153 INTRODUCTION In order to understand behavioural strategies in free-living birds, especially during breeding periods, it is important to be well informed about the energy costs related to various activities such as foraging, parental care, incubation, territory defence and self- maintenance. The calculation of energy budgets from time budgets directly implies the measurement of energy expenditure (EE) associated with each specific activity. Such measurements are challenging to obtain under field conditions, which explains why the vast majority of energy budgets have been estimated from EE measured under laboratory conditions. Unfortunately, such EE estimates often differ widely from those obtained in the field (Flint and Nagy, 1984). During the last decade, numerous studies have estimated field EE from heart rate (f H ) measurements, especially in birds (Woakes and Butler, 1983; Nolet et al., 1992; Bevan et al., 1994; Bevan et al., 1995; Boyd et al., 1999; Ward et al., 2002; Weimerskirch et al., 2002; Green et al., 2003; Fahlman et al., 2004; Froget et al., 2004). The use of f H as a predictor of EE relies on the relationship between the O 2 consumption rate (V O2 ) and f H as formulated in the Fick equation for O 2 convection in the cardiovascular system (Dejours, 1981). The recording of f H provides an accurate correlation of O 2 consumption (Boyd et al., 1995; Butler et al., 2004), and is also a relatively non-invasive technique offering the possibility to monitor metabolism with a fine time-resolution. Precaution is however necessary, as this relationship is species-specific, and may be affected by several factors such as gender (Froget et al., 2001; Green et al., 2001), type and level of activity (Nolet et al., 1992; Butler et al., 2000; Froget et al., 2002), physiological and nutritional state (Froget et al., 2001; Green et al., 2001) and seasonal changes (Holter et al., 1976; Speakman, 1997). The relationship between O 2 consumption and f H , as well as the influence of various factors on this relationship have been investigated in great detail for the king penguin Aptenodytes patagonicus (Froget et al., 2001; Froget et al., 2002; Fahlman et al., 2004). These previous studies, carried out on regularly manipulated captive king penguins, have shown that the relationship slope (i.e. the O 2 pulse, the volume of O 2 used per one heart beat), which depends on body condition (Froget et al., 2001), is three times higher during walking than while resting (Froget et al., 2002). Whether the relationship is affected by gender, however, was not clearly established (Froget et al., 2001). These studies also showed that fasting affects the V O2 /f H relationship during exercise but not during resting periods (Fahlman et al., 2004). For V O2 measurements, birds were kept in a metabolic chamber and forced to walk on a treadmill. As stress-related f H variations do not always correspond to metabolic variations (McPhee et al., 2003), these V O2 /f H relationships may be affected by handling and confinement. Moreover, the ‘HRDDL Loggers’ used to measure f H in previous studies (Woakes et al., 1995), have yet to be validated in king penguins, and a limited number of individuals have been used for calibration in this species. The use of previously established relationships to estimate EE from f H in free-living king penguins The Journal of Experimental Biology 213, 153-160 Published by The Company of Biologists 2010 doi:10.1242/jeb.033720 Heart rate as a predictor of energy expenditure in undisturbed fasting and incubating penguins R. Groscolas 1, *, V. Viera 1,2 , N. Guerin 1 , Y. Handrich 1 and S. D. Côté 2 1 Institut Pluridisciplinaire Hubert Curien, Département d’Ecologie, Physiologie et Ethologie, UMR 7178, 23 rue Becquerel, 67087 Strasbourg Cedex 02, France and 2 Département de Biologie and Centre d’études nordiques, Université Laval, Québec, Québec, Canada G1V 0A6 *Author for correspondence (rene.groscolas@c-strasbourg.fr) Accepted 17 September 2009 SUMMARY Heart rate (f H ) measurement offers the possibility to monitor energy expenditure (EE) in wild animals if the EE/f H relationship for the species, physiological stages and activities of interest is known. This relationship has been extensively studied using oxygen consumption rate (V O 2 ) measurement in captive, repeatedly handled king penguins (Aptenodytes patagonicus). Unfortunately, the potential effects of stress on the observed relationships resulting from handling and confinement were not considered. This study is the first involving undisturbed animals, and determines the EE/f H relationship in naturally fasting and freely incubating or captivity-acclimatized male and female king penguins. EE determination was based on (1) the measurement of body mass loss during periods of phase II fasting, and (2) the calculation of its energy equivalent from changes in body composition, i.e. 23.9 kJ g –1 . f H levels in freely incubating and captivity-acclimatized birds were found to be 50–70% lower than those previously reported for resting king penguins during V O 2 measurements. Significant EE/f H relationships were found in freely incubating and captive males and females (R 2 0.59 to 0.84), with no difference observed between genders. The best overall relationship was obtained by including fasting duration (t, days) in the model: EE818+43.7f H +36.3t–1.4tf H (R 2 0.91). This equation yielded EE estimates approximately 26% higher than the previously reported ‘best’ predictive equation in king penguins, and even more so when f H was low. This result suggests that stress induces a disproportionate increase of f H vs O 2 consumption, and that the use of EE/f H relationships obtained in stressed birds could lead to underestimated EE values. Key words: heart rate, energy expenditure, energy pulse, seabird, fasting, incubation, stress. THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY