum through a jugular catheter at 0 (before LPS infusion start), 1, 2, 3, 4, 6, 12, and 24 h following the start of LPS infusion. To collect blood samples from CTRL animals during the infusion period, the saline infusion was stopped and blood allowed to circulate for 2 min before blood was sampled. After sampling, the infusion was restarted. After the 48-h sampling, catheters were removed and blood was collected by venipuncture of the coccygeal vessels at 72 h relative to challenge into evacuated tubes described above. After collection, whole blood was allowed to clot for 30 min at ambient temperature and heparin tubes were immediately placed on ice. Serum and plasma were separated by centrifugation at 2,800 × g for 15 min at 4°C, aliquoted, frozen at −20°C, and then stored at −80°C until analysis.
At 0, 1, 4, 12, 24, and 72 h relative to the start of LPS challenge, blood was collected into evacuated tubes (Monoject, Covidien) containing K3 EDTA and submitted to the New York State Animal Health Diagnostic Center (Cornell University, Ithaca, NY) for automated complete blood cell counts (Advia 2120, Siemens). Blood smears were prepared from EDTA-blood and submitted to the Animal Health Diagnostic Center (Ithaca, NY) for manual leukocyte differential cell counts.
Serum glucose, nonesterified fatty acids (NEFA), BHB, and urea nitrogen concentrations were measured using commercially available kits (BHB: D-3 hydroxybutyrate Ranbut, Randox Laboratories; NEFA: HR Series NEFA-HR, Wako Diagnostics; glucose and urea nitrogen: Roche Diagnostics) on a Roche Cobas 6000 series, c501 Clinical Chemistry Automated Analyzer (Roche Diagnostics) at the New York State Animal Health Diagnostic Center. Serum cortisol was measured by solid-phase, competitive chemiluminescent enzyme immunoassay (Immulite 1000 Chemiluminescent Cortisol Assay, Siemens) in the Endocrinology Laboratory of the New York State Animal Health Diagnostic Center. Serum haptoglobin concentrations were determined in duplicate using the peroxidase activity colorimetric method (Phase Haptoglobin Assay, TP-801, Tridelta Development Ltd.) with a modification of reducing overall assay volume to 200 µL per reaction. Serum amyloid A (SAA) concentrations were determined using a commercial analysis kit (Multispecies SAA, Tridelta Development Ltd.) with at least 1,000-fold dilution of samples (
Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ; concentrations in plasma and cell culture supernatant.
. For quality control of all in-house assays, low and high concentrations of controls were run on every plate.
Analytical Approach
Sample size calculation was performed in JMP v. 14.0 (SAS Institute Inc.) and was based on expected differences in circulating plasma TNF concentration following LPS challenge at the time of maximal response. We desired to detect a difference in circulating TNF concentration of 30% at 2 h post LPS challenge in animals under conditions of eucalcemia compared with CTRL cows. Based on previously reported plasma concentrations of TNF (68 ng/mL) after an IV LPS challenge in early postpartum cows (
), we expected the eucalcemia treatment group to have a mean 30% greater than CTRL, or 88 ng/mL. Applying a power of 95%, a significance level of 0.05, and standard deviation of TNF concentration of 10 ng/mL resulted in 7 animals per treatment group.
Hourly THI data during the experimental period were summarized by PROC UNIVARIATE of SAS (SAS 9.4, SAS Institute Inc.). Repeated measures ANOVA was performed for outcomes equally spaced over time (iCa, body temperature, hourly rumination time, plasma cytokines, serum haptoglobin) using PROC MIXED in SAS (SAS 9.4). Fixed effects were treatment, time, and treatment × time interaction. A random effect of pair nested in LPS dose was included in the model with a REPEATED statement for time, subject of cow, and either a heterogeneous first-order autoregressive, heterogeneous compound symmetry, or unstructured covariance structure chosen based on the lowest Akaike information criterion. When measurable, 0-h values were included as baseline covariates for each outcome. Before analysis, body temperature data collected at each minute were condensed to 15-min intervals by averaging the previous 15 min of data. Body temperature at 0 h, condensed from the previous 15-min interval before the start of infusion, was included as a baseline covariate in the body temperature model. Blood iCa concentrations measured at −24, −2, and 0 h relative to the start of LPS infusion were averaged and included as baseline covariate in the iCa model. Baseline rumination time was calculated as the sum of each 1-h interval on d −1 relative to LPS challenge. On the day of challenge, total rumination time was calculated as the sum of each 1-h interval within the first 12 h and the total 24 h following the start of LPS challenge. Difference in total rumination time between treatment groups during the 12 and 24 h following challenge was analyzed using mixed-effects ANOVA with fixed effect of treatment and total rumination time the day before challenge as a baseline covariate, as well as random effect of pair nested in LPS dose. All models were corrected for BW, parity, and DIM at enrollment. Because plasma cytokines at 0 h were below the lower limit of detection for the multiplex assay, cytokine models did not include baseline covariates. Differences in baseline covariates between treatment groups were explored using mixed-effects ANOVA and corrected for BW, parity, and DIM at enrollment, with random effect of pair.
