DISCUSSION

Novelty and significance

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 as a model for seasonal Ca homeostasis in conservation-dependent birds

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.

Mechanistic basis of mineral regulation during eggshell formation

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.

Figure 5: Conceptual model describing calcium (Ca) and phosphorus (P) regulation during eggshell formation in captive Houbara bustards. These pathways influence medullary bone mobilization and intestinal Ca absorption. In high-performing layers, coordinated endocrine signaling promotes efficient uterine Ca transport through calbindin and plasma membrane Ca ATPase (PMCA) pathways, resulting in normal eggshell formation. In contrast, soft-shelled egg and retained egg cases may involve a functional dissociation between systemic Ca mobilization and uterine deposition efficiency, leading to elevated circulating Ca concentrations but impaired eggshell mineralization. This conceptual framework suggests that eggshell abnormalities are more closely associated with utilization inefficiency than with Ca deficiency.

Seasonal variation in plasma Ca and P

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].

Mineral changes associated with eggshell defects

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.

Stability of the Ca:P ratio and physiological regulation

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.

Conservation and biological significance

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.