Postmortem Diagnostic Profile of Laying Hens with Hypocalcemia

Hypocalcemia is one of the leading causes of death in commercial layers in Michigan, USA, which manifests as weakness, lameness, or sudden death of laying hens (1). The hen loses approximately 2 g of calcium in each eggshell, which needs to be replenished daily (2). The major source of calcium comes from intestinal absorption of calcium in the diet, which provides approximately two-thirds of the calcium delivered to the uterus to form the eggshell (3). Recommended daily intake for hens at peak production is 4.35% calcium consisting of a mix of coarse (e.g., oyster shell or large-particle-size limestone) and fine-particle-size calcium (4). The coarse calcium allows for continued intestinal absorption during the night when the hen doesn’t feed but is forming the eggshell (5). Another major source of calcium is from medullary bone. Calcium (and phosphate) is rapidly resorbed from medullary bone, located in the marrow cavity of long bones (e.g., femur or tibia), and provides approximately 30%–40% of the calcium required for daily eggshell formation (3,6). In aged hens, bone resorption of mature cortical bone, which provides the mechanical support of long bones, serves as another source of calcium (6). However, this predisposes to long bone fractures and lameness, commonly referred to as cage layer fatigue.

Calcium (and phosphorus) homeostasis is tightly regulated by parathyroid hormone (PTH) and calcitriol (active form of vitamin D). When ionized calcium is low, the parathyroid gland is stimulated to produce and secrete PTH, which exerts direct effects on bone and kidney, but also stimulates the production of calcitriol, which exerts effects on bone, intestines, and kidney, as follows (7):

The daily egg production by laying hens works like clockwork. Hens lay their eggs 2–6 hr after the lights are turned on, followed by ovulation, 1 hr later (8). Shell calcification begins 5–9 hr post ovulation, takes approximately 18 hr, and occurs overnight (8). Blood levels of ionized calcium, phosphorus, PTH, and calcitriol fluctuate over the course of the 24–25 hr egg laying cycle. Mean concentrations of ionized calcium gradually decrease during eggshell calcification and rebound at the end of calcification, while PTH levels, in response to low blood calcium, peak at the midpoint of eggshell calcification (9). Mean plasma levels of calcitriol and phosphorus follow a similar pattern to PTH levels and progressively increase during eggshell calcification (8). Due to these fluctuations, careful and consistent timing of blood sampling is critical for obtaining meaningful results.

Antemortem diagnosis of hypocalcemia is made based on clinical signs (e.g., weakness, lameness) and improved clinical signs following calcium supplementation (10). Postmortem diagnosis is a diagnosis by exclusion in which the hen has an active ovary, an egg in the eggshell gland, and no lesions to account for its death (1,10). However, without a definitive test for hypocalcemia, other causes of death (infectious, metabolic, toxic) that do not cause overt lesions may be overlooked.

The goals of this study were 1) to develop a hypocalcemia model in laying hens by feeding calcium-altered diets, 2) to validate the model by measuring serum mineral and calcitriol levels, and by histopathology and morphometry of bones, and 3) to quantify macromineral levels in bones to determine cutoff values that discriminate between normocalcemic and hypocalcemic hens.

Forty 35-wk-old white leghorn hens were housed in individual, 22 inch × 24 inch × 16 inch (0.56 × 0.61 × 0.41 m) conventional cages, in rows on two levels, at the Michigan State University (MSU) Poultry Teaching and Research Center. Hens were identified with metal leg bands, acclimatized for 2 wk, fed a standard 4.3% calcium, 0.55% phosphorus, 16.5% protein diet ad libitum (Webberville Feed and Grain Co., Webberville, MI), given water ad libitum, and kept on a 16L:8D lighting program. An exhaust wall fan maintained the temperature between 18 and 27 C (July to October 2022). Inclusion criteria for this study were healthy and in production (laid more than one egg during the experimental period).

Hens were randomly assigned to one of three groups, fed either 6.3% (n = 14), 4.0% (n = 13), or 1.5% (n = 13) calcium diets, ad libitum, starting at 37 wk of age (week 0) until 47 wk of age (week 10) (11). Diets were formulated with similar nutritional values (2940 kcal ME/kg, 0.55% phosphorus, ∼17.5% protein) and prepared by Webberville Feed and Grain Co. (Webberville, MI; Table 1). Diets were analyzed for macromineral (calcium, magnesium, phosphorus, potassium, sodium, sulfur) content. Each formulated diet was homogenized using a Precellys bead homogenizer, and 500 mg was weighed and digested in nitric acid (10× dry sample mass) overnight at 95 C in duplicate (n = 6 replicates per diet). Replicates were analyzed using an Agilent 7900 Inductively Coupled Plasma Mass Spectrometer (ICP-MS), and the mean of the two values, the coefficient of variation, and standard deviation were reported.

