Near infrared reflectance spectroscopy (NIRS) for predicting glucocorticoid metabolites in lyophilised and oven-dried faeces of red deer

Interest in measuring faecal glucocorticoid metabolites (FGM) as indicators of physiological homeostasis and performance in wildlife is increasing. However, current reference techniques, specifically enzyme immunoassays (EIAs) and radioimmunoassays (RIAs), are expensive, time-consuming, reagent-based, and the samples are destroyed during their application. Conversely, near infrared reflectance spectroscopy (NIRS) is a rapid, reagent-free and non-destructive technique, which, once calibrated by standard laboratory methods, can be used at a low cost. The objectives of this study were to evaluate the feasibility of using NIRS to predict glucocorticoid metabolite concentrations in red deer (Cervus elaphus) faeces, as well as the effect of lyophilisation and oven drying on FGM quantification. Seventy-eight fresh faecal samples were collected directly from the rectum of hunter-harvested red deer and then divided into two equal portions; one portion of each individual sample was lyophilised and the other portion was oven-dried. After dehydration, all faecal samples were ground and then analysed by RIA (standard laboratory technique) and scanned with an NIR spectrophotometer. Modified partial least squares regression was used to generate NIRS calibration equations for both lyophilised and oven-dried samples and a cross-validation procedure was employed for their optimisation. Near infrared reflectance spectroscopy proved to be a feasible, acceptably accurate and reliable technique for predicting FGM concentrations in red deer faeces subjected either to lyophilisation or to oven drying. Calibration and cross-validation results indicated that predictive equations for lyophilised faeces were slightly more precise and robust than for the oven-dried ones (lyophilised: R2 = 0.90, r2 cv = 0.81, RPD = 2.72; oven-dried: R2 = 0.88, r2 cv = 0.79, RPD = 2.26; CV: cross-validation, RPD: ratio of performance to deviation). Nevertheless, oven-dried faeces may be used as an alternative to lyophilised ones to quantify FGM levels accurately, provided that an appropriate combination of dehydration time and temperature is used during the desiccation process. High degrees of association and statistically significant positive correlations (p < 0.001) were found between the lyophilised and oven-dried samples regarding their FGM content, both for RIA assays and NIRS analyses. This study provides a new approach for assessing stress levels in free-ranging populations and has practical implications concerning wildlife monitoring as it makes it possible to improve the efficiency and reduce the cost and time constraints of current analytical techniques.This study was possible thanks to the CGL2009-11631 research project and the Spanish MICINN, and had the support of Santiago Lavín, head of Servei d’Ecopatologia de Fauna Salvatge (SEFaS) from the Universitat Autònoma de Barcelona (UAB). The present work also benefited from the financial aid of the research grant Ministerio de Ciencia e Innovación ref. AGL2010-20730-C02-01. J.P.V. Santos was supported by a PhD Grant (SFRH/BD/65880/2009) from Fundação para a Ciência e a Tecnologia (FCT) co-financed by the European Social Fund POPH-QREN programme and E. Serrano by a Postdoctoral grant (SFRH/BPD/96637/2013) from FCT, Portugal.Peer Reviewe