The study was approved by the Institutional Animal Ethics Committee of the International Foundation for Ecological Research (IFER), Errachidia, Morocco, under approval number IFER-2025-01. All procedures were conducted in accordance with institutional animal welfare guidelines for the handling and management of captive avian species. Blood sampling was performed under the supervision of a licensed veterinarian using minimally invasive procedures to minimize stress, pain, and discomfort. Before sampling, all birds underwent clinical examination, and only clinically healthy adult female Houbara bustards were included. Birds were monitored throughout the study period to ensure their health and welfare, and all husbandry, restraint, and sampling procedures followed established avian welfare and captive breeding protocols.

This study was conducted in 2025 (During breeding season, 15^th May to 15^th June; out of breeding season, 1^st December to 30^th December) at the IFER, Errachidia, Morocco. Sample collection was performed during and outside the breeding season in captive female Houbara bustards (Chlamydotis undulata). All birds were maintained under standardized husbandry conditions within the captive breeding facility.

A cross-sectional study was conducted using 239 blood samples collected from captive female Houbara bustards aged 2–5 years. Of these, 136 samples were obtained during the breeding season, whereas 103 samples were collected outside the breeding season. Sampling was performed during the final third of the breeding season to evaluate mineral profiles in relation to reproductive performance and eggshell quality. The detailed experimental design, group classification, and methodological workflow are illustrated in Figure 1.

Birds sampled during the breeding season were classified into four groups according to reproductive performance and egg characteristics:

1. Good layers (n = 70): Females that produced normal eggs with regular laying intervals.

2. Non-laying group (n = 21): Females of optimal reproductive age that did not produce eggs during the breeding season.

3. Retained egg (stuck egg) group (n = 19): Females diagnosed with egg retention through physical examination and, when necessary, radiographic evaluation.

4. Soft-shelled egg group (n = 26): Females that produced soft-shelled eggs or eggs with shell abnormalities unsuitable for incubation.

Outside the breeding season, samples were collected from clinically healthy females (n = 103) and served as a reference group for comparison with birds during the breeding season.

Blood samples were collected between 8:00 and 10:00 A.M. and within 30 min of capture. Sampling was performed during the latter part of the breeding season, specifically toward the end of the egg-laying period, to minimize disturbance to reproductive activity and reduce stress-related physiological variation. This timing enabled assessment of plasma Ca and P concentrations with minimal impact on egg production.

Before sampling, all birds underwent a clinical examination by a veterinarian and were confirmed to be clinically healthy. Approximately 3 mL of whole blood was collected from the brachial vein into lithium-heparinized blood collection tubes (Vacutest KIMA; KIMA, Arzergrande, Italy) following standard avian hematologic procedures [14]. Samples were subsequently centrifuged at 2,500 × g for 10 min using a bench-top centrifuge (Magafuge 8; Thermo Scientific, Waltham, MA, USA).

Plasma was separated immediately after centrifugation and analyzed for total Ca and P concentrations using a fully automated point-of-care biochemistry analyzer (Catalyst One; IDEXX Laboratories, Westbrook, ME, USA) as previously described for avian species [15, 16]. Subsequent analyses were performed using the Catalyst One system (IDEXX Laboratories).

Ca concentration was determined using a colorimetric method based on the formation of a colored complex with o-cresolphthalein in an alkaline medium. P concentration was measured enzymatically by reacting inorganic phosphate with ammonium molybdate and a reducing agent to produce a colored end product [17]. All samples were analyzed in duplicate to ensure analytical accuracy and reproducibility.

The Ca and P values obtained from the good layer group during the breeding season were used to establish reproductive performance-based reference intervals and to compare birds exhibiting eggshell abnormalities. The interpretation of biochemical variation and the establishment of reference intervals followed established veterinary clinical pathology guidelines [18, 19].

The Catalyst One system was routinely calibrated according to the manufacturer's instructions using IDEXX calibrators. Internal quality control procedures were performed throughout the study to ensure analytical reliability and consistency of results.

