Abstract
Background
The transition period in dairy cows is associated with major metabolic adaptations, including increased calcium demand and altered bone metabolism. Osteoimmunological markers may reflect these changes and contribute to the prediction of periparturient hypocalcemia.
Hypothesis/Objectives
Evaluate osteoimmunological markers during the periparturient period and their association with parity and hypocalcemia in dairy cows.
Animals
Sixty-six Fleckvieh, Holstein-Friesian, and New Zealand Friesian dairy cows and heifers from a research herd.
Methods
Blood samples were collected at −21, −14, −7, and −0 days before calving, at calving, and at +0, +1, +3, +7, and + 14 days postpartum. Serum concentrations of total calcium, alkaline phosphatase (AP), C-terminal cross-linked telopeptide of type I collagen (CTX-I), osteocalcin (OC), receptor activator of nuclear factor κB (RANK), receptor activator of nuclear factor κB ligand (RANKL), and osteoprotegerin (OPG) were measured. Cows were classified according to postpartum serum calcium concentrations as normocalcemic, subclinical hypocalcemic, or low-calcium. Receiver operating characteristic (ROC) analysis was used to evaluate predictive performance.
Results
The CTX-I concentrations were higher in primiparous and normocalcemic cows compared with low-calcium cows. Activity of AP was increased in normocalcemic cows during early lactation. OC, RANKL, and OPG showed no consistent association with calcium status. Concentration of CTX-I demonstrated high predictive value for hypocalcemia before calving (AUC, 0.75-0.90), whereas AP showed moderate and RANK limited predictive value.
Conclusions and clinical importance
Markers of bone resorption, particularly CTX-I, were associated with calcium homeostasis during the transition period and may help identify cows at risk for periparturient hypocalcemia before clinical manifestations.
Keywords: bone metabolism, CTX-I, RANK, AP, prediction, transition period
Introduction
Pregnancy and parturition in dairy cows are characterized by marked physiological changes affecting overall performance, including metabolic, immune, and skeletal functions. Immunological processes play a central role during this period and are closely linked to bone metabolism, which is increasingly recognized being modulated by immune function.1 This relationship raises the question of whether osteoimmunological interactions contribute to metabolic disorders such as hypocalcemia, which is one of the most prevalent diseases in periparturient dairy cows.2,3 Understanding these interactions could provide new insight into the pathogenesis of hypocalcemia.
In human medicine, immune mediated processes substantially influence bone remodeling in bone-associated diseases (eg, osteoporosis, rheumatoid arthritis).4 Osteoimmunology, the study of the interaction between the immune system and the bone metabolism, is established in human medicine but also has gained some attention in veterinary medicine.1,5 Immunological processes during the periparturient period in cattle have been described to some extent. For example, one study demonstrated a decrease in neutrophil and lymphocyte function around the time of calving.6 In cattle, immune and inflammatory processes play important roles during pregnancy and parturition, because on one hand the fetus must be protected from the immune system of the dam to maintain pregnancy and on the other hand detachment and expulsion of the fetal membranes are inflammatory processes.7 Bovine trophoblast cells express major histocompatibility complex antigens in the third trimester of pregnancy, resulting in inflammatory processes influencing the maturation and later the expulsion of the placenta in cattle.8 An increase in pro-inflammatory cytokine production by peripheral blood mononuclear cells and in the uterus also occur during and after parturition in cattle.9,10
Bone remodeling is influenced by various factors. The receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL), and the osteoprotegerin (OPG) signaling system (RANK/RANKL/OPG), which are 3 key molecules belonging to the tumor necrosis factor (TNF) superfamily, represents one of these systems. RANKL, which is expressed by osteoblasts and activated T-cells, binds to its receptor RANK located on osteoclast precursor cells. Binding stimulates osteoclast differentiation and activity. OPG, a soluble decoy receptor produced by osteoblast-lineage cells, neutralizes RANKL, thereby inhibiting osteoclastogenesis.11 This triad forms the core regulatory mechanism of bone remodeling, balancing resorption and formation, and responds to hormonal and cytokine-mediated signals.4
The periparturient period of cattle is characterized by substantial changes in metabolism and the immune system. These changes may alter the dynamics of bone turnover. Several studies in cattle have documented changes in bone metabolism markers during the periparturient period.1,12–15
One study found that multiparous cows had higher OPG concentrations than primiparous cows approximately 3 weeks before calving, which decreased after calving, suggesting increased osteoclastic activity at this time.13 Concurrently, markers of collagen degradation such as cross-linked carboxyterminal telopeptide of collagen type I (ICTP) increase after calving, suggesting intensified bone resorption. Similarly, C-terminal cross-linked telopeptide of collagen type I or CrossLaps (CTX-I) a specific indicator of type I collagen breakdown, peaks during early lactation and decrease later.16
In contrast, osteocalcin (OC) has been studied less frequently in cattle. Evidence from humans and rodent models suggests that increased OC concentrations occur during lactation and reflect compensatory osteoblast activation.17 However, it is not yet known whether similar adaptations occur in bone physiology of cattle.
