Extração de lipídeos por Soxhlet para quantificação de corticosterona em penas e avaliação da variação hormonal entre diferentes penas de Harpia harpyja

Abstract

Corticosterone, the primary glucocorticoid produced by birds, is an essential biomarker for stress assessment in veterinary endocrinology. Monitoring corticosterone in feathers has emerged as a promising alternative for studies on threatened and hard-to-access species, such as the harpy eagle (Harpia harpyja). However, the extraction and analysis of corticosterone from feathers face technical and validation challenges, including uncertainties regarding extraction efficiency, patterns of hormonal deposition, the influence of morphological characteristics, and the interpretation of data as biological and environmental indicators. This study aimed to compare two lipid extraction methods for the quantification of corticosterone in feathers from a captive adult, healthy female Harpia harpyja. In addition, it sought to evaluate intraindividual variation in corticosterone levels among different types of feathers from the same individual. Nineteen feathers—flight and body feathers—collected between July 2024 and March 2025 were analyzed. After preparation and weighing, the samples were subjected to methanol extraction according to each of the tested methodologies, followed by solvent evaporation and reconstitution in phosphate buffer for both. Corticosterone quantification was performed using an enzyme-linked immunosorbent assay (ELISA). The results revealed greater efficiency of the Soxhlet method, indicating that repeated contact of the sample with fresh solvent and the higher processing temperature yield superior hormone recovery. Moreover, the method reduced total extraction time by approximately 50% and proved less costly. Intraindividual analysis revealed variations in corticosterone levels among feathers from different body regions of the same bird. Tertiary/scapular feathers, followed by remiges and rectrices, showed the highest hormone concentrations, whereas head coverts and flank coverts exhibited the lowest, demonstrating that morphological characteristics influence corticosterone incorporation and should be considered when interpreting data. This is the first study to quantify corticosterone in feathers of Harpia harpyja, as well as the first to apply the Soxhlet extraction methodology for this purpose. The superiority of Soxhlet extraction for corticosterone, alongside other additional benefits of the technique, was confirmed. The wide variability in hormonal content among feathers from the same individual suggests the need for sample standardization by size, weight, coloration, and function. The findings of this research broaden methodological perspectives in conservation endocrinology, with particular benefits when dealing with rare samples, such as those from Harpia harpyja.