Repeated measures ANOVA was performed for outcomes with measurements non-equally spaced over time during the LPS challenge (cortisol, heart rate, respiratory rate, rumen motility, complete and differential white blood cell counts, glucose, NEFA, BHB, blood urea nitrogen, and SAA) using PROC MIXED in SAS with fixed effects of treatment, time, and treatment × time interaction. A random effect of pair nested in LPS dose was included in the model with a REPEATED statement for time, subject of cow, and either a first-order ante-dependence, spatial power, spatial exponential, unstructured, or heterogeneous compound symmetry covariance structure depending on the lowest Akaike information criterion. When measurable, 0-h values were included as baseline covariates for each outcome. All models were corrected for BW, parity, and DIM at enrollment. Differences in baseline covariates between treatment groups were explored using mixed-effects ANOVA and corrected for BW, parity, and DIM at enrollment, with random effect of pair.
Normality and homoscedasticity of residuals were visually assessed for all models. To fulfill model assumptions, the dependent variables cortisol, NEFA, TNF, and IL-10 were log-transformed. The INFLUENCE statement was used to test for outliers using Cook's distance defined as a value >0.5. If the F-test for the treatment × time interaction effect was ≤0.05, the SLICE option was used to produce pairwise comparisons between treatment groups within time point, and P-values were adjusted for multiple comparisons using the Bonferroni correction. Values are reported as least squares means and largest standard error of the mean unless otherwise specified. The authors used an α of 0.05 to declare statistical significance.
RESULTS
During the experiment, average ± SD (range) ambient temperature was 15 ± 5°C (3 to 29°C), relative humidity was 74 ± 11% (38 to 94%), and THI was 59 ± 7 (45 to 79). On the days of LPS challenge, ambient temperature averaged 14 ± 5°C (4 to 29°C), relative humidity averaged 76 ± 11% (46 to 91%), and THI was 58 ± 7 (46 to 79).
Description of Study Population
Ten (71%) and 4 (29%) cows were in their second and third lactation, respectively, and parity averaged (± SD) 2.3 ± 0.5 for both CTRL and IVCa. The average (±SD) DIM at enrollment was 8.0 ± 1.3 and 8.3 ± 1.1 for CTRL and IVCa, respectively. Body weight average (range) at enrollment for CTRL was 700 (606 to 798) kg and for IVCa was 714 (650 to 820) kg. Body condition score was similar between treatment groups; most animals had a BCS of 3.0 (n = 4, 28%), 3.25 (n = 4, 28%), or 3.75 (n = 3, 21%) and one cow each had a BCS of 2.75, 3.5, or 4.0. No animals were diagnosed with displaced abomasum, clinical mastitis, or clinical metritis during the experiment. At enrollment, BHB was (mean ± SD) 1.0 ± 0.4 and 0.8 ± 0.2 mM for CTRL and IVCa, respectively. One animal (in CTRL) was treated for hyperketonemia diagnosed at 48 h following challenge; however, the animal was not detected as an outlier for outcomes of interest and was retained in the data set. During the experiment, 4 pairs of cows received 0.040 μg of LPS/kg of BW and 3 pairs of cows received 0.045 μg of LPS/kg of BW. During LPS challenge, 2 CTRL animals (both received 0.040 µg of LPS/kg of BW) did not demonstrate a cytokine or clinical response in the 12 h following challenge (i.e., cytokines were not measurable and increase in body temperature was absent). Because we were interested in comparing the inflammatory response between groups, the data from these 2 animals were removed from the analysis, and the final data analysis was performed using data collected from 5 animals in CTRL and 7 animals in IVCa. Data from all other animals were included in the analysis. For baseline values measured at 0 h and included in models as baseline covariates, treatment effects were P > 0.10 for all outcomes except for BHB, which was 0.9 ± 0.04 mM for CTRL and 0.7 ± 0.04 mM for IVCa (P = 0.04), blood urea nitrogen, which was 2.0 ± 0.1 mM for CTRL and 2.5 ± 0.1 mM for IVCa (P = 0.03), and percent of neutrophils and lymphocytes in differential white blood cell count, which was 53 ± 4% in CTRL and 44 ± 3% for IVCa (P = 0.09), and 30 ± 3% for CTRL and 42 ± 2% for IVCa (P = 0.02), respectively.