The interaction between immune mediators and bone metabolism is influenced by cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-17 (IL-17).18 These cytokines are known to modulate the RANK/RANKL/OPG axis and enhance osteoclastogenesis.19 Given the immune activation and metabolic stress that are characteristic for the periparturient phase, such cytokine-driven mechanisms may contribute to transient imbalances in bone turnover. This situation can influence systemic calcium availability and potentially contribute to periparturient hypocalcemia.13 Also, the cytokine pattern differs between first calf heifers and multiparous cows and might be one reason for the different frequency of periparturient hypocalcemia between those groups.18
In human medicine, alkaline phosphatase (AP) is used to evaluate bone metabolism and diseases such as osteomalacia, rickets, and Paget’s disease.17 Recent research in cattle has investigated the prognostic value of antepartum biochemical variables, including AP and calcium concentrations, for predicting the risk of periparturient hypocalcemia in dairy cows with variable results.20,21
Considered as a whole, the periparturient period is a critical time when bone metabolism and immune function are closely linked. Monitoring changes in CTX-I, OC, RANK, RANKL, and OPG during this period could offer insights into osteoimmune regulation and its impact on calcium homeostasis in dairy cows. We aimed to determine whether these variables are associated with the periparturient stage, thereby improving our understanding of the molecular mechanisms that underlie skeletal adaptation and the risk of metabolic disease during this demanding physiological period.
The following hypotheses were proposed:
Compared to primiparous cows, multiparous cows will show significantly higher variability and different baseline concentrations of osteoimmunological markers (CTX-I, OC, RANK, RANKL, OPG) during the periparturient period, reflecting age-associated changes.
Cows that develop postpartum hypocalcemia will show significantly different periparturient profiles of CTX-I, OC, RANK, RANKL, and OPG compared with normocalcemic cows, indicating dysregulation of osteoclastogenic signaling and bone turnover.
Concentrations of osteoimmunological biomarkers before parturition may be feasible to predict the risk of postpartum hypocalcemia.
Materials and methods
Our study was conducted at the Agricultural Research and Education Center (AREC) Raumberg-Gumpenstein (Irdning-Donnersbachtal, Austria) between November 2022 and March 2024 using the same cohort of cows described previously.22 A total of 85 calvings from 66 Fleckvieh, Holstein-Friesian, and New Zealand Friesian cows and heifers were included. Animals were grouped according to parity (1, 2-3, and ≥4 lactations). Blood samples were collected according to the expected calving date. Sampling time points were retrospectively assigned relative to the recorded time of the actual calving (Table 1). Serum total calcium concentration (tCa), AP, CTX-I, OC, RANK, RANKL, and OPG were measured.
Table 1.