All birds were maintained in open housing systems and exposed to natural environmental conditions; therefore, ambient temperature was not experimentally controlled. Each bird was housed individually, with one bird per enclosure. Birds received a balanced breeding diet formulated according to the nutritional requirements of captive Houbara bustards [20]. The diet contained 3.8% Ca and 0.75% P. Vitamin D supplementation was provided through a commercial vitamin–mineral premix incorporated into the diet according to breeding season recommendations.

Management practices were standardized across all study groups. Birds exhibiting clinical signs of disease or infection were excluded from the study to minimize potential confounding effects on biochemical measurements.

The Ca:P ratio was calculated for each sample, and plasma Ca and P concentrations were expressed as mg/dL. Statistical analyses were performed using jamovi software (Version 2.6) [21], which is built on the R statistical environment [22].

Descriptive statistics, including mean, standard deviation, median, IQR, minimum, and maximum values, were calculated for each study variable. Data distribution and potential outliers were evaluated using boxplots and distribution plots. Normality was assessed using the Shapiro–Wilk test for all biochemical variables within each group. Based on the distribution of the data, appropriate statistical tests were selected.

The Kruskal–Wallis test was used to analyze non-normally distributed variables. When significant differences were detected, post hoc pairwise comparisons were performed using the Dwass–Steel–Critchlow–Fligner test with adjusted p-values to control the type I error rate associated with multiple comparisons [23]. Statistical significance was established at p < 0.05.

Adult female Houbara bustards outside the egg production season (n = 103) exhibited a median plasma Ca concentration of 10.7 mg/dL, a median plasma P concentration of 4.0 mg/dL, and a median Ca:P ratio of 2.68. Plasma Ca concentrations ranged from 8.6 to 11.8 mg/dL, whereas plasma P concentrations ranged from 2.0 to 8.3 mg/dL. These findings indicate a relatively stable mineral profile outside the reproductive season and provide valuable baseline reference values for comparison with birds during the breeding season. The observed stability in mineral homeostasis may serve as a useful physiological benchmark for health assessment, nutritional management, and reproductive monitoring in captive female Houbara bustards (Table 1). Spearman correlation analysis demonstrated a significant positive correlation between plasma Ca and P concentrations (r = 0.315, p < 0.001) (Figure 2).

Descriptive statistics for plasma Ca, P, and Ca:P ratio in the good layer, soft-shelled egg, retained egg (stuck egg), and non-laying groups are summarized in Table 2. Higher mean plasma Ca concentrations were observed in the soft-shelled egg and retained egg groups compared with the good layer and non-laying groups. The retained egg group also exhibited the highest mean P concentration and Ca:P ratio. Greater variability in the Ca:P ratio was observed in the retained egg and non-laying groups, as reflected by larger SD values and wider concentration ranges. Median values and IQRs demonstrated higher plasma Ca concentrations in the soft-shelled egg and retained egg groups than in the other groups. The highest plasma P concentrations were observed in the retained egg group. The Ca:P ratio exhibited greater variability and a wider distribution in the retained egg and non-laying groups (Figure 3).

Reference intervals for plasma Ca, P, and Ca:P ratio were established using the good layer group, as summarized in Table 3. The intervals were calculated using the non-parametric 2.5th–97.5th percentile method and may serve as reproductive performance-based reference values for captive female Houbara bustards.

The Kruskal–Wallis test revealed significant differences among reproductive groups for plasma Ca (χ² = 16.27, p < 0.001) and plasma P (χ² = 11.12, p = 0.011). In contrast, the Ca:P ratio did not differ significantly among groups (χ² = 1.61, p = 0.657) (Table 4).

Dwass–Steel–Critchlow–Fligner post hoc analysis confirmed the Kruskal–Wallis results. Plasma Ca concentrations were significantly higher in both the soft-shelled egg and retained egg groups than in the good layer group. Plasma P concentrations were significantly higher in the soft-shelled egg group than in the good layer group (Table 5). Higher plasma Ca and P concentrations were generally observed in the soft-shelled egg and retained egg groups than in the good layer group (Figure 4).