Ionized Ca and Clinical Response to LPS Challenge
Results of repeated measures analysis of blood iCa concentration and the rate of Ca infusion and total Ca infused in IVCa animals following LPS challenge are shown in Figure 1. Blood iCa concentration decreased by 30% in CTRL and reached a nadir at 0.87 ± 0.03 mM from 6 to 7 h following the start of LPS infusion. Blood iCa concentration was maintained in IVCa and remained within 3.0% of baseline values, meeting the pre-determined goal of less than ±5% of variation from baseline. This difference in blood iCa concentration resulted in a treatment × time interaction (P < 0.001). To maintain eucalcemia in IVCa animals for the 12 h following the start of LPS infusion, the average (range) grams of total Ca infused was 15 (10 to 24) g. Intravenous Ca infusion was started on average (range) at 34 (22 to 45) min relative to the start of LPS challenge in IVCa. Results of repeated measures analysis of intravaginal temperature and hourly rumination time are shown in Figure 2. Body temperature of all cows increased within the first 2 h following the start of LPS infusion (P < 0.001) and both treatment groups demonstrated a biphasic response pattern of body temperature over time with an initial peak at 1.5 h and a greater peak at 5 h. Maximum body temperature was 40.4 ± 0.19°C versus 40.6 ± 0.16°C for CTRL and IVCa, respectively, and did not differ between groups (P = 0.48). In a subset of animals that were equipped with ear tag data loggers before enrollment (CTRL, n = 4; IVCa, n = 6), hourly rumination time decreased (P < 0.01) from baseline at 1 h but did not differ (P = 0.07) from 1 to 12 h following LPS challenge and was greater (P = 0.03) in IVCa versus CTRL. Total rumination time in the 12 h following challenge was 172 ± 27 min in CTRL and 241 ± 27 min in IVCa (P = 0.08). Total rumination time in the 24 h following challenge did not differ (P = 0.85) between groups and was 590 ± 45 min in CTRL and 577 ± 34 min in IVCa. Results of repeated measures analysis of cortisol, heart rate, respiratory rate, and rumen contractions are shown in Figure 3. Cortisol concentration increased in both groups following LPS challenge, but a treatment × time interaction was identified (P < 0.001) as cortisol was lower (P = 0.003) in IVCa compared with CTRL at 6 h. Heart rate and respiratory rate increased (P < 0.001), but the frequency of rumen contractions decreased (P < 0.001) following LPS infusion, and no treatment effects or treatment × time interactions were identified (P ≥ 0.06).
Figure 1Ionized Ca (iCa) concentration, Ca infusion rate, and total grams of Ca infused following an intravenous LPS challenge (0.040 or 0.045 µg of LPS/kg of BW over 1 h) in early postpartum cows (8 ± 1 DIM) infused with a control solution of 0.9% NaCl (CTRL, n = 5), or maintained at eucalcemia (IVCa, n = 7) in a eucalcemic clamp (23% Ca gluconate) for 12 h. Data for iCa are presented as LSM ± SE. Data for Ca infusion rate and total Ca infused are presented as average and SD. Baseline iCa values included in model as covariate. Least squares means for baseline (0 h) from mixed-effects ANOVA. Baseline iCa presented at 0 h represents mean of blood iCa concentration measured at −24, −2, and 0 h relative to start of LPS infusion. Trt = treatment. *Pairwise comparisons between treatment groups within time point with Bonferroni correction for multiple comparisons (P < 0.05).