Egg count and egg quality were recorded daily between 8:30 AM and 9:30 AM weekdays and 1:00 PM and 2:30 PM on weekends. An ingested egg was defined as the presence of only a partial eggshell in the cage or on the ground. At 0, 5, and 10 wk, hens were examined and weighed, blood was sampled, and eggs were collected. Between 6:00 and 9:00 AM, 1.5–3.0 ml of blood was sampled from the brachial vein starting with 4% group, the 6.3% group, and then the 1.5% group, transferred into serum separator tubes, allowed to clot at room temperature for 30–60 min, and centrifuged for 10 min at 1200 × g. Serum was pipetted into 1.5 ml microcentrifuge tubes and kept at 4 C until analysis. Eggs were weighed, and eggshell thickness was measured by cracking the egg in half, and for each half measuring the shell thickness (including the inner shell membrane) using a digital caliper. The mean thickness was calculated for each shell. Hens were humanely euthanized at 10 wk, and tissue samples were weighed, measured, placed in 10% neutral buffered formalin for histopathology and morphometry, or frozen at −20 C for mineral analysis. Bone weights, lengths, cortical thickness at mid-diaphysis, and relative bone index, calculated as the bone weight (g)/body weight (kg), were measured (11).

Ionized calcium assay was performed on 300 µl of serum by the direct ion selective electrode method using a clinical electrolyte analyzer, Nova Stat Profile® Prime^TM (Nova Biomedical, Waltham, MA) by the endocrinology laboratory at the MSU Veterinary Diagnostic Laboratory (VDL). A calcitriol (1,25-dihydroxyvitamin D) assay was performed on 300 µl of serum by radioimmunoassay (Immunodiagnostics Systems, Boldon, Tyne & Wear, UK) by the endocrinology laboratory at MSU VDL. Phosphorus was analyzed on 100 µl of serum with the Beckman inorganic phosphorus method using a Beckman AU 680 analyzer (Beckman Coulter, Brea, California) by the clinical pathology laboratory at MSU VDL. Hemolyzed serum samples flagged by the analyzer and verified by visual examination were excluded. Parathyroid hormone assay was attempted on serum samples with a chemiluminescent immunoassay (Immunodiagnostics Systems), but the kit antibody did not recognize chicken PTH antigen.

Each right femur and tibia were collected and demuscled, and 1.0-cm-long cross sections were sampled from the mid-diaphysis, and fixed in 10% neutral buffered formalin. One centimeter from the caudal border of the keel was removed, and then 1.0 cm was collected for histopathology (fixed in 10% formalin). Samples of femur, tibia, and keel were decalcified in Decalcifier II (Leica Biosystems Richmond Inc., Richmond, IL) for 3 hr (1.5% group), 6 hr (4.0% group), and 14 hr (6.3% group) prior to sectioning and routine processing. The right thyroid and parathyroid gland were sectioned in half and routinely processed. Five-micrometer-thick sections were routinely stained with hematoxylin and eosin (H&E). Deeper sections were examined if there was insufficient amount of tissue on the first examined section.

QuPath 0.5.1 open-source software was used for morphometry (12). For measuring cortical thickness of femur and tibia, two measurements were taken of the bone cortex at the mid-diaphysis at approximately 90° from one another and averaged. Parathyroid gland size was measured as the area (µm²) in a representative section. Osteoclast number was counted in sections of tibia by manually annotating the number of osteoclasts, defined as a multinucleated (2+ nuclei) cell in contact with the medullary bone surface within a 0.5 mm² grid. The amount of tibial medullary bone was measured using the magic wand tool within a 0.5 mm² grid. Similarly, the amount of mineralized medullary bone in tibia was determined within the same 0.5 mm² grid by selecting for hematoxylin (blue). The amount of unmineralized medullary bone was calculated by subtracting mineralized medullary bone from total medullary bone for each sample. To determine the number of resorption pits and clefts, a semiquantitative scoring system was devised: 0 = no pits or lacunae, 1 = 1–5 pits, 2 = 6–10 pits, 3 > 10 pits or clefts, and evaluated within a representative circumferential length of 5 mm of cortical bone.