Spearman correlation analysis demonstrated a significant positive correlation between plasma Ca and P concentrations (r = 0.690, p < 0.001) (Table 6). A moderate positive correlation was observed between plasma Ca concentration and the occurrence of soft-shelled eggs (r = 0.320, p = 0.001), whereas plasma P concentration exhibited a weak but significant positive correlation with soft-shelled egg production (r = 0.229, p = 0.025). Plasma Ca concentration also showed a weak but significant positive correlation with retained egg occurrence (r = 0.240, p = 0.024). No significant correlations were detected between mineral concentrations and the non-laying group.

These findings suggest that Ca may play a more prominent role than P in reproductive abnormalities, particularly in soft-shelled egg production and egg retention, although the observed associations were weak to moderate.

This study provides the first comprehensive evaluation of reproductive performance by quantifying plasma Ca, P, and the Ca:P ratio in captive female Houbara bustards (Chlamydotis undulata). Unlike previous studies that were limited to general biochemical characterization, the present work directly links mineral homeostasis with reproductive outcomes, including soft-shelled eggs and retained (stuck) eggs. The findings suggest that eggshell abnormalities are associated with impaired mineral utilization and deposition efficiency rather than simple mineral deficiency. Consequently, this study provides a mechanistic framework with direct relevance to reproductive monitoring, nutritional management, and conservation breeding of endangered avian species.

C. undulata is a valuable model for understanding seasonal Ca and P homeostasis due to its distinct endocrine cycles. Unlike continuously laying birds such as domestic hens, it exhibits pronounced physiological transitions between reproductive and non-reproductive periods, enabling investigation of adaptive mineral mobilization associated with egg production. These characteristics are particularly relevant to conservation breeding programs, including those conducted at IFER, where eggshell quality directly influences reproductive success and hatchability.

Establishing associations between plasma mineral status and reproductive outcomes may help reduce embryo loss and improve breeding efficiency. Furthermore, the findings have practical implications for both captive management and potential in situ conservation strategies. The relatively large dataset used in this study (n = 239, including 136 breeding season birds) further strengthens the utility of the C. undulata as a model species for conservation physiology and reproductive biology.

In avian species, Ca homeostasis during egg formation is tightly regulated through integrated endocrine and cellular mechanisms involving estrogen, PTH, and vitamin D₃ metabolites (Figure 5). Estrogen stimulates the formation of medullary bone, which serves as a readily mobilizable Ca reserve, whereas 1,25-dihydroxyvitamin D₃ enhances intestinal Ca absorption by increasing the expression and activity of Ca-binding proteins such as calbindin-D28k and PMCA transporters in enterocytes and uterine epithelial cells [4, 24].

During eggshell formation, Ca is actively transported across the uterine epithelium into the shell gland lumen through transcellular pathways [25]. This process is energy-dependent and synchronized with circadian mechanisms regulating oviposition. Any impairment in epithelial transporter function or uterine glandular activity may result in a mismatch between systemic Ca availability and eggshell deposition, thereby producing eggshell defects despite adequate or elevated circulating Ca concentrations [26].

In the present study, elevated plasma Ca concentrations in birds producing defective eggs likely reflect sustained medullary bone mobilization under preserved endocrine stimulation but reduced efficiency of uterine incorporation and shell mineralization.

Outside the laying season, plasma Ca and P concentrations were consistent with previously reported reference intervals for adult Houbara bustards [13]. Bailey et al. [12] also reported age-related variations in biochemical parameters among chicks, juveniles, and adults, indicating an association between mineral status and physiological development. The baseline values observed in the present study likely reflect the endocrine quiescence characteristic of seasonal breeders [9], during which Ca homeostasis depends primarily on dietary absorption and skeletal equilibrium, in the absence of the substantial mineral demands of eggshell formation. The relatively narrow variation observed supports the stability of mineral regulation during the non-reproductive period.