Figure 2Intravaginal temperature (Temp) and hourly rumination time following an intravenous LPS challenge (0.040 or 0.045 µg of LPS/kg of BW over 1 h) in early postpartum cows (8 ± 1 DIM) infused with a control solution of 0.9% NaCl (CTRL, n = 5), or maintained at eucalcemia (IVCa, n = 7) in a eucalcemic clamp (23% Ca gluconate) for 12 h. Rumination time was collected from a subset of animals equipped with ear tag monitors (CTRL, n = 4; IVCa, n = 6). Data are presented as LSM ± SE. Baseline values included in model as covariate. Least squares means for baseline (0 h) from mixed-effects ANOVA. Trt = treatment.
Figure 3Serum cortisol concentration, heart rate, respiratory rate, and rumen contractions following an intravenous LPS challenge (0.040 or 0.045 µg of LPS/kg of BW over 1 h) in early postpartum cows (8 ± 1 DIM) infused with a control solution of 0.9% NaCl (CTRL, n = 5), or maintained at eucalcemia (IVCa, n = 7) in a eucalcemic clamp (23% Ca gluconate) for 12 h. Cortisol data are presented as geometric mean and back-transformed 95% CI; all other data are presented as LSM ± SE. Baseline values included in model as covariate. Least squares means for baseline (0 h) from mixed-effects ANOVA. Trt = treatment. *Pairwise comparisons between treatment groups within time point with Bonferroni correction for multiple comparisons (P < 0.05).
Results of repeated measures analysis of serum glucose, NEFA, BHB, and urea nitrogen concentrations during LPS challenge are shown in Figure 4. Serum glucose concentration decreased from baseline at 2 h (P = 0.03) but did not differ over time (P = 0.36) following challenge. Serum NEFA concentration decreased (P = 0.002), but serum BHB concentration increased (P = 0.04) at 24 h following challenge. Urea nitrogen concentration increased (P < 0.001) following challenge. Serum glucose, NEFA, BHB, and urea nitrogen concentrations did not differ between groups, and no treatment × time interactions were identified (P ≥ 0.32).
Figure 4Serum concentrations of glucose, nonesterified fatty acids (NEFA), BHB, and urea nitrogen following an intravenous LPS challenge (0.040 or 0.045 µg of LPS/kg of BW over 1 h) in early postpartum cows (8 ± 1 DIM) infused with a control solution of 0.9% NaCl (CTRL, n = 5), or maintained at eucalcemia (IVCa, n = 7) in a eucalcemic clamp (23% Ca gluconate) for 12 h. Data for NEFA presented as geometric mean and back-transformed 95% CI; all other data are presented as LSM ± SE. Baseline values included in model as covariate. Least squares means for baseline (0 h) from mixed-effects ANOVA. Trt = treatment. Serum glucose concentration at 2 h differed from baseline (P = 0.03).
Results of repeated measures analysis of automated complete white blood cell counts and manual differential white blood cell counts during LPS challenge are presented in Figure 5. Circulating leukocyte counts were decreased at 4 h following challenge (P < 0.01). By 24 h, lymphocyte and monocyte counts had returned to pre-challenge values. Segmented neutrophil and total white blood cell counts returned to pre-challenge values by 12 h but increased subsequently and were greater at 24 h compared with 0 h (P < 0.01) before decreasing to pre-challenge counts by 72 h. Eosinophil counts did not differ between groups or over time (P ≥ 0.40). At 0 h, platelet counts for CTRL were 402 ± 24 × 103 cells/µL and for IVCa were 356 ± 22 × 103 cells/µL (P = 0.22), and platelets decreased from baseline at 4 h (P < 0.001) but did not differ (P = 0.39) over time between 4 and 72 h following challenge and did not differ (P = 0.21) between groups. No treatment effects or treatment × time interactions were identified (P ≥ 0.08) for complete blood cell counts. In manual differential white blood cell counts, immature neutrophils were not detected at 0 h, but were present at 4 h relative to the start of LPS challenge and were still present at 72 h postchallenge. Percent of lymphocytes and eosinophils increased (P < 0.001), and percent of segmented neutrophils and monocytes decreased (P < 0.001) within 4 h of the start of LPS infusion, but returned to baseline values within 72 h. No treatment effects or treatment × time interactions were identified (P ≥ 0.06) for manual differential blood cell counts.