Blood samples and measurement of the laboratory parameters total calcium, C-terminal cross-linked telopeptide of collagen type I, osteocalcin, receptor activator of nuclear factor κ-B, receptor activator of nuclear factor κ-B ligand, osteoprotegerin, alkaline phosphatase.
| Time point | Sampling time |
|---|---|
| −21 | 21.9 ± 5 days before calving |
| −14 | 14.6 ± 4.7 days before calving |
| −7 | 7.8 ± 4.5 days before calving |
| −0 | 2.8 ± 1.6 hours before calving |
| 0 | Calving |
| +0 | 2.8 ± 1.6 hours after calving |
| +1 | 1 day postpartum |
| +3 | 3 days postpartum |
| +7 | 7 days postpartum |
| +14 | 14 days postpartum |
Based on serum tCa concentrations after parturition, cows were retrospectively classified as normocalcemic (>2.20 mmol/L), subclinical hypocalcemic (1.80-2.20 mmol/L), or low-calcium (<1.80 mmol/L).3,22–24 Serum tCa concentration and AP activity were analyzed at a commercial diagnostic laboratory (Laboklin, Linz, Austria).
Commercial enzyme-linked immunosorbent assay (ELISA) kits were used for osteoimmunological markers. CrossLaps was measured using a non-species-specific sandwich ELISA (Serum CrossLaps CTX-I; Immunodiagnostic Systems Ltd., Boldon, UK). Osteocalcin, RANK, RANKL, and OPG were determined using ELISA kits (MyBioSource Inc., San Diego, CA, USA) designed for use in cattle. Analyses were performed according to the manufacturers’ instructions at the Clinical Center for Ruminants and Camelid Medicine, University of Veterinary Medicine, Vienna. Additional assay characteristics are provided in Appendix.
Statistical analyses were performed using R software (version 4.4.1; R Foundation for Statistical Computing, Vienna, Austria). Group differences were assessed using analysis of variance with Bonferroni-adjusted post hoc comparisons. Calcium status and parity group were included as fixed effects, whereas time was treated as a repeated factor. Receiver operating characteristic (ROC) analysis was performed using the cutpointr package to evaluate the predictive value of biomarkers for hypocalcemia. Optimal cut-off values were determined by maximizing Youden’s J statistics.
Results
The analyses included 85 calvings from heifers and cows (20 heifers and 56 cows). Table 2 shows the distribution of the 3 groups according to their serum tCa concentrations.
Table 2.
Distribution of calvings (n = 85) by total serum calcium concentration postpartum (timepoint 0).
| n | % | Lactation number | |||
|---|---|---|---|---|---|
| Total serum calcium concentration | 1 | 2-3 | ≥ 4 | ||
| NC (≥2.2 mmol/L) | 33 | 38.82 | 11 | 18 | 4 |
| SCH (<2.2-1.8 mmol/L) | 46 | 54.12 | 9 | 19 | 18 |
| LC (<1.8 mmol/L) | 6 | 7.06 | 1 | 5 | |
Association of CTX-I, OC, RANK, RANKL, OPG, and AP to the lactation number (age) of the animals.
Normocalcemic (NC), subclinical hypocalcemia (SCH), and low-calcium (LC).22
Concentrations of CTX-I increased after parturition reaching the highest concentrations in early lactation and gradually declined thereafter. First lactation cows generally exhibited higher CTX-I concentrations than multiparous animals at all examination times (P < .001), but the difference between cows of 2 and 3 lactation and older cows (4 and higher lactation number) was not significant (P < .05). Figure 1 shows the serum CTX-I concentrations of cows of different parities.
Figure 1.
C-terminal cross-linked telopeptide of collagen type I (CTX-I, ng/mL) concentrations grouped by lactation number.
Figure 2 shows the OC, RANK, RANKL, and OPG concentrations and AP activity in the serum of cows in different parity groups during the periparturient period. No differences were identified between the parity groups for OC, RANK, RANKL, and OPG. Multiparous cows (≥4 lactations) however exhibited higher overall OC concentrations and had several outliers. No increases or decreases in OC, RANKL, and OPG concentrations were observed during the study period. In contrast, concentrations of RANK decreased in all parity groups before calving (−7) and remained at these concentrations until the end of the study (+14; Figure 2B). First lactation cows had higher AP activities before calving than multiparous cows (P = .001-.04; Figure 2E).
