Researchers at the University of Veterinary Medicine in Vienna, working with the Agricultural Research and Education Center Raumberg-Gumpenstein in Austria, followed 66 Fleckvieh, Holstein-Friesian, and New Zealand Friesian cows and heifers through 85 calvings between November 2022 and March 2024. Blood samples were collected at a dense series of time points: roughly 21, 14, and 7 days before the expected calving date, just hours before and after parturition, and then at 1, 3, 7, and 14 days postpartum. From each sample, the team measured serum total calcium alongside a panel of osteoimmunological markers, proteins and enzymes that reflect the ongoing dialogue between the immune system and bone tissue.

The central focus was the RANK/RANKL/OPG signaling system, a triad of molecules from the tumor necrosis factor superfamily that governs bone remodeling. RANK ligand, produced by osteoblasts and activated T cells, binds to the RANK receptor on osteoclast precursor cells and drives their differentiation into mature bone-resorbing cells. Osteoprotegerin acts as a soluble decoy receptor, neutralizing RANKL and thereby restraining osteoclast formation. This regulatory axis responds to hormonal and cytokine signals, including interleukin-6, tumor necrosis factor-alpha, and interleukin-17, which are known to shift during the inflammatory events of pregnancy and calving. Because parturition itself is an inflammatory process, involving detachment and expulsion of the fetal membranes, the researchers reasoned that immune activation around calving might perturb bone turnover and influence systemic calcium availability.

To track bone resorption directly, the team measured C-terminal cross-linked telopeptide of type I collagen, or CTX-I, a fragment released when type I collagen is degraded, widely accepted as a sensitive marker of osteoclastic activity in both human and veterinary medicine. They also measured osteocalcin, a marker of osteoblast formation activity, and alkaline phosphatase, an enzyme long used in human medicine to evaluate bone diseases such as osteomalacia and Paget’s disease. After calving, cows were retrospectively classified by their serum calcium concentrations as normocalcemic, above 2.20 millimoles per liter; subclinical hypocalcemic, between 1.80 and 2.20; or low-calcium, below 1.80 millimoles per liter.

The distribution of calcium status revealed how widespread the problem is even in a well-managed research herd. Of the 85 calvings, only about 39 percent remained normocalcemic, while 54 percent fell into the subclinical hypocalcemic range and 7 percent into the low-calcium category. Notably, the burden fell disproportionately on older animals: among cows in their fourth or later lactation, 18 of 27 calvings were subclinical and 5 were frankly low-calcium, whereas most heifers maintained normal calcium concentrations.

CTX-I emerged as the standout marker. Its concentrations rose after parturition, peaked in early lactation, and then gradually declined, consistent with a transient stimulation of bone resorption to meet the demands of lactation. Crucially, first-lactation animals showed significantly higher CTX-I concentrations than multiparous cows at every examination time, and cows that went on to develop low calcium had the lowest CTX-I concentrations at nearly all time points, with significant differences from normocalcemic cows stretching from two weeks before calving through two weeks after. The interpretation is striking: cows that could not mobilize calcium from their skeletons effectively were the ones that became hypocalcemic, and their impaired resorptive capacity was visible in the blood before calving.

The predictive power was quantified using receiver operating characteristic analysis, which evaluates how well a biomarker separates two groups across all possible threshold values. Prepartum CTX-I achieved area under the curve values between 0.75 and 0.90, with Youden indices between 0.75 and 0.82 at several time points. At 14 days before calving, a cut-off of 0.17 nanograms per milliliter yielded 100 percent specificity and roughly 81 percent sensitivity for identifying cows that would later become hypocalcemic. In practical terms, a single blood test in late gestation could flag most at-risk cows while essentially never falsely accusing a cow that would remain healthy. Alkaline phosphatase showed moderate predictive value before calving, and RANK, whose concentrations declined in all parity groups before calving and then remained low, was more useful for tracking postpartum regulatory processes than for prepartum prediction. No reliable models could be built for osteocalcin, RANKL, or osteoprotegerin, likely because RANKL and OPG act predominantly within the local bone microenvironment and are not fully reflected in circulating concentrations.

The parity findings carry their own biological weight. The consistently lower CTX-I concentrations in older cows suggest an age-related decline in bone remodeling, echoing human studies showing that osteoclast precursors become less responsive to RANKL stimulation with age. This diminished capacity to activate osteoclast-mediated calcium mobilization offers a mechanistic explanation for why older cows are far more susceptible to milk fever: when the sudden calcium drain of lactation begins, their skeletons simply cannot respond as vigorously as those of younger animals. Markers of bone formation, by contrast, showed far less pronounced age-related variation, indicating that the parity effect is concentrated on the resorptive side of skeletal metabolism.

The authors are careful to frame the findings appropriately. The low-calcium group contained only six calvings, limiting statistical power, and the study was conducted under specific herd and management conditions without controlled nutritional factors, which are known to influence calcium metabolism. The commercial ELISA kits used may introduce analytical variability, and the observational design precludes causal inference. The proposed cut-off values require validation in independent cohorts before any clinical application, and the researchers explicitly caution that biomarker screening should complement, not replace, established herd-level prevention strategies such as dietary calcium management around calving.

Even with those caveats, the study opens a genuinely new window on an old problem. Milk fever has been managed for decades through nutritional strategies, yet individual risk prediction has remained crude. The demonstration that a bone resorption marker measured weeks before calving can identify cows destined for hypocalcemia suggests that the disease process begins long before the calcium crash, rooted in the skeleton’s diminished ability to respond. If validated at scale, CTX-I testing in late gestation could allow farmers and veterinarians to target preventive interventions precisely at the cows that need them most, turning a metabolic emergency into a manageable prediction problem.

Subject of Research: Osteoimmunological biomarkers for predicting periparturient hypocalcemia in dairy cows

Article Title: Periparturient changes in osteoimmunological markers and their association with hypocalcemia in dairy cows

Article References: Gleissenberger, J., Gleissenberger, M., Becker, S., Schraml, M., Eingang, D., Khol, J. L., & Wittek, T. (2026). Periparturient changes in osteoimmunological markers and their association with hypocalcemia in dairy cows. Journal of Veterinary Internal Medicine, 40(5), Article aalag244. https://doi.org/10.1093/jvimsj/aalag244

Image Credits: AI Generated

DOI: 10.1093/jvimsj/aalag244

Keywords: dairy cows, hypocalcemia, bone resorption, CTX-I, RANK/RANKL/OPG, osteocalcin, alkaline phosphatase, transition period, milk fever, osteoimmunology, biomarkers, veterinary medicine