In contrast, the laying season was associated with marked increases in circulating Ca concentrations, particularly in females that produced soft-shelled eggs or experienced egg retention. Hypercalcemia during the laying period represents a normal physiological adaptation and is primarily mediated by estrogen-induced medullary bone formation and enhanced intestinal Ca absorption [4, 27]. These mechanisms facilitate rapid mobilization of ionized Ca to meet the substantial demands of shell calcification. Therefore, the elevated Ca concentrations observed in laying Houbara bustards are consistent with physiological adaptation rather than pathological disturbance, in agreement with observations reported in other avian species [28].

Eggshell abnormalities contribute indirectly to reproductive failure because defective eggs are often unsuitable for incubation and may result in embryo loss.

One of the most notable findings of this study was that females producing soft-shelled eggs and females experiencing egg retention exhibited significantly higher plasma Ca concentrations than good layers. This observation challenges the traditional assumption that eggshell abnormalities primarily result from Ca deficiency. Previous studies in commercial laying hens have similarly reported eggshell defects in the presence of hypercalcemia when mineral deposition efficiency within the shell gland is impaired [29, 30].

Eggshell calcification is a highly regulated and time-dependent process occurring within the uterine portion of the oviduct. Successful shell formation depends not only on systemic availability of Ca and P but also on efficient epithelial transport, matrix protein secretion, carbonic anhydrase activity, and precise circadian regulation [3, 31, 32]. Consequently, elevated plasma Ca concentrations in females producing defective eggs may indicate intensified skeletal mobilization exceeding local deposition capacity. Under such circumstances, systemic mobilization remains active, whereas uterine incorporation becomes inefficient, leading to transient accumulation of Ca within the circulation.

This dissociation between mineral mobilization and deposition may result from subtle alterations in uterine physiology, endocrine timing, or stress-related modulation under captive conditions. Although direct assessment of ionized Ca, estrogen concentrations, and uterine transporter expression was beyond the scope of this study, the observed patterns are consistent with reduced deposition efficiency despite preserved systemic mobilization [33, 34].

Plasma P concentrations further support this interpretation. Although group differences were less pronounced than those observed for Ca, females producing soft-shelled eggs exhibited significantly higher plasma P concentrations than good layers. P plays a critical role in bone remodeling and mineral homeostasis [6]. During enhanced mobilization of medullary bone reserves, P is released simultaneously with Ca, reflecting increased skeletal turnover [24, 34, 35]. Therefore, elevated plasma P concentrations may represent a secondary consequence of intensified mineral mobilization rather than an independent pathological process.

Despite significant differences in absolute Ca and P concentrations among reproductive groups, the Ca:P ratio remained relatively stable. In avian species, Ca and P homeostasis is coordinated through integrated endocrine regulation involving PTH, estrogen, and vitamin D metabolites [4, 27]. Maintenance of a relatively stable Ca:P ratio suggests that systemic regulatory mechanisms remain functional despite fluctuations in individual mineral concentrations.

Therefore, the present findings indicate quantitative shifts in circulating mineral concentrations within an otherwise preserved physiological regulatory framework rather than evidence of generalized mineral imbalance. Furthermore, egg retention may prolong estrogenic stimulation and skeletal Ca mobilization beyond the normal period of shell deposition. Continued mobilization without successful oviposition may contribute to sustained elevations in circulating Ca concentrations. Although the precise mechanisms require further investigation, this interpretation is consistent with established models of avian reproductive endocrinology.

From a conservation perspective, reproductive success in captive breeding programs is influenced by numerous interacting physiological, nutritional, environmental, and infectious factors. Diseases, including E. coli infections [36], together with eggshell abnormalities and egg retention, represent important obstacles to successful reproduction in captive C. undulata.

The observed associations between plasma mineral profiles and reproductive outcomes indicate that biochemical monitoring may provide a useful approach for the early identification of females at risk of reproductive failure. Routine assessment of plasma Ca and P concentrations could therefore complement traditional reproductive evaluations and assist in optimizing nutritional and management strategies.

These findings highlight the value of integrating physiological monitoring with reproductive assessment in conservation breeding programs. Such an approach may not only improve productivity in captive populations but also support long-term ex situ conservation efforts for this vulnerable species.