For determining mineral concentrations of calcium, phosphorus, magnesium, and manganese, remaining specimens of keel, right femur, and right tibia were frozen at −20 C. Two-centimeter-long samples of proximal femur and tibia diaphysis (just distal to the metaphysis) or 2 cm of the cranial border of the keel were defatted using a Soxhlet extraction method with petroleum ether overnight, dried in a drying oven, pulverized, 500 mg weighed and digested in nitric acid (10× dry sample mass), diluted with water to 50× bone mass, and analyzed on an Agilent 7900 ICP-MS in duplicate. Molarity for each mineral and molar ratio of Ca:P were calculated. These analyses were performed in the Toxicology laboratory at MSU VDL.

Data are presented as means ± SEM. Statistical differences between each diet at each timepoint was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Cutoff values for Ca, P, and Ca:P in samples of keel and tibia were determined using the receiver operating characteristic (ROC) curve, comparing 4.0% diet (control) to 1.5% diet (test). Sensitivity was determined based on the number of values below the established cutoff value; specificity was determined based on the number of values above the established cutoff value. A value of P ≤ 0.05 was considered statistically significant. Analyses were performed using Prism software, v. 6.01 (GraphPad Software, Boston, MA).

Hens fed a 1.5% calcium diet had significantly lower levels of ionized calcium at 5 wk (1.30 ± 0.07 mmol/L; P = 0.027) and 10 wk (1.05 ± 0.06 mmol/L; P = 0.0001) relative to 4.0% (1.43 ± 0.02 mmol/L at 5 wk; 1.31 ± 0.03 mmol/L at 10 wk) and 6.3% (1.43 ± 0.02 mmol/L at 5 wk; 1.29 ± 0.02 mmol/L at 10 wk) groups (Fig. 1A). Levels of phosphorus and calcitriol were mostly similar between groups (Fig. 1B,C).

Postmortem examination revealed parathyroid glands that were enlarged in hens fed 1.5% diet relative to 4.0% and 6.3% diet (Fig. 1D). In normocalcemic hens, parathyroid glands are grossly poorly visible. By morphometry, parathyroid glands were significantly larger (4.12 ± 0.63 mm²) in hens fed 1.5% diet relative to 4.0% (2.14 ± 0.51 mm²) and 6.4% (2.18 ± 0.25 mm²) (P = 0.0139; Fig. 1E). Histologically, enlarged parathyroid glands consisted of hyperplastic chief cells (not shown).

Hen weight was significantly decreased at 5 wk (P = 0.0035) and 10 wk (P = 0.0005) in the 1.5% group (1.4 ± 0.04 kg, 1.39 ± 0.05 kg, respectively) relative to the 4.0% group (1.57 ± 0.03 kg; 1.59 ± 0.03 kg) and 6.3% group (1.62 ± 0.05 kg; 1.65 ± 0.04 kg; Fig. 1F). Food consumption was similar among groups (data not shown).

During the 10-wk period, hens fed 1.5% diet laid an average of 0.54 ± 0.09 eggs daily compared to 0.97 ± 0.01 eggs (4.0% diet) and 0.97 ± 0.01 eggs (6.3% diet; P < 0.0001; Fig. 2A). Weekly egg production was lower starting at week 1 in hens fed 1.5% calcium (5.7 ± 0.60 eggs) relative to 4.0% (6.92 ± 0.14 eggs) and every week thereafter, relative to 4.0% and 6.3% (P = 0.0226; Fig. 2B). One exception was week 7, in which there was no difference in egg production. At 0, 5, and 10 wk, egg weight did not differ between groups (Fig. 2C). Eggshell thickness was different between groups at 10 wk: 0.69 ± 0.01 mm relative to 0.82 ± 0.02 mm and 0.79 ± 0.01 mm (1.5% vs. 4.0% and 6.3%, respectively; P = 0.0004; Fig. 2D). Hens from all three groups laid a low number of soft eggshells every week (Supplemental Table S1). In the 1.5% group, the number of broken eggshells was increased (relative to 4.0% and 6.3%), and only hens from this group ingested their own eggshells (Supplemental Table S1). The clutch size (number of developing ovarian follicles and follicle in the oviduct or shell gland) was significantly lower in 1.5% group (1.78 ± 0.72 yolk) relative to 4.0% (5.23 ± 0.30 yolk) and 6.3% (5.15 ± 0.19 yolk) groups (P < 0.0001).

A total of five hens were excluded from the study. Two hens became lame and were humanely euthanized (at week 1 and 8 from the 1.5% group). Three hens were out of production starting at day 0 and over the course of 10 wk (from the 6.3% and 1.5% groups).

