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Background Haematological and clinical biochemistry analyses are essential tools for evaluating the health status of avian species, including the endangered Bonelli’s eagle. Despite their importance, existing reference intervals (RIs) for such parameters in raptor species are frequently constrained by limited sample sizes, thus complicating clinical interpretations. This research followed the American Society for Veterinary Clinical Pathology (ASVCP) guidelines to establish haematological and clinical biochemistry reference intervals for the Bonelli’s eagle, utilizing an indirect approach. Conservation initiatives of the Bonelli’s eagle have provided a unique opportunity to gather a high number of samples under various conditions, facilitating a comprehensive comparison between wild and captive populations. Over the course of six years, from 2016 to 2021, our research analyzed 516 blood samples collected within the framework of a European Life Project. After rigorous data cleaning and stringent selection criteria application, a representative sample of 184 birds was determined. Various physiological parameters and blood lead levels were quantified in healthy individuals. Additionally, we used Principal Component Analysis to discern distinctions between populations and to investigate potential interrelationships among the diverse parameters. Results We established Reference Intervals for three distinct reference groups of birds: wild nestlings ( n = 72 ), captive nestlings ( n = 45 ), and adults ( n = 49 ). Our findings indicate that age significantly affects many blood parameters. Meanwhile, gender impacts only a few parameters in adult birds. Notably, wild nestlings exhibited higher levels of AST, K, TP, and Pb in their blood compared to those bred in captivity. Conclusions This paper provides the first reliable RI for physiological, haematological, and clinical biochemistry parameters in both nestling and adult Bonelli’s eagles. The data will augment the knowledge of the physiology of this endangered raptor, contributing significantly to the understanding and monitoring of both free-living birds and captivity programs.

Radiographs are an important diagnostic tool available in wildlife hospitals to evaluate the size of the avian heart. Despite the large variety wild birds in the Iberian peninsula, clinical studies addressing these species are lacking. To establish reference values for cardiac size in the Bonelli's eagle (Aquila fasciata), ventrodorsal radiographs of 20 healthy birds were obtained, and the width of the cardiac silhouette, sternum, thorax, coracoid, and hepatic silhouette were measured. The ratios between cardiac width and other mentioned indices were calculated. Results showed that cardiac silhouette width should occupy 81%–93% of sternal width, 48%–57% of thoracic width, and 506%–673% of coracoid width. The cardiac silhouette width was strongly correlated with sternal and thoracic widths. A moderate correlation was found between the width of the heart and the width of the coracoid. No significant correlation was found between width of the cardiac silhouette and the hepatic silhouette. These results support that sternal and thoracic widths should be used preferentially when evaluating the width of the cardiac silhouette in this species. The values obtained in this study can be used as a reference for normal cardiac size when evaluating radiographs of Bonelli's eagles.