MATERIAL AND METHODS

Ethical approval

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.

Study period and location

This study was conducted in 2025 (During breeding season, 15th May to 15th June; out of breeding season, 1st December to 30th 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.

Study design

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.

Figure 1: Schematic workflow of the experimental design, sample collection, biochemical profiling, and data analysis in breeding and non-breeding Houbara bustard females.

Group classification

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.

Sample collection

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).

Biochemical analysis

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].

Quality control

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.

Diet and management

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.

Statistical analysis

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.