For tibias, there was a significant decrease in mid-diaphyseal cortical thickness in the 1.5% group (362.00 ± 19.10 µm) relative to the 4.0% (474.00 ± 27.46 µm) and the 6.3% (504.30 ± 37.10 µm) groups (P = 0.0055; Fig. 3C). There was an increase in tibial index, calculated as the tibial weight (g)/body weight (kg) in the 1.5% group (8.80 ± 0.40) relative to 4.0% (7.03 ± 0.13) and 6.3% (7.56 ± 0.24) groups (P = 0.0001; Fig. 3F). There was no difference in tibial length or weight (Fig. 3D,E).

For femurs, there was no difference in length, weight, or cortical thickness between groups (Fig. 3I–K). There was an increase in femoral index in the 1.5% group (6.88 ± 0.36) relative to 4.0% (5.55 ± 0.27) and 6.3% (5.64 ± 0.18) groups (P = 0.0036; Fig. 3L).

For tibias, medullary bone from hens fed 1.5% calcium contained a significantly larger amount of total medullary bone (P = 0.0131), consisting of a greater amount of unmineralized osteoid (P = 0.0002) and a lesser amount of mineralized bone (P < 0.0001), relative to 4.0% and 6.3% (Fig. 4C–E). The number of osteoclasts within resorption lacunae was similar between groups (Fig. 4F). Osteoblasts were very attenuated (poorly visible) lining medullary bone trabeculae in hens fed 4.0% and 6.3% diets; osteoblasts were hypertrophied and disorganized in hens fed 1.5% diet (Fig. 4A,B). Using a semiquantitative scoring system, hens fed 1.5% diet had higher scores for resorption pits and clefts relative to 4.0% and 6.3% diets (Fig. 4G).

All hens from the 1.5% group had keel deviations, and 66.7% had evidence of fractures (Supplemental Fig. S1A,B). Deviations were also detected in 4.0% and 6.3% groups. Keel length was lower in hens fed 1.5% diet (9.2 ± 0.19 cm) relative to 4.0% diet (10.19 ± 0.09 cm) and 6.3% diet (10.11 ± 0.15 cm; P < 0.0001; Supplemental Fig. S1C). Keel weight was also lower in 1.5% group (13.88 ± 0.77 g) and 4.0% group (12.82 ± 0.54 g) relative to 6.3% group (16.86 ± 0.85 g; P = 0.0008; Supplemental Fig. S1D). Keel index was higher in 1.5% group (10.15 ± 0.72) and 6.3% (10.23 ± 0.45) relative to 4.0% (8.06 ± 0.31) groups (P = 0.0029; Supplemental Fig. S1E). The intercostal joint of the seventh right rib was markedly thickened, measuring 7.25 ± 0.97 mm, relative to 3.61 ± 0.13 mm and 3.85 ± 0.19 mm (1.5%, vs 4.0% and 6.3%, respectively; P < 0.0001; Supplemental Fig. S1F). Other necropsy findings included remnant cystic right oviduct (n = 2) and a pale tan liver (n = 1).

For tibia, hens fed 1.5% diet had a significant decrease in [Ca] and [P] relative to 4.0% and 6.3% groups ([Ca] was 5.0 ± 0.094 M, 6.33 ± 0.06 M, and 6.39 ± 0.13 M, respectively (P < 0.0001); [P] was 3.08 ± 0.05 M, 3.79 ± 0.034 M, and 3.75 ± 0.11 M (P < 0.0001; Fig. 5A). [Mg] and [Mn] were increased in 4.0% group (1.59 ± 0.026 M and 0.0027 ± 0.00021 M, respectively) relative to 1.5% (0.15 ± 0.0023 M and 0.00031 ± 0.000021 M) and 6.3% (0.15 ± 0.005 M and 0.00025 ± 0.000023 M) groups (P < 0.0001 for both; Fig. 5A).

For keel, hens fed 1.5% diet had a significant decrease in [Ca] and [P] relative to 4.0% and 6.3% groups ([Ca] was 2.85 ± 0.27 M, 4.05 ± 0.25 M, and 4.33 ± 0.16 M, respectively (P = 0.0016); [P] was 1.82 ± 0.17 M, 2.41 ± 0.14 M, and 2.64 ± 0.073 M (P = 0.0028; Fig. 5B). [Ca]:[P] ratio was decreased in 1.5% group (1.56 ± 0.017) relative to 4.0% group (1.68 ± 0.015) and 6.3% group (1.64 ± 0.016; P = 0.0006). [Mg] was similar among groups (P = 0.46). [Mn] was increased in 4.0% and 1.5% groups relative to 6.3% (0.00037 ± 0.000035 M, 0.00046 ± 0.000042 M, and 0.0002 ± 0.000011 M, respectively; P = 0.0004; Fig. 5B).