Figure 2.
(A) Osteocalcin (OC) concentrations grouped on lactation numbers. (B) Receptor activator of nuclear factor κB (RANK) concentrations grouped on lactation numbers. (C) Receptor activator of nuclear factor κB (RANKL) concentrations grouped on lactation numbers. (D) Osteoprotegerin (OPG) concentrations grouped on lactation numbers. (E) Alkaline phosphate (AP) activity grouped on lactation numbers.
Low-calcium cows had the lowest CTX-1 concentrations at nearly all timepoints (Figure 3). On days −21 (P = .07), −14 (P = .02), −7 (P = .01), 0 (P = .01), +0 (P = .03), +1 (P = .01), +7 (P = .02), and +14 (P = .03) CTX-1 showed significant differences between the normocalcemic and LC group. After parturition, CTX-I concentrations increased in all 3 groups.
Figure 3.
C-terminal cross-linked telopeptide of collagen type I (CTX-I) concentrations for the groups: normocalcemic, subclinical, and low calcium.
The cows in the SCH and LC groups tended to have lower OC concentrations than normocalcemic cows, but these differences were not significant. Only at day −0 the concentration of OC increased significantly in the LC group (P < .05; Figure 4A). Similar to CTX-I, RANK concentrations were significantly different between the LC and normocalcemic groups on days −14 (P = .03), +0 (P = .003), +1 (P = .02), +7 (P = .03), and +14 (P = .003; Figure 4B). No significant changes in RANKL and OPG were observed during the study period.
Figure 4.
(A) Osteocalcin (OC) concentrations for the groups: normocalcemic, subclinical, and low calcium. (B) Receptor activator of nuclear factor κB (RANK) concentrations for the groups: normocalcemic, subclinical, and low calcium. (C) Receptor activator of nuclear factor κB ligand (RANKL) concentrations for the groups: normocalcemic, subclinical and low calcium. (D) Osteoprotegerin (OPG) concentrations for the groups: normocalcemic, subclinical, and low calcium. (E) Alkaline phosphate (AP) concentrations for the groups: normocalcemic, subclinical, and low calcium.
Significant differences between groups before and around parturition, with higher AP activities, were observed in normocalcemic cows compared with low-calcium cows at −21 (P = .04), −14 (P = .03), −0 (P = .03), 0 (P = .02), +0 (P = .04), and +1 (P = .02; Figure 4E).
Normocalcemic cows had significantly higher CTX-I concentrations at days −21 (P = .07), −14 (P = .02), −7 (P = .01), and 0 (P = .01; Table 3). These time points also had high area under the curve (AUC) values (0.75-0.89) and Youden indices between 0.75 and 0.82, suggesting that the biomarker provides reliable prognostic and diagnostic capacity before and after calving.
Table 3.
Threshold model for predicting hypocalcemia with C-terminal cross-linked telopeptide of collagen type I (CTX-I); specificity, sensitivity, area under the curve, Youden and P-values (P < .05) for all timepoints.