Figure 5Automated complete (left) and manual differential (right) white blood cell (WBC) counts following an intravenous LPS challenge (0.040 or 0.045 µg of LPS/kg of BW over 1 h) in early postpartum cows (8 ± 1 DIM) infused with a control solution of 0.9% NaCl (CTRL, n = 5), or maintained at eucalcemia (IVCa, n = 7) in a eucalcemic clamp (23% Ca gluconate) for 12 h. Data presented as LSM ± SE. Baseline values included in model as covariate. Least squares means for baseline (0 h) from mixed-effects ANOVA. Trt = treatment.
Results of repeated measures analysis of concentrations of plasma TNF and IL-10, and serum haptoglobin and SAA are presented in Figure 6. Plasma TNF and IL-10 concentrations increased (P < 0.010) in both groups following challenge, peaking at 2 h and did not differ (P ≥ 0.37) between groups. Intra- and interassay coefficients of variation (CV) for TNF were 9% and 11% and for IL-10 were 3% and 11%, respectively. Haptoglobin concentration increased (P = 0.001) over 2-fold in both groups, peaked at 48 h following LPS infusion, and remained increased at 72 h, but did not differ between groups (P = 0.17). Serum amyloid A concentration increased (P < 0.001) within 12 h following challenge and increased over 5-fold to peak at 24 h (Figure 6). Concentrations of SAA remained increased until 72 h but did not differ between groups (P = 0.90). No treatment × time interactions were identified for plasma cytokines or acute-phase proteins (P ≥ 0.26). Haptoglobin intraassay CV ranged from 2.6 to 7.5% and interassay CV from 1.9 to 4.3% for low and high controls, respectively. Serum Amyloid A intraassay CV ranged from 2.4 to 2.8% and interassay CV from 2.7 to 4.8% for low and high controls, respectively.
Figure 6Plasma tumor necrosis factor (TNF) and IL-10 concentration, and serum haptoglobin and serum amyloid A concentration following an intravenous LPS challenge (0.040 or 0.045 µg of LPS/kg of BW over 1 h) in early postpartum cows (8 ± 1 DIM) infused with a control solution of 0.9% NaCl (CTRL, n = 5), or maintained at eucalcemia (IVCa, n = 7) in a eucalcemic clamp (23% Ca gluconate) for 12 h. Plasma cytokine data are presented as geometric mean and back-transformed 95% CI. Serum amyloid A and haptoglobin presented as LSM ± SE, and baseline values included in model as covariate. Least squares means for baseline (0 h) from mixed-effects ANOVA. Trt = treatment.
Our objective was to compare the clinical, inflammatory, and metabolic response to an IV LPS challenge between early postpartum cows that experienced either hypocalcemia or eucalcemia following immune activation. As expected, blood iCa concentration decreased to values considered hypocalcemic (
) in CTRL between 4 and 10 h following challenge and the observed decrease was similar to that observed in mid- and late-lactation LPS challenge models (
). Despite fasting for 12 h, animals were able to recover iCa concentrations within 24 h. Using the current protocol, eucalcemia was maintained in IVCa within a tight range of baseline values. Given the rapid onset of events that determine the direction and magnitude of the inflammatory response, including cytokine release (
), it was our primary objective to maintain iCa concentrations in IVCa immediately following LPS infusion, and as a result, Ca infusion began within 1 h of the start of LPS challenge in all IVCa cows. While Ca infusion preceded changes in outcomes measured here, differences in iCa concentrations between groups were not detected until 2 h following the start of LPS challenge when CTRL cows had declining concentrations. The amount of total Ca infused to maintain eucalcemia was similar to that in mid-lactation cows at a similar level of production (
), despite 30% lower intake in the postpartum cows studied here.
