Assessment of the Stress Response in Columbian Ground Squirrels: Laboratory and Field Validation of an Enzyme Immunoassay for Fecal Cortisol Metabolites

Abstract

Stress responses play a critical role in the ecology and demography of wild animals, and the analysis of fecal hormone metabolites is a powerful noninvasive method to assess the role of stress. We characterized the metabolites of injected radiolabeled cortisol in the urine and feces of Columbian ground squirrels and validated an enzyme immunoassay for measuring fecal cortisol metabolites (FCM) with a 5 alpha-3beta,11 beta-diol structure by stimulation and suppression of adrenocortical activity and by evaluation of the circadian pattern of FCM excretion. In addition, we also evaluated the impact of capture, handling, and acclimation to the laboratory on FCM. Cortisol is highly metabolized, with virtually none being excreted, and of the radiolabeled cortisol injected, 31% was recovered in urine and 6.5% in feces. The lag time between cortisol injection and its appearance in urine and feces was 4.5 +/- 0.82 (SE) h and 7.0 +/- 0.53 (SE) h, respectively. FCM levels varied over the day, reflecting circadian variation in endogenous cortisol. Dexamethasone decreased FCM levels by 33%, and ACTH increased them by 255%. Trapping and housing initially increased FCM levels and decreased body mass, but these reversed within 3-7 d, indicating acclimation. Finally, FCM levels were modestly repeatable over time (r=0.57) in wild, live trapped, nonbreeding animals, indicating that FCMs provide a measure of the squirrel's stress-axis state. This assay provides a robust noninvasive assessment of the stress response of the Columbian ground squirrel and will facilitate an integration of its life history and physiology.

Excretion and measurement of corticosterone and testosterone metabolites in bank voles (Myodes glareolus)

“…For measuring fecal corticosterone metabolites (FCM) we used a 5α-pregnane-3β,11β,21-triol-20-one EIA, which measures steroid metabolites with 5α-3β,11β-diol structure and was first developed and validated for mice (Mus musculus f. domesticus) (Touma et al 2003, Touma et al 2004. It has been shown to be also suited for rabbits (Oryctolagus cuniculus; Monclús et al 2006), rats (Rattus norvegicus; Lepschy et al 2007), hamsters (Mesocricetus auratus;Chelini et al 2010) and several squirrel species (Bosson et al 2009, Dantzer et al 2010, Bosson et al 2013, Hammond et al 2015. For fecal testosterone metabolites (FTM) we used a testosterone EIA (measuring 17β-hydroxyandrogens) first described by Palme and Möstl (1994).…”

Sex‐specific variation in the onset of reproduction and reproductive trade‐offs in a boreal small mammal

“…For measuring corticosterone metabolites (CM) we used a 5 -pregnane-3 ,11 ,21-triol-20-one EIA, which measures metabolites with 5 -3 ,11 ,-diol structure. It has been shown to be suitable for mice (Mus musculus f. domesticus) , Touma et al 2004, rabbits (Oryctolagus cuniculus) (Monclús et al 2006), rats (Rattus norvegicus) (Lepschy et al 2007), hamsters (Mesocricetus auratus) (Chelini et al 2010) and several squirrel species (Bosson et al 2009, Dantzer et al 2010, Bosson et al 2013 For testosterone metabolites (TM) we used a testosterone EIA, measuring 17ß-hydroxyandrogens, first described by Palme & Möstl (1994). This particular EIA has been validated also for assessing androgen metabolite concentrations in female and male red squirrels (Tamiasciurus hudsonicus) (Dantzer et al 2011a, Dantzer et al 2011b).…”

Excretion of Steroid Hormones in Rodents: An Overview on Species Differences for New Biomedical Animal Research Models

“…Therefore, a non-invasive technique to monitor stress hormones in these animals is highly desirable (Siswanto et al, 2008;Touma et al, 2004;Thanos et al, 2009;Kalliokoski et al, 2010). Likewise, Harper and Austad (2000) developed a noninvasive method for measuring adrenal activity in house mice, deer mice, and red-back voles (members of Muridae and Cricetidae families), as other authors did in old field mice (Cricetidae family; Good et al, 2003), in spiny mice (Acomys cahirinus; Muridade family) (Nováková et al, 2008;Frynta et al, 2009), in agouti and non-agouti deer mice (Peromyscus maniculatus; Cricetidae family) (Hayssen et al, 2002), and in Columbian ground squirrel (Sciuridae family) (Bosson et al, 2009). Similarly, although most of those authors did not perform a previous radioinfusion study, a noninvasive assessment proved to be useful also in different reproductive studies conducted in others small rodents (DeCantanzaro et al, 2003;Kuznetzov et al, 2004;Cavigelli et al, 2005;Chelini et al, 2005).…”

“…Colors of circles are associated with steroids; in addition, circles as well as references were located according to the main route of excretion of each hormone of interest in the studied species, i.e. in Sicurognathi: feces: corticosterone in rat: similar results are revealed by different reports, e.g., Taylor, 1971;Bamberg et al, 2001;Lepschy et al, 2007 andAbelson et al, 2009. Recently, cortisol radioinfusions were informed for squirrel species (Bosson et al, 2009;Dantzer et al, 2010) as well the application of noninvasive monitoring in Cape ground squirrel (Pettitt et al, 2007). In Columbian ground squirrel, this glucocorticoid is highly metabolized, with virtually none being excreted.…”

“…According to the latest publications, taxonomic relatedness does little to predict the precise nature of metabolites or their relative routes of excretion (urine versus feces; Palme et al 2005;Schwanzenberger, 2007). The best way to clarify these questions is to perform radiometabolism experiments and a physiological and biological validation of the immunoassay, such as the work conducted in Columbian ground squirrel (Bosson et al, 2009). However, at present, radioinfusion as an experimental approach to the noninvasive study of adrenal and/or gonadal activity seems "old-fashioned".…”