Ecological Indicators 45 (2014) 522–528 Contents lists available at ScienceDirect Ecological Indicators jo ur nal ho me page: www.elsevier.com/locate/ ecolind Near infrared reflectance spectroscopy (NIRS) for predicting glucocorticoid metabolites in lyophilised and oven-dried faeces of red deer João P.V. Santos a,b,∗ , Joaquín Vicente b , Miriam Villamuelas c , Elena Albanell d , Emmanuel Serrano a,c , João Carvalho a , Carlos Fonseca a,e , Christian Gortázar b , Jorge Ramón López-Olvera c a Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal b Sanidad y Biotecnología (SaBio), Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13071 Ciudad Real, Spain c Servei d’ Ecopatologia de Fauna Salvatge (SEFaS), Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Barcelona, Spain d Grup de Recerca en Remugants, Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Barcelona, Spain e Universidade Lúrio, Campus de Marrere, Nampula, Mozambique a r t i c l e i n f o Article history: Received 29 January 2014 Received in revised form 8 May 2014 Accepted 21 May 2014 Keywords: Cervus elaphus Faecal indicators Glucocorticoid metabolites NIRS Stress a b s t r a c t 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 spec- troscopy (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 labo- ratory 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: R 2 = 0.90, r 2 cv = 0.81, RPD = 2.72; oven-dried: R 2 = 0.88, r 2 cv = 0.79, RPD = 2.26; CV: cross-validation, RPD: ratio of performance to deviation). Nevertheless, oven-dried fae- ces 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 pro- cess. 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 popula- tions 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. © 2014 Elsevier Ltd. All rights reserved. ∗ Corresponding author at: Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. Tel.: +351 234370350; fax: +351 234372587. E-mail address: joaovalente@ua.pt (J.P.V. Santos). http://dx.doi.org/10.1016/j.ecolind.2014.05.021 1470-160X/© 2014 Elsevier Ltd. All rights reserved.