Research investigating the direct immunomodulatory role of Ca administration in dairy cows is limited, particularly during the critical postpartum period. Using an LPS challenge model that bolus infused 0.5 µg of LPS/kg of BW in late-lactation cows and a eucalcemic clamp to maintain iCa concentration,
showed that eucalcemia was associated with increased body temperature, increased concentration of LPS-binding protein, and delayed recovery of milk production (
). This could suggest an altered inflammatory response with Ca infusion, but cytokine profile was not characterized in their study. As mediators of the acute-phase and clinical response to LPS challenge, we expected a greater pro-inflammatory cytokine response in IVCa animals; however, we did not find evidence that maintaining eucalcemia during immune activation altered the IL-10 or TNF cytokine response here. The lack of an effect of treatment on cytokines measured here may have been related to the time course of Ca infusion and iCa concentrations, as differences in iCa between groups were not apparent until 2 h after the start of LPS infusion, at which time plasma cytokine concentrations were already increased but iCa was not yet decreased in CTRL. Concentrations of the longer-lived and nonspecific markers for systemic inflammation, SAA, and haptoglobin, whose secretion is mediated by pro-inflammatory cytokines (
), also did not differ between groups. Any differences in SAA directly or indirectly induced by maintaining eucalcemia in IVCa were not apparent here or in a similar experiment in mid-lactation cows (
), and likely preceded Ca infusion in the current experiment. Immediate increases in cortisol concentration before elevations in cytokines here and elsewhere (
) by mechanisms independent of the classical hypothalamic-pituitary-adrenal axis. Later cortisol release was likely sustained by cytokines and downstream signaling of the hypothalamic-pituitary-adrenal axis as previously reviewed (
) when iCa differed between groups. Cortisol clearance from circulation by hepatic metabolism would have also occurred when iCa concentrations were greater in IVCa, but it is unknown if cortisol clearance was altered here. Greater cortisol concentration at 6 h in CTRL may have resulted from yet-unknown mechanisms due to hypocalcemia, which may directly or indirectly regulate downstream cortisol release as observed in goats (
Independent of a relationship between cortisol and Ca, cortisol exerts potent anti-inflammatory effects during the systemic inflammatory response by inhibiting the production of pro-inflammatory cytokines and blocking inflammatory processes in which cytokines participate (
). Decreased cortisol concentration in IVCa at 6 h was not accompanied by differences in circulating cytokine concentrations, but IL-10 and TNF cytokine concentrations had nearly returned to baseline before differences in cortisol concentration between groups were apparent. Cortisol is a major glucocorticoid of the stress response and acts to increase hepatic gluconeogenesis and glycogenolysis and NEFA release from adipose tissue (
). In contrast, we observed decreased glucose and NEFA concentrations following challenge. Glucocorticoid-induced glucose release in these early postpartum cows may have been confounded by already high rates of metabolism and depleted hepatic glycogen stores, as well as maximal hepatic gluconeogenic rate to support lactation demands as previously discussed (
). Cortisol also increases respiration and enhances vascular reactivity by increasing the ability of vascular smooth muscle to contract in response to norepinephrine, thereby improving cardiovascular performance (
). Despite decreased cortisol concentration at 6 h in IVCa, we did not observe a difference in heart rate or respiratory rate at that time between groups. Immature neutrophils were not detected in manual differential cell counts for all cows before the start of LPS challenge, but we noted that these cells increased within 4 h, suggesting release from medullary stores or the marginated pool within the early stages of the inflammatory response. Cortisol can stimulate neutrophil release from bone marrow into blood (
). Despite reduced rumen contractions, it is unclear how maintaining eucalcemia would have increased rumination time in IVCa animals in the first 12 h following challenge. Decreased rumination time is detectable in cows experiencing infectious disease (
). Given our experimental protocol, we cannot delineate inflammatory and fasting responses but these would happen concurrently as animals are hypophagic and DMI is decreased following LPS challenge (
), but lying time was not measured here. Animals in CTRL appeared to recover rumination in the last 12 h of the challenge period as total rumination time did not differ between groups in the 24 h following challenge. A similar pattern of recovered rumination in the later hours following LPS challenge was observed in cows intramammary-challenged with LPS (
We previously used a dose of 0.0625 µg of LPS/kg of BW to produce an acute inflammatory response predictable in magnitude and duration in postpartum cows at 8 DIM, but noted a high degree of between-animal variation in the inflammatory cytokine response (
). We elected to use a dose that was 36% lower in the current experiment to reduce between-animal variation in the cytokine response. To avoid concurrent immune activation, our enrollment criteria included only clinically healthy postpartum cows (i.e., that are not already experiencing inflammation due to naturally occurring disease); however, 2 CTRL animals did not demonstrate a cytokine or clinical response to LPS. The reason for a lack of a response is unclear but could be related to the lower dose of LPS or previous exposure to LPS that resulted in a refractory state that has been described in dairy cows following intramammary LPS exposure (
The eucalcemic clamp used in the current study does not directly reflect common Ca therapy employed in clinical settings or on farm, including oral, IV, or subcutaneous Ca during infectious disease in cows. For example, cows are often bolus-infused Ca or given an oral Ca bolus rather than infused over an extended period to maintain eucalcemia. In a previous study in late-lactation cows, oral Ca bolus supplementation before and during IV LPS challenge ameliorated hypocalcemia and improved recovery of animals based on production and intake outcomes (
Effects of an oral supplement containing calcium and live yeast on post-absorptive metabolism, inflammation and production following intravenous lipopolysaccharide infusion in dairy cows.
