analyser (Sable Systems, Las Vegas, NV, USA) then recorded CO2 content of the sample air. Resting metabolic rate was measured as the rate of carbon dioxide production (V_ CO2 ) and was calculated using Equation 10.5 from Lighton [14]: 1 ð Þ V_ CO2 ~ðFeCO2{FiCO2Þ FR= ð1{FeCO2 ð1{ð1{RERÞÞÞ where FiCO2 and FeCO2 are the concentrations of carbon dioxide flowing into the respirometry chamber and after leaving the chamber, respectively. FR is the flow rate of air into the chamber after mathematical correction for the presence of water vapour (using Eq. 8.6 of Lighton [14]) and RER is respiratory exchange ratio (V_ CO2 :V_ O2 ). It is not clear how resting posture may affect RER since the underlying physiological mechanisms are not fully understood [15]. Therefore, since oxygen consumption (V_ O2 ) was not measured in these experiments, an RER of 0.85 was assumed in order to minimise error in the subsequent metabolic calculation [16]. An RER of 0.85 is also consistent with previous reports of resting metabolism in the barnacle goose (Nolet [17]: 0.77; Nudds [18]: 0.79; P. G. Tickle (unpublished data): 0.87). V_ CO2 was converted to mass specific power (W kg21) using the thermal equivalents in table 12.1 of Brody [19]. (c) Experimental procedure Prior to the trials presented in this paper, geese had been used for separate experiments [13,18]. Consequently, these birds were very familiar with being housed inside the chamber and showed no discernable stress during the sitting and standing experiments. The temperature inside the chamber (20.1uC60.2) was maintained within their thermoneutral zone [20]. Trials were conducted in daylight hours and lasted 121.369.2 minutes (mean 6 SE). By standardising laboratory conditions (temperature, light intensity and humidity) we controlled for factors that can potentially influence choice of posture [9]. Food was not provided within the respirometry chamber. Straw bedding, however, identical to that found in the housing area, was placed inside the respirometry chamber to encourage natural resting behaviour. During each trial the goose was allowed to walk into the respirometry chamber and then left alone in the experimental room. Behaviour was monitored remotely using a webcam connected to a computer that was programmed to take a photograph at 10-second intervals. Goose posture was then established from the photographs and synchronised to the corresponding respirometry data. Steady periods of V_ CO2 stable for at least 180 seconds, after accounting for the time difference (ca. 20 s) between instantaneous behaviour record and detection in the analyser), corresponding to sitting and motionless standing posture were considered representative of resting metabolism. In addition, the different postures were displayed in no particular order. (d) Data analyses Data from a total of 17 trials (each trial represents data from 1 bird) using 10 birds were analysed in this paper, representing a larger sample size than used in previous studies of postural energetics [4,5,6,8]. In all cases (sitting and standing), data was taken from the trace as the 3-minute period with least variation in _ V CO2 . A repeated measures ANOVA was used to test whether posture (sitting or standing) and the individual goose (random factor) affected V_ CO2 . All means are displayed as 6 standard error. The statistical analyses were performed using the statistics toolbox Posture Energetics in MATLABH R2007b (The MathWorks, Inc., 3 Apple Hill Drive, Natick, MA). Results Standing was 25% more metabolically costly than sitting (Fig. 1) a23n=d 9V_.5C4O,2 rw2a=s 0a.l1so1, afpfe=ct0e.d00b5y; tghoeoisned, ivFidu9a,l 2g3o=os5e.6(5p,osrt2ur=e,0.F611,, p,0.001). There was no interaction between goose and posture (F 9, 14 = 0.43, p = 0.899) so this interaction term was removed from the final two-way ANOVA described in the preceding sentence. Converting V_ CO2 to metabolic power yields 5.5060.46 Wkg21 for standing and 4.4160.36 Wkg21 for sitting, both lower than the 6.73 Wkg21 estimated of a zero walking speed (i.e., standing cost) by Nudds et al. [18]. Discussion Here we demonstrate a significant effect of posture upon resting metabolic rate. The observed increase in metabolism associated with standing is consistent with an earlier report in guinea fowl [8], guillemots [6,7] and domestic white leghorn hens [5]. The magnitude of the increase here (25%), however, exceeds the 16% reported for domestic white leghorn hens [5] and 7–9% in guillemots [6,7]. A 25% increase in metabolic costs of standing over sitting was found when the leghorns were not settled and were indulging in extraneous activities (e.g., pecking or fluffing out their feathers). The geese in this study did not perform any auxiliary behaviour when standing in the respirometry chamber. Therefore, it is likely that the increased costs in the geese are due to morphology. Specifically, the barnacle geese have large pectoralis and supracoracoideus muscles to permit sustained flight, equiva- lent to 17.6–17.8% body mass [21]. White leghorn chickens have comparatively smaller flight musculature, accounting for 12.3% of total body mass [22], while guillemot breast muscle equates to 10.4–11.1% of body mass [23]. The extra weight upon the sternum in the goose is likely to increase the cost of respiration when standing, because it must be moved up and down with the sternum during a breathing cycle [12]. The statistical effect of individual goose on the magnitude of postural dependent change in metabolism is intriguing. As the birds were not fasted prior to experimentation it may be that the postprandial energetic costs [19] varied according to previous feeding behaviour [24]. Interspecific analyses often use criteria to ensure common experimental procedures have been implemented across data sources, such as the maintenance of species-specific thermoneutral zones and circadian rhythms, since these factors can affect resting metabolic rate [2,3]. In light of our experiments, careful consideration should be given to how resting posture could influence estimates of basal and resting energy metabolism. Research on terrestrial locomotion in birds has identified a discrepancy between projected resting metabolism, calculated by extrapolating the straight line relationship between speed and metabolism, and measured resting rate [25]. This is assumed to represent the cost of maintaining posture [25]. Our calculations of the resting cost for standing and sitting are both lower than the metabolic rate estimated in Nudds et al [18] (standing is 18.3% lower, sitting is 34.5% lower), indicating a significant metabolic effect of stress and alertness associated with treadmill locomotion. Our results support the hypothesis that experimental stress represents an alternative mechanism to account for the high rate of resting metabolism when derived from walking/running costs [26]. PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46950