Experimental Physiology : Translation and Integration Objective, quantitative and practicable measures of stress are pivotal to studies in many branches of vertebrate biology, including human biology, animal husbandry and wildlife ecology (e.g. Dawkins, 1980; Bateson & Bradshaw, 1997; Palme & Möstl, 1997; Creel, 2001; Goymann et al. 2001). The stress response in animals is currently assessed using a variety of techniques, including measurement of cortisol levels (e.g. Beerda et al. 1996; Palme & Möstl, 1997; Harper & Austad, 2000) and haematological values (e.g. Millspaugh et al. 2000), and observations of behaviour (reviewed by Rushen, 2000). Here we present a new method for quantifying stress, based on direct measures of an immune response. We state the benefits of the technique, and discuss the circumstances under which its use is most valuable. The method is based on the ability of individuals to mount a challenge-induced immune response after a defined, potentially stressful event. Each individual’s capacity to respond to immune challenge is compared with its own baseline level of immune system activity. After the putatively stressful event, we measure the capacity of the individual’s leukocytes to produce a quantifiable immune response known as the respiratory burst. During the respiratory burst, oxygen uptake by leukocytes is accelerated in order to produce oxygen free radicals that destroy bacteria (a process reviewed by Halliwell & Gutteridge, 2000). Leukocytes are known to produce oxygen free radicals in response to agonists such as bacterial peptides binding to receptors on their cell membranes (Dietert et al. 1996), the activation of protein kinase C with phorbol myristate acetate (PMA; Hu et al. 1999) and stress (Ellard et al. 2001). It has also been demonstrated that stress affects the respiratory burst: leukocytes isolated from the head kidney of salmon (Salmo salar) showed a reduced respiratory burst (40 % reduction in oxygen free radical production) after the fish were subjected to a 2 h period of confinement stress (Thompson et al. 1993). Therefore, there is evidence that the respiratory burst activity of leukocytes is affected by stress. In particular we wanted to determine whether a reduction in the respiratory burst of circulating leukocytes (which can be measured in whole blood without using isolation techniques) could be used as a measure of stress. In our in vitro challenge–coping approach, after a stressful event, we chemically stimulate a respiratory burst in whole blood in vitro using PMA, and measure the capacity of Leukocyte coping capacity: a novel technique for measuring the stress response in vertebrates G. W. McLaren *, D. W. Macdonald, C. Georgiou †, F. Mathews, C. Newman and R. Mian † Wildlife Conservation Research Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS and †Department of Biomedical Science, School of Science and the Environment, Coventry University, Cox Street, Coventry CV1 5FB, UK (Manuscript resubmitted 26 February 2003; accepted 12 May 2003) Methods used to quantify the stress response in animals are vital tools in many areas of biology. Here we describe a new method of measuring the stress response, which provides rapid results and can be used in the field or laboratory. After a stressful event, we measure the capacity of circulating leukocytes to produce a respiratory burst in vitro in response to challenge by phorbol myristate acetate (PMA). During the respiratory burst leukocytes produce oxygen free radicals, and the level of production can be measured directly as chemiluminescence. When in vitro PMA-stimulated whole blood chemiluminescence is measured directly after a stressful event, we define the response as the leukocyte coping capacity (LCC). In an experiment badgers (Meles meles), which were caught as part of an on-going population study, were either transported to a central site prior to blood sampling or blood was collected at their site of capture. Transported animals had a significantly lower LCC and showed changes in leukocyte composition that were indicative of stress. We conclude that the stress of transport reduced LCC in badgers and that LCC serves as a quantitative measure of stress. Potential applications of this method are discussed. Experimental Physiology (2003) 88.4, 541–546. 2571 Publication of The Physiological Society * Corresponding author: graeme.mclaren@zoo.ox.ac.uk ) by guest on July 14, 2011 ep.physoc.org Downloaded from Exp Physiol (