). Effects of Ca administration on inflammatory and clinical responses may be distinct when supplied orally or through rapid infusion compared with a eucalcemic IV clamp following an LPS challenge, and it remains to be determined conclusively if the potential beneficial effects on intake and production outweigh potential effects on inflammation.
CONCLUSIONS
We did not find evidence that eucalcemia during immune activation altered the metabolic or the inflammatory cytokine response in this early postpartum IV LPS challenge model. It followed that concentrations of systemic acute-phase markers of inflammation did not differ between groups. Maintaining eucalcemia was associated with a more rapid return of cortisol concentration to baseline, and a clinical response as rumination time in the 12 h following challenge was greater in IVCa, but the mechanisms for these effects are not clear. Relative to the discouraged use in other species, we did not find evidence that maintaining eucalcemia following LPS challenge exacerbated the inflammatory response in early postpartum cows in this study; however, it remains to be determined if common Ca administration strategies alter inflammation in early postpartum cows.
ACKNOWLEDGMENTS
This project was supported by the Agriculture Food Research Initiative Postdoctoral Fellowship competitive grant no. 2020-67034-31775 from the USDA National Institute of Food and Agriculture (Washington, DC). Any opinions, findings, conclusions, or recommendations expression in this publication are those of the authors and do not necessarily reflect the view of the National Institute of Food and Agriculture of the USDA. This project was also supported in part by the Research Grants Program in Animal Health, a grant made available to the College of Veterinary Medicine, Cornell University (Ithaca, NY). The authors thank Jessica McArt (Cornell University) for use of the VetScan i-STAT analyzer, and Suzanne Klaessig and Charlene Ryan (Cornell University, Ithaca, NY) for their help during the experiment. We acknowledge the support of the Endocrinology Laboratory of the New York State Animal Health Diagnostic Center (Ithaca, NY). We thank Bettina Wagner (Cornell University) for her expertise in developing and validating the multiplex cytokine assay used in this study. We also thank the staff at the Cornell University Ruminant Center (Harford, NY) for the care of the animals used in this experiment. We thank Zoetis Cattle Division (Parsippany, NJ) for the donation of blank CIDR devices used in this study. The authors have not stated any conflicts of interest.
Effects of an oral supplement containing calcium and live yeast on post-absorptive metabolism, inflammation and production following intravenous lipopolysaccharide infusion in dairy cows.
Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ; concentrations in plasma and cell culture supernatant.
Hypocalcemia induced by immune activation is a conserved response among mammals. Early postpartum cows will experience decreased circulating Ca concentrations following acute immune activation; however, the cause for decreased Ca concentration is unknown. Our objectives were to (1) describe Ca dynamics following an intravenous (IV) LPS challenge in early postpartum cows, and (2) compare inflammatory-induced changes in Ca dynamics between IV Ca-treated cows and control cows. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) in a eucalcemic clamp for 12 h, or 0.9% NaCl (CTRL) following an IV LPS infusion (0.040 or 0.045 µg of LPS/kg of body weight over 1 h).
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