For femurs, there was no difference among groups for [Ca] (P = 0.39; Fig. 5C). [P] was decreased in the 1.5% group relative to 4.0% and 6.3% groups (3.5 ± 0.082 M, 3.69 ± 0.036 M, and 3.74 ± 0.049 M, respectively; P = 0.035; Fig. 5C). [Mg] and [Mn] were increased in the 1.5% group relative to 4.0% and 6.3% groups ([Mg] was 0.19 ± 0.0072 M, 0.17 ± 0.0026 M, and 0.17 ± 0.0027 M, respectively (P = 0.044; [Mn] was 0.00037 ± 0.000039 M, 0.00021 ± 0.000025 M, and 0.00012 ± 0.000012 M (P = 0.0001; Fig. 5C).

A ROC curve was used to determine cutoff values, with 4.0% group as “normal” and 1.5% group as “test.” For tibia, cutoff values were 5.71 M for Ca (100% sensitivity, 100% specificity), 3.49 M for P (100% sensitivity, 100% specificity), and 0.84 M for Mg (100% sensitivity, 100% specificity). For keel, cutoff values were 2.99 M for Ca (80% sensitivity, 100% specificity), 1.86 M for P (80% sensitivity, 100% specificity), and 1.64 for Ca:P (100% sensitivity, 100% specificity; Supplemental Table S2).

In this study, hens fed the low-calcium diet were in a metabolically reduced state of calcium, which was portrayed by significantly lower ionized blood calcium, enlarged parathyroid glands, decreased egg production and eggshell quality, thinner tibial cortical bone, and an increased amount of unmineralized tibial medullary bone, attributed to decreased intestinal calcium absorption. Mineral analysis of tibia and keel combined with ROC curve analysis determined cutoff values for Ca and P, below which, values were consistent with hypocalcemia.

Blood calcium, phosphorus, parathyroid hormone, and calcitriol varied during the 24–25-hr egg cycle depicting the various stages of egg formation and calcification. At each timepoint (0, 5, 10 wk), blood was sampled starting at 6 AM with the 4.0% group, around the time when eggshell calcification is ending (when calcium is at its lowest in circulation) and ending at 9 AM with the 1.5% group, around the time of oviposition (when calcium rebounds in circulation) (13). A difference in ionized calcium was detected in hens fed 1.5% calcium diet relative to 4.0% and 6.3% at 5 wk and 10 wk. This finding illustrated the effectiveness of the low calcium diet despite the fact that when blood was sampled around 8–9 AM from the 1.5% group, this should correspond to increased serum calcium levels. There was a slightly higher level of phosphorus and calcitriol in the 1.5% group relative to 4.0% group at 10 wk; however, the difference was not of statistical significance. Phosphorus and calcitriol have been found to follow PTH levels, which progressively increase during eggshell calcification in response to decreasing levels of calcium (8). The enlarged parathyroid glands in the 1.5% group can be interpreted as an indirect measure of PTH levels. Histologically, the enlarged parathyroid glands consisted of hyperplastic chief cells, which are the principal cells that synthesize and secrete parathyroid hormone, suggesting that they were proliferating in response to low blood calcium (not shown).

Egg production and eggshell quality were significantly lower in the 1.5% group relative to the 4.0% and 6.3% groups. Weekly egg production started to decline in the 1.5% group already after 1 wk on the low-calcium diet, but this average reflects the decreased egg production by only 4/10 hens. In contrast, 4/10 hens in the 1.5% calcium group maintained a high level of weekly egg production over the course of 10 wk. Interestingly, at 7 wk, 6/10 hens had an increase or peak in weekly egg production. This suggests that over the course of 7 wk, hens became more efficient at mobilizing medullary and cortical bone despite low intestinal calcium absorption. There was an increase in consumption of eggshells in the 1.5% group, which can be attributed to 4/10 hens. While this may have initially been a response to low dietary calcium, once this behavior is started, it becomes a bad habit.

