RESULTS

Plasma Ca, P, and Ca:P ratio outside the breeding season

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).

Comparison of plasma Ca, P, and Ca:P ratio among reproductive groups

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).

Variable | Mean ± SD (mg/dL) | Median [IQR] (mg/dL) | Min–Max (mg/dL) | Normality p-value
Ca | 10.5 ± 0.695 | 10.7 | 8.6–11.8 | 0.006**
P | 3.98 ± 0.945 | 4.0 | 2.0–8.3 | <0.001***
Ca:P ratio | 2.78 ± 0.58 | 2.68 | 1.36–4.6 | 0.013*

Figure 2: Scatterplot displaying the relationship between plasma calcium (Ca; mg/dL) and phosphorus (P; mg/dL) concentrations in adult female Houbara bustards outside the egg-laying season (n = 103). Marginal boxplots illustrate the distribution of each mineral, with boxes representing the IQR, horizontal lines indicating the median, whiskers representing the minimum and maximum, and individual dots indicating outliers.

Group | Variable | Mean ± SD (mg/dL) | Median [IQR] (mg/dL) | Min–Max (mg/dL) | Normality p-value | Suggested analysis
Good layers (n = 70) | Ca | 15.9 ± 5.39 | 13.4 (12.0–18.4) | 7.2–31.3 | <0.001 | Non-parametric
 | P | 4.31 ± 1.14 | 3.9 (3.6–4.8) | 2.1–7.1 | 0.002 | Non-parametric
 | Ca:P | 3.72 ± 0.859 | 3.56 (3.1–4.12) | 2.38–6.27 | 0.001 | Non-parametric
Soft-shelled egg group (n = 26) | Ca | 19.0 ± 4.73 | 18.4 (15.5–21.8) | 11.5–27.5 | 0.322 | Parametric
 | P | 5.0 ± 1.38 | 4.6 (3.95–6.27) | 2.8–7.5 | 0.165 | Parametric
 | Ca:P | 3.89 ± 0.746 | 3.72 (3.38–4.32) | 2.8–5.85 | 0.061 | Borderline normality
Non-laying group (n = 21) | Ca | 15.0 ± 5.63 | 12.7 (11.7–13.9) | 10.9–30.3 | <0.001 | Non-parametric
 | P | 3.94 ± 1.11 | 3.7 (3.3–4.1) | 2.5–6.9 | <0.001 | Non-parametric
 | Ca:P | 3.87 ± 1.23 | 3.46 (3.16–4.04) | 2.66–8.19 | <0.001 | Non-parametric
Retained egg group (n = 19) | Ca | 19.3 ± 5.8 | 18.8 (14.4–23.3) | 11.1–30.9 | 0.428 | Parametric
 | P | 4.92 ± 1.75 | 4.9 (3.85–5.75) | 1.9–8.8 | 0.226 | Parametric
 | Ca:P | 4.23 ± 1.76 | 3.74 (3.09–4.57) | 2.52–10.3 | <0.001 | Non-parametric

Reference intervals for plasma Ca, P, and Ca:P ratio in good layers

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.

Differences among reproductive groups

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).

Post hoc pairwise comparisons

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).

Figure 3: Boxplots representing the distribution of plasma Ca, P, and Ca:P ratio in adult female Houbara bustards outside the egg production season (n = 103) and during the breeding season across four reproductive groups (n = 136). The boxes represent the IQR, horizontal lines indicate median values, whiskers indicate minimum and maximum values, and dots indicate outliers. Ca = Calcium, P = Phosphorus.

Parameter | Median (50 th percentile) (mg/dL) | Reference interval (2.5 th –97.5 th percentile) (mg/dL)
Ca | 13.4 | 10.8–27.8
P | 3.9 | 2.6–6.58
Ca:P | 3.56 | 2.48–5.91

Variable | χ² | p-value
Ca | 16.27 | <0.001***
P | 11.12 | 0.011**
Ca:P | 1.61 | 0.657 (NS)

Variable | Pairwise comparison | W | p-value
Ca | Soft-shelled egg vs. good layer | 4.41 | 0.002**
 | Retained egg vs. good layer | 3.18 | 0.025*
 | Non-laying vs. good layer | −1.48 | 0.296 (NS)
P | Soft-shelled egg vs. good layer | 3.16 | 0.025*
 | Retained egg vs. good layer | 2.08 | 0.141 (NS)
 | Non-laying vs. good layer | −2.31 | 0.103 (NS)

Figure 4: Scatterplot illustrating the relationship between plasma calcium (Ca) and phosphorus (P) concentrations during the breeding season in captive female Houbara bustards (n = 136). Each point represents an individual bird. Colors indicate reproductive groups, including good layers, soft-shelled egg producers, retained egg cases, and non-laying females.

Correlation analysis

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.

Variables | Spearman's rho (r) | p-value | Interpretation
Ca vs. P | 0.690 | <0.001 | Significant positive correlation
Ca vs. soft-shelled eggs | 0.320 | 0.001 | Moderate significant positive correlation
P vs. soft-shelled eggs | 0.229 | 0.025 | Weak significant positive correlation
Ca vs. non-laying status | −0.110 | 0.298 | No significant correlation
P vs. non-laying status | −0.172 | 0.103 | No significant correlation
Ca vs. retained eggs | 0.240 | 0.024 | Weak significant positive correlation
P vs. retained eggs | 0.157 | 0.142 | No significant correlation