| Timepoint | Specificity | Sensitivity | AUC | Youden | P-value | Cut off (ng/mL) |
|---|---|---|---|---|---|---|
| −21 | 100 | 76.19 | 0.90 | 0.76 | .07 | 0.20 |
| −14 | 100 | 80.95 | 0.89 | 0.81 | .02 | 0.17 |
| −7 | 100 | 75.00 | 0.75 | 0.75 | .01 | 0.19 |
| −0 | 100 | 77.78 | 0.85 | 0.78 | .15 | 0.23 |
| 0 | 100 | 80.00 | 0.88 | 0.80 | .01 | 0.28 |
| +0 | 100 | 80.95 | 0.87 | 0.81 | .03 | 0.31 |
| +1 | 100 | 77.27 | 0.86 | 0.77 | .01 | 0.40 |
| +3 | 83.3 | 77.27 | 0.76 | 0.61 | .08 | 0.51 |
| +7 | 100 | 81.82 | 0.88 | 0.82 | .02 | 0.61 |
| +14 | 66.7 | 72.73 | 0.66 | 0.39 | .031 | 0.61 |
RANK had only limited predictive value for hypocalcemia before parturition, with a significant difference observed only at day −14 (P = .03). In contrast, significant differences between calcium status groups were observed predominantly in the postpartum period (+0, +1, +7, and +14; Table 4).
Table 4.
Threshold model for predicting hypocalcemia with receptor activator of nuclear factor κB (RANK); specificity, sensitivity, area under the curve, Youden and P-values (P < .05) for all time points.
| Timepoint | Specificity | Sensitivity | AUC | Youden | P-value | Cut off (ng/mL) |
|---|---|---|---|---|---|---|
| −21 | 83.33 | 68.18 | 0.67 | 0.52 | .23 | 43.28 |
| −14 | 100 | 50.00 | 0.79 | 0.50 | .03 | 54.05 |
| −7 | 80.00 | 76.19 | 0.74 | 0.56 | .1 | 34.47 |
| −0 | 100 | 50.00 | 0.75 | 0.50 | .14 | 35.47 |
| 0 | 100 | 47.62 | 0.71 | 0.48 | .14 | 37.37 |
| +0 | 100 | 68.18 | 0.89 | 0.68 | .003 | 26.98 |
| +1 | 83.33 | 81.82 | 0.85 | 0.65 | .01 | 16.67 |
| +3 | 66.67 | 77.27 | 0.67 | 0.44 | .24 | 17.39 |
| +7 | 83.33 | 77.27 | 0.79 | 0.61 | .03 | 17.43 |
| +14 | 83.33 | 86.36 | 0.81 | 0.70 | .003 | 17.26 |
No prediction model could be calculated for OC, RANKL, and OPG, because no significant differences were observed among normocalcemic, subclinical, and low-calcium groups for these variables.
Alkaline phosphatase before calving (−21, −14, −0, and 0) showed moderately significant (P-values −21 = .04, −14 = .03, −0 = .03, and 0 = .02) predictive value for hypocalcemia at parturition with specificities between 66.76% and 100% and sensitivities between 63.64 and 85.71%, respectively (Table 5).
Table 5.
Threshold model for predicting hypocalcemia with alkaline phosphate (AP); specificity, sensitivity, area under the curve, Youden and P-values (P < .05) for all time points.
| Timepoint | Specificity | Sensitivity | AUC | Youden | P-value | Cut off (U/L) |
|---|---|---|---|---|---|---|
| −21 | 83.33 | 72.73 | 0.78 | 0.56 | .04 | 64 |
| −14 | 83.33 | 63.64 | 0.78 | 0.47 | .03 | 67 |
| −7 | 100 | 47.62 | 0.74 | 0.48 | .11 | 88 |
| −0 | 100 | 66.67 | 0.85 | 0.67 | .03 | 59 |
| 0 | 66.67 | 85.71 | 0.81 | 0.52 | .02 | 56 |
| +0 | 66.67 | 81.82 | 0.78 | 0.48 | .04 | 61 |
| +1 | 66.67 | 90.91 | 0.82 | 0.58 | .02 | 47 |
| +3 | 66.67 | 50 | 0.75 | 0.17 | .07 | 56 |
| +7 | 50 | 95.45 | 0.62 | 0.45 | .4 | 30 |
| +14 | 50 | 95.45 | 0.65 | 0.45 | .29 | 27 |
Discussion
We investigated the association between osteoimmunological markers and calcium concentrations during the periparturient period in dairy cows.
