Within-sample variation in snowshoe hare faecal glucocorticoid metabolite measurements.

Diana J R Lafferty,Sarah Whitcher,Alexander V Kumar,L Scott Mills,Klaus Hackländer,Steven Cooke

doi:10.1093/conphys/cox068

Abstract

Faecal glucocorticoid metabolite (FGM) concentrations are used increasingly as a non-invasive measure to index physiological stress experienced by diverse taxa. However, FGM may not be evenly distributed throughout a faecal mass or faecal pellet group. Moreover, within-sample variation in FGM measurements associated with different sampling and/or processing techniques is rarely reported despite potentially having important implications for inferring stress levels in free-ranging wildlife. Using a captive collection of snowshoe hares (Lepus americanus), we (i) assessed repeatability of FGM measurements (i.e. precision) from two processing techniques (measurements derived from dividing whole pellet groups into equal proportions prior to processing [G1], measurements from subsamples derived from thoroughly homogenized whole pellet groups [G2]) and (ii) conducted a power analysis to estimate sample-size requirements for detecting statistically significant differences in FGM concentrations at a population level. Our results indicate that the mean percent coefficient of variation (%CV) for within-sample FGM variation was slightly higher for G1 (%CV = 35, range 13.45-65.37) than for G2 (%CV = 23, range 7.26-47.94), though not statistically significant (two sample t-test, n = 8, t = 1.57, P = 0.16). Thus, FGM is relatively evenly distributed within snowshoe hare faecal pellet groups. However, subsampling from homogenized whole pellet groups may be more appropriate when the sampling time frame is less controlled (e.g. multiple defecation events) because a subsample derived from a homogenized whole pellet group might be more representative of the animal's 'average' physiological state compared to FGM concentrations derived from a few haphazardly selected faecal pellets. Power analysis results demonstrated the importance of a priori consideration of sample sizes. Relatively small effect sizes (e.g. <20%) may require sampling that is logistically and/or cost prohibitive. Yet for many situations of ecological or conservation interest, treatment effects may be substantial (>25%) and thus moderate sample sizes may be sufficient for testing research hypotheses regarding changes FGM concentrations.

Highlights

A major focus of conservation physiology is to identify the causes and consequences of adrenocortical activity in wildlife populations and to determine whether the stress responses of individuals scales up to effect populationlevel health and dynamics
Using a captive collection of snowshoe hares our objectives were to (i) assess the repeatability of Faecal glucocorticoid metabolite (FGM) measurements from two processing techniques (FGM measurements derived from dividing whole pellet groups into equal proportions prior to processing [Group 1 (G1)]; FGM measurements from subsamples derived from thoroughly homogenized whole pellet groups [Group 2 (G2)]) and (ii) conduct a power analysis to estimate sample-size requirements to detect differences (e.g. 10%, 20% and 40%) in FGM concentrations at a population level following a perturbation
Within-sample variation in FGM measurements represented by mean %coefficient of variation (CV) was slightly lower for sample processing technique G2 (%CV 23, range 7.26–47.94) compared to G1 (%CV = 35, range 13.45–65.37), the mean difference was not significant

Summary

Introduction

A major focus of conservation physiology is to identify the causes and consequences of adrenocortical activity (hereafter stress) in wildlife populations and to determine whether the stress responses of individuals scales up to effect populationlevel health and dynamics. Measures of glucocorticoid (GC) hormones (i.e. cortisol or corticosterone depending upon the species) or their metabolites are being used increasingly to index stress in free-ranging and captive wildlife and as a biomarker to infer population-level health (Möstl and Palme, 2002; Millspaugh and Washburn, 2004; Palme et al, 2005; Sheriff et al, 2011a; Goymann, 2012; Dantzer et al, 2014). Two important aspect of study design regarding the use of non-invasive sampling of faeces that have received little attention are the repeatability of FGM measurements from a single sample (i.e. precision) and sample-size requirements needed for being able to detect meaningful differences (e.g. 10%, 20% and 40%) in FGM concentrations at a population level

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Conclusion