Establishing hematological and biochemical reference intervals in Spix's macaws as tools for environmental and conservation biomonitoring

The Spix's macaw Cyanopsitta spixii, iconic symbol of global avian conservation, is currently classified as "Extinct in the Wild," with only 403 individuals surviving in captivity. As reintroduction programs progress, there is an urgent need for robust physiological baselines capable of contributing to health assessment, detecting environmental stress, and supporting evidence-based conservation interventions. Yet, no comprehensive hematological or biochemical reference values exist for this species, limiting the capacity to monitor its resilience in human-managed and restored environments. This study establishes species-specific reference intervals for hematological and biochemical measurands in 141 clinically healthy C. spixii housed at the Association for the Conservation of Threatened Parrots (Tasdorf, Germany). Analyses followed American Society for Veterinary Clinical Pathology recommendations and incorporated a multivariate framework to quantify the influence of sex, age, and rearing method on physiological variation. Subgroup-specific intervals were generated when covariate effects were significant, improving diagnostic precision and minimizing misinterpretation. The resulting reference values form a foundational dataset for conservation physiology, providing a critical benchmark to evaluate individual condition prior to release, guide adaptive management of captive populations, and enable long-term biomonitoring of individuals exposed to natural environmental pressures after reintroduction. By integrating biological and management-related determinants of variation, this study delivers a comprehensive physiological baseline for C. spixii, offering a key tool to assess environmental stressors, anticipate emerging threats, and strengthen the ecological and health surveillance of one of the world's most endangered bird species.