Parity-related effects
The observed differences in CTX-I concentrations indicate that bone resorption activity decreases with increasing parity. Primiparous cows exhibited higher CTX-I concentrations than multiparous cows, suggesting higher capacity for osteoclast-mediated calcium mobilization in younger cows. In contrast, older cows (≥4th lactation) exhibited consistently lower CTX-I concentrations, indicating decreased ability to activate bone resorption. This finding is consistent with studies in humans demonstrating age-related decreases in bone remodeling and decreased responsiveness of osteoclast precursors to RANKL stimulation.11,19 The decreased CTX-I response in multiparous cows may contribute to their increased susceptibility to periparturient hypocalcemia, because less calcium can be mobilized from the skeleton at calving.
In contrast, markers of bone formation exhibited less pronounced parity-related effects. Although primiparous cows had higher OC concentrations, no significant differences between parity groups were observed. Similar age-related patterns have been described in other species, where OC concentrations decrease with increasing age,25,26 but this effect appears less pronounced in periparturient dairy cows.
Comparable findings were observed for AP, which was higher in heifers before calving but did not differ among parity groups postpartum, consistent with previous reports.18 In addition, OPG did not show any association with parity, supporting earlier findings in dairy cows.27
Taken together, these results suggest that parity-related differences in skeletal metabolism are primarily reflected in markers of bone resorption, whereas markers of bone formation and systemic osteoimmunological regulators appear to be less sensitive to age-related variation during the transition period.
Postpartum calcium concentration-related effects
Significant differences in several osteoimmunological markers were observed among groups of different calcium status, indicating that alterations in calcium homeostasis around parturition are closely linked to changes in bone metabolism.
C-terminal cross-linked telopeptide of type I collagen
C-terminal cross-linked telopeptide of collagen type I is widely accepted as a sensitive marker of bone resorption in both humans and animals.28 The increase in CTX-I concentrations observed after parturition is consistent with previous reports describing a transient stimulation of bone resorption in early lactation.1,13,16 In contrast, decreased bone resorption activity, reflected by lower CTX-I concentrations, has been associated with an increased risk of hypocalcemia in dairy cows.21,27 These findings support the concept that impaired osteoclast-mediated calcium mobilization is a key factor in the development of hypocalcemia.
Receptor activator of nuclear factor κB
Lower RANK availability or altered RANK signaling limits the responsiveness of osteoclast precursors to RANKL stimulation, thereby decreasing bone resorption and calcium release from the skeleton. In humans and experimental animal models, disturbances in this pathway are known to impair bone remodeling and mineral mobilization.19,29 Our data suggest that similar mechanisms operate in dairy cows during the periparturient period.
OC, RANKL, OPG
In contrast to the findings observed for CTX-I, AP, and, to a lesser extent, RANK, no consistent or significant differences between calcium-status groups were identified for OC, RANKL, and OPG. OC, a marker of osteoblastic activity,17 exhibited a modest decrease during the periparturient period across all groups. This finding aligns with the transient suppression of bone formation previously described,13,17 suggesting a physiological shift towards bone resorption to meet the increased calcium demand after calving.3,30
The absence of distinct differences in circulating RANKL and OPG concentrations may be explained by their predominantly local action within the bone microenvironment, which is not fully reflected in systemic measurements.13,19 These findings indicate that markers of bone formation and systemic osteoimmunological regulators are less sensitive indicators of calcium status than markers of bone resorption. This observation is consistent with the results of earlier studies, which indicated that systemic immune-related variables may vary according to parity, but this situation is not necessarily reflected in circulating osteoimmunological markers.18
Alkaline phosphatase
Normocalcemic cows showed higher AP activity than low-calcium cows up to day +7. As a marker of osteoblastic activity and bone turnover, decreased AP activity in low-calcium cows may indicate a generally suppressed skeletal turnover state. Similar results have been reported previously.27
Predictive value of CTX-I, RANK, and AP for periparturient hypocalcemia
The ROC analysis demonstrated that prepartum CTX-I concentrations have high predictive value for periparturient hypocalcemia. Values for AUC ranging from 0.75 to 0.90, in conjunction with high specificity and moderate sensitivity, suggest that CTX-I reliably differentiates between normocalcemic and low-calcium cows before the onset of clinical signs. The hypothesis that impaired osteoclast-mediated bone resorption is a key mechanism underlying hypocalcemia is supported by the consistently lower CTX-I concentrations observed in low-calcium cows. Our findings are consistent with those of earlier studies, which reported analogous predictive performance of bone resorption markers.21
In contrast, RANK is more closely associated with ongoing regulatory processes after calving rather than serving as a reliable prepartum predictor of hypocalcemia risk.
