INTRODUCTION

The Houbara bustard (Chlamydotis_ undulata_) is a seasonal breeding bird characterized by endocrine cycles extending from late winter through summer. Unlike continuously laying birds such as domestic hens, C. undulata exhibits pronounced reproductive-associated physiological fluctuations, making it an excellent model for investigating adaptive mineral mobilization and reproductive physiology. This species is of considerable conservation concern [1, 2] and is classified as Vulnerable on the International Union for Conservation of Nature Red List, with an estimated global population of approximately 49,000–62,000 individuals. Consequently, extensive captive breeding programs have been established in several countries to reduce the risk of extinction and support population reinforcement and reintroduction efforts. Despite these conservation initiatives, reproductive disorders, particularly eggshell abnormalities and egg retention, remain significant challenges that can compromise breeding success and reduce productivity in conservation breeding programs.

In laying birds, eggshell calcification imposes a substantial physiological demand on calcium (Ca) homeostasis because of the continuous requirement for ionized Ca during shell formation [3]. Ca is the principal structural component of the avian eggshell and is primarily deposited as Ca carbonate, whereas P plays essential roles in energy metabolism, skeletal maintenance, and mineral mobilization associated with eggshell formation [4, 6]. Dietary mineral imbalance, particularly excessive P relative to Ca, can impair Ca utilization and result in thinner, weaker eggshells that are more susceptible to cracking and structural defects [7, 8]. Therefore, assessment of reproductive performance is closely associated with circulating Ca and phosphorus (P) concentrations. In addition, the Ca:P ratio is considered an important indicator of mineral balance and endocrine regulation during eggshell formation in laying birds [5]. Recent evidence has further demonstrated that circadian and hormonal regulation of Ca and P metabolism is critical for synchronizing mineral availability with the oviposition cycle [3].

Limited information is available regarding the seasonal reproductive physiology of C. undulata, although previous studies have documented distinct patterns of breeding activity and associated endocrine changes during the laying period [9]. High-quality eggshells are essential for protecting the developing embryo during incubation and directly influence hatching success [10, 11]. This aspect is particularly important in C. undulata, where reproductive efficiency is a critical determinant of the success of conservation breeding programs [12]. Previous investigations have reported baseline hematologic and biochemical values in Houbara bustards [13], providing useful physiological reference data for health assessment and nutritional management.

Although baseline blood chemistry values have been established for Houbara bustards [13], existing studies have primarily focused on general physiological characterization rather than reproductive performance. To date, no study has investigated the relationship between plasma Ca and P concentrations, the Ca:P ratio, and specific reproductive outcomes in captive female Houbara bustards. Furthermore, the potential associations between mineral profiles and reproductive disorders such as soft-shelled eggs, retained eggs (stuck eggs), and non-laying status remain unknown. The absence of reproductive performance-based reference intervals for Ca and P represents a significant knowledge gap, limiting veterinarians' and conservation managers' ability to use biochemical indicators to early identify females at risk of reproductive failure. Addressing this gap is essential for improving reproductive monitoring, optimizing nutritional management, and enhancing breeding success in conservation programs for this vulnerable species.

We hypothesized that females producing defective eggs would exhibit altered plasma Ca, P, and Ca:P ratios compared with reproductively successful females. Therefore, this study aimed to establish the first reproductive performance-based reference intervals for plasma Ca and P concentrations in captive female Houbara bustards during and outside the breeding season. In addition, the study evaluated variations in the Ca:P ratio among females with different reproductive outcomes, including good layers, soft-shelled egg producers, retained egg cases, and non-laying females. Furthermore, the study investigated associations between plasma mineral profiles and eggshell abnormalities, including soft-shelled, stuck, and malformed eggs, and provided mechanistic insights into mineral homeostasis in a conservation-dependent avian species. Ultimately, the findings are expected to support evidence-based nutritional and management strategies that may reduce eggshell defects, improve reproductive performance, and strengthen the effectiveness of captive breeding programs for C. undulata.