Tibias from the 1.5% group had significant differences in cortical bone thickness, amount of unmineralized medullary bone, and amount of macrominerals in comparison to 4.0% and 6.3% groups. Cortical bone is resorbed when medullary bone cannot supply sufficient calcium for daily egg production (6). In the tibia, but not femur, cortical thickness was decreased after 10 wk on the 1.5% calcium diet, suggesting that tibial cortical bone is more readily labile than femur. Also, femur may play a more important role in weight bearing, which requires maintaining the integrity of cortical bone. Tibias had an increased amount of total medullary bone, which consisted almost exclusively of unmineralized bone in hens fed 1.5% diet. Samples were collected between 9 AM and 3 PM, at which point, hens are actively feeding and calcium is being mobilized to be stored as medullary bone. The histologic appearance of the unmineralized medullary bone indicates that there was insufficient calcium for mineralization to occur. New bone formation is carried out by osteoblasts, which were difficult to evaluate histologically (3). There was a qualitative difference between osteoblasts in the 1.5% group in that osteoblasts were hypertrophied (large and cuboidal) lining bone spicules, indicating active new bone formation, rather than flat with indistinct cell borders (as seen in the 4.0% and 6.3% groups) (3). For this reason, osteoblasts could not be quantified. Bone resorption is primarily carried out by osteoclasts, which produce enzymes that break down mineralized bone and liberate calcium and phosphorus into the blood (6). The time period of collection may explain why there was no difference in the number of osteoclasts between groups given that resorption of mineralized bone does not occur at this time of day (it occurs during eggshell calcification at night) (8). In addition, osteoclasts can only bind to exposed mineralized bone, which was deficient in the 1.5% group. The increased total amount of tibial medullary bone observed in the 1.5% group likely represents a physiological response to maintain a constant bone weight, to compensate for the loss of cortical bone, even though there was insufficient calcium for mineralization of bone. There was an increase in tibia and femur index in the 1.5% group, which reflects the significant decrease in hen body weight rather than a change in bone weight.

Challenges in this study included differences in bone mineralization and preparation of custom diets. Bones from each of the three groups had palpable differences in cortical bone thickness. For this reason, decalcification times were different among groups to avoid excessive decalcification, which results in loss of cellular detail. The diets were initially intended to contain 1.5% (low), 3.0% (low-normal), and 4.3% (normal) calcium but feed analysis revealed that the diets contained 1.5%, 4.0%, and 6.35% calcium. This was likely due to the challenge of mixing small batches of feed (1 ton per diet).

The ultimate goal of the study was to compare amounts of bone macrominerals between hypocalcemic and normocalcemic hens. The large differences in tibial and keel calcium and phosphorus concentrations between the two groups (1.5% and 4.0% calcium diets) allowed us to establish cutoff values, below which, values were consistent with hypocalcemia. In the diagnostic setting, mineral analysis would be indicated only once the other diagnostic criteria were met, including a history of sudden death, presence of an active ovary, an egg in the shell gland, an enlarged parathyroid gland, thin tibial cortical bone, and no other gross or microscopic lesions to account the death of the hen.

This is the first study to establish cutoff values for mineral concentrations in tibia and keel for the purpose of diagnosing hypocalcemia in laying hens postmortem. The combination of clinical history (sudden death in a laying hen), gross findings (an active ovary, egg in the shell gland, thin tibial cortical bone, enlarged parathyroid glands), and concentrations of calcium and phosphorus in keel and/or tibia below the established cutoff values is diagnostic for hypocalcemia. Development of a postmortem diagnostic test for hypocalcemia is critical so that the appropriate interventions can be put into place to prevent further losses to the flock.

Supplemental data associated with this article can be found at https://doi.org/10.1637/aviandiseases-D-24-00043.s1.

Thank you to Herbruck’s Poultry Ranch (Saranac, MI) for generously donating the hens, MSU Poultry Teaching and Research Center, Dr. Megan Crawford and Dr. Katie Bederka for sample collection, and the histology, endocrinology, clinical pathology, and toxicology laboratories at MSU. This project was funded by a grant from the Michigan Alliance for Animal Agriculture (AA-22-0036) and by start-up funds allocated to MFT by the Department of Pathobiology and Diagnostic Investigation at MSU. All procedures were approved by the Institutional Animal Care and Use Committee at MSU (PROTO202200162).

Abbreviations

H&E =

hematoxylin and eosin;

ICP-MS =

Inductively Coupled Plasma Mass Spectrometer;

M =

molar concentration;

MSU =

Michigan State University;

PTH =

parathyroid hormone;

ROC =

receiver operating characteristic;

VDL =

Veterinary Diagnostic Laboratory