Alkaline phosphatase exhibited moderate predictive capacity before calving, with sensitivity and specificity comparable to previous reports.27 However, variation among studies and inconsistent findings across cohorts suggest that its predictive value may depend on herd-specific and methodological factors. No reliable prediction models could be established for OC, RANKL, or OPG, emphasizing their limited diagnostic value.
Study limitations
Sample size, particularly in the low-calcium group, was relatively small, which may have resulted in limited statistical power and the generalizability of the findings. In addition, the study was conducted under specific herd and management conditions. Nutritional factors influencing calcium metabolism were not controlled in the study setting, which could have affected the results. Furthermore, the discriminatory value of circulating concentrations of osteoimmunological markers, especially RANKL and OPG, may be limited because they do not fully reflect local bone regulatory processes. The use of commercial ELISA kits also may result in analytical variability despite acceptable assay precision. These findings should not be interpreted as suggesting that screening based on biomarkers should replace established herd-level prevention strategies. Instead, osteoimmunological markers could complement current management by improving understanding of the biological mechanisms that underlie susceptibility to hypocalcemia. Finally, the observational design and lack of external validation of the ROC-based models mean that causal inference is not possible, and the proposed cut-off values must be confirmed in independent cohorts.
Conclusions
We demonstrated that periparturient calcium homeostasis in dairy cows is closely linked to bone resorption activity and strongly influenced by parity. Among the investigated osteoimmunological markers, CTX-I emerged as the most robust indicator of skeletal calcium mobilization, showing consistent associations with parity and calcium status and providing the highest predictive accuracy for hypocalcemia before and around parturition. Alkaline phosphatase and RANK showed additional, but weaker, associations with calcium status, whereas OC, RANKL, and OPG displayed limited diagnostic and predictive value. These findings support the concept that impaired activation of bone resorption is a key mechanism underlying periparturient hypocalcemia, particularly in older cows. Measurement of CTX-I and RANK in late gestation therefore may represent a promising tool for early identification of cows at risk of milk fever, although validation in larger, independent populations is required before clinical application.
Acknowledgments
The authors gratefully acknowledge the Agricultural Research and Education Centre Raumberg-Gumpenstein for providing the animals, infrastructure, and technical resources necessary for this study. Open Access Funding by the University of Veterinary Medicine Vienna.
Abbreviations
- AP
alkaline phosphatase
- AREC
Agricultural Research and Education Center
- AUC
area under the curve
- CTX-I
C-terminal cross-linked telopeptide of type I collagen
- CV
coefficient of variation
- ELISA
enzyme-linked immunosorbent assay
- IL
Interleukin
- LC
low-calcium
- OC
Osteocalcin
- OPG
osteoprotegerin
- RANK
receptor activator of nuclear factor κB
- RANKL
receptor activator of nuclear factor κB ligand
- ROC
receiver operating characteristic
- SCH
subclinical hypocalcemia
- tCa
total calcium
- TNF
tumor necrosis factor
Appendix—Detailed ELISA procedures
Serum samples were slowly thawed at room temperature and subsequently stored at 4 °C until analysis within 48 h. Osteoimmunological markers were analyzed at the Clinical Center for Ruminants and Camelid Medicine, University of Veterinary Medicine Vienna, using commercially available ELISA kits according to the manufacturers’ instructions.
CTX-I was measured using the Serum CrossLaps (CTX-I) ELISA kit (Immunodiagnostic Systems Ltd., Boldon, UK; catalog no. AC-02F1). The assay is a sandwich ELISA directed against the dimerized linear octapeptide β(EKAHD-β-GGR) and can be used with serum or plasma. The analytical measurement range was 0.02-2.585 ng/mL. The intra- and interassay coefficients of variation (CV) were 2.4% and 7.7%.
OC concentrations were determined using a bovine-specific sandwich ELISA kit (MyBioSource Inc., San Diego, CA, USA; catalog no. MBS2884640-96). The assay measurement range was extended from 1.25-40.0 ng/mL to 0.6125-40.0 ng/mL because preliminary analyses indicated that several samples were below the lower detection limit. The assay sensitivity was < 0.625 ng/mL.
RANK was measured using a competitive ELISA kit (MyBioSource Inc.; catalog no. MBS7236119). Signal intensity was inversely proportional to the concentration of RANK in the sample. The assay detection limit was 0.1 ng/mL, with intra- and interassay CV of <10% and <12%, respectively.
Receptor activator of nuclear factor RANKL and OPG were measured using bovine ELISA kits (MyBioSource Inc.; catalog nos. MBS2609728 and MBS2609588, respectively). Both assays were based on antibody-mediated colorimetric detection using tetramethylbenzidine substrate. Detection limits were 2000 pg/mL for RANKL and 10 ng/mL for OPG. Intraassay CV were ≤8% and interassay CV were ≤12%.
Contributor Information
Julia Gleissenberger, Clinical Center for Ruminants, Camelids and Herd Health Management, Vetmeduni Vienna, A-1210 Wien, Austria.
Matthias Gleissenberger, Clinical Center for Ruminants, Camelids and Herd Health Management, Vetmeduni Vienna, A-1210 Wien, Austria.
Sebastian Becker, Clinical Center for Ruminants, Camelids and Herd Health Management, Vetmeduni Vienna, A-1210 Wien, Austria.
Manuel Schraml, Clinical Center for Ruminants, Camelids and Herd Health Management, Vetmeduni Vienna, A-1210 Wien, Austria.
Daniel Eingang, Institute of Livestock Research, Agricultural Research and Education Centre Raumberg-Gumpenstein, A-8952 Irdning-Donnersbachtal, Austria.
Johannes L Khol, Clinical Center for Ruminants, Camelids and Herd Health Management, Vetmeduni Vienna, A-1210 Wien, Austria.
Thomas Wittek, Clinical Center for Ruminants, Camelids and Herd Health Management, Vetmeduni Vienna, A-1210 Wien, Austria.
Author contributions
Julia Gleissenberger (Conceptualization, Investigation, Project administration, Visualization, Writing—original draft), Matthias Gleissenberger (Data curation, Formal analysis, Software, Validation, Visualization, Writing—original draft), Sebastian Becker (Investigation), Manuel Schraml (Investigation), Daniel Eingang (Investigation, Resources), Johannes Lorenz Khol (Supervision, Writing—review & editing), and Thomas Wittek (Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing—review & editing)
Conflicts of interest
All authors declare that they do not have any conflicts of interest.
Funding
This project was funded by the Federal Ministry of Agriculture and Forestry, Climate and Environmental Protection, Regions and Water Management via dafne.at. The BMLUK supports applied, problem-oriented research in its area of expertise. Grant number: 101820. Open Access Funding by the University of Veterinary Medicine Vienna.
Off-label antimicrobial declaration
Authors declare no off-label use of antimicrobials.
Institutional animal care and use committee or other approval declaration
The experimental protocols were approved by the institutional ethic committees and by the national authority according to §26 of the Law for Animal Experiments, Tierversuchsgesetz TVG (GZ: 2021-0.744.378).
Human ethics approval declaration
Authors declare human ethics approval was not needed.
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