Comparison of different enzyme-immunoassays for assessment of adrenocortical activity in primates based on fecal analysis

Most studies published to date that used fecal glucocorticoid measurements to assess adrenocortical activity in primate (and many nonprimate) species applied a specific cortisol or corticosterone assay. However, since these native glucocorticoids are virtually absent in the feces of most vertebrates, including primates, the validity of this approach has recently been questioned. Therefore, the overall aim of the present study was to assess the validity of four enzyme-immunoassays (EIAs) using antibodies raised against cortisol, corticosterone, and reduced cortisol metabolites (two group-specific antibodies) for assessing adrenocortical activity using fecal glucocorticoid metabolite (GCM) measurements in selected primate species (marmoset, long-tailed macaque, Barbary macaque, chimpanzee, and gorilla). Using physiological stimulation of the hypothalamo-pituitary-adrenocortical (HPA) axis by administering exogenous ACTH or anesthesia, we demonstrated that at least two assays detected the predicted increase in fecal GCM levels in response to treatment in each species. However, the magnitude of response varied between assays and species, and no one assay was applicable to all species. While the corticosterone assay generally was of only limited suitability for assessing glucocorticoid output, the specific cortisol assay was valuable for those species that (according to high-performance liquid chromatography (HPLC) analysis data) excreted clearly detectable amounts of authentic cortisol into the feces. In contrast, in species in which cortisol was virtually absent in the feces, group-specific assays provided a much stronger signal, and these assays also performed well in the other primate species tested (except the marmoset). Collectively, the data suggest that the reliability of a given fecal glucocorticoid assay in reflecting activity of the HPA axis in primates clearly depends on the species in question. Although to date there is no single assay system that can be used successfully across species, our data suggest that group-specific assays have a high potential for cross-species application. Nevertheless, regardless of which GC antibody is chosen, our study clearly reinforces the necessity of appropriately validating the respective assay system before it is used.     

A validation of extraction methods for noninvasive sampling of glucocorticoids in free-living ground squirrels

Fecal hormone assays provide a powerful tool for noninvasive monitoring of endocrine status in wild animals. In this study we validated a protocol for extracting and measuring glucocorticoids in free-living and captive Belding's ground squirrels (Spermophilus beldingi). We first compared two commonly used extraction protocols to determine which performed better with commercially available antibodies. We next verified the preferred extraction method by correlating circulating and fecal glucocorticoid measures from a group of individuals over time. For this comparison, we used both a cortisol and a corticosterone antibody to determine which had greater affinity to the fecal metabolites. Cortisol was the primary circulating glucocorticoid, but both hormones were present in well above detectable concentrations in the blood, which does not occur in other sciurids. In addition, the cortisol antibody showed greater binding with the fecal extracts than did the corticosterone antibody. Finally, we used adrenocorticotropic hormone and dexamethasone challenges to demonstrate that changes in adrenal functioning are reflected in changing fecal corticoid levels. These results suggest that our extraction protocol provides a fast, reliable assay of stress hormones in free-living ground squirrels without the confounding influence of short-term rises in glucocorticoid concentrations caused by handling and restraint stress and that it can facilitate ecological and evolutionary studies of stress in wild species.     

Determination of Fecal Glucocorticoid Metabolites to Evaluate Stress Response in <Emphasis Type="Italic">Alouatta pigra</Emphasis>

Measuring fecal glucocorticoid metabolites is now a common practice to assess the stress response in primates. Nevertheless, it is important to validate the utilized immunoassay for each primate species before the technique is applied to populations in the wild. We determined the stress response of black howlers (Alouatta pigra) via 2 different group-specific enzyme immunoassays (EIAs). 11-oxoetiocholanolone EIAs are suited to assess the stress response of black howlers via fecal glucocorticoid metabolites. Levels of fecal glucocorticoid metabolites increased after we applied a stressor, i.e. anesthesia, reaching peak concentrations 24–96 h poststressor. Both basal and stress-induced fecal glucocorticoid metabolite levels showed individual variations. The increase of fecal glucocorticoid metabolites after the stressor (paralleling increases in serum) indicates that one can effectively measure adrenocortical activity in Alouatta pigra via these 2 enzyme immunoassays. However, it is important to consider individual variations in the excretion of fecal glucocorticoid metabolites when planning field endocrinological research on Alouatta pigra. Fecal glucocorticoid metabolite excretion takes 1–3 d poststressor depending on the individual. Further, there is an important individual variability in the concentrations of glucocorticoid metabolites, which might reflect differences in stress reactivity or fecal glucocorticoid metabolite metabolism and excretion.     

Non-invasive monitoring of adrenocortical activity in free-ranging fallow deer (<Emphasis Type="Italic">Dama dama</Emphasis> L.)

Measurement of faecal glucocorticoid metabolites is increasingly used as a non-invasive tool to examine disturbances in various domestic and wild animals. Because measurements of faecal glucocorticoid metabolites has previously never been reported in fallow deer, we determined 11,17-dioxoandrostanes (11,17-DOA), a group of cortisol metabolites, in the faeces of four fallow deer yearlings after an adrenocorticotropic hormone (ACTH) challenge or control saline injection by an 11-oxoaetiocholanolone enzyme immunoassay (EIA), to validate a method. A 2.9- to 4.3-fold increase in measured cortisol metabolites in challenged animals after approximately 22 h demonstrated the suitability of this group-specific EIA to monitor adrenocortical activity in respective deer species. To determine faecal cortisol metabolites in fallow deer from a Mediterranean habitat, we collected samples during a 1-year study at Veliki Brijuni Island. The study confirmed seasonal pattern of cortisol release in fallow deer. Higher 11,17-DOA concentrations (median; min–max) were determined for November (99; 50–2,035), March (112; 25–315) and May (92; 40–196 ng/g faeces). Significantly lower concentrations were measured during July (30; 10–195 ng/g faeces). This study indicates that the analysis of faecal glucocorticoid metabolites is a valuable non-invasive technique for monitoring adrenocortical activity in fallow deer. This, together with information about the seasonal pattern of glucocorticoid excretion, could help to improve fallow deer management and welfare, especially in the case of farmed and park animals.     

Validation of a Fecal Glucocorticoid Metabolite Assay to Assess Stress in the Budgerigar (Melopsittacus undulatus)

The budgerigar (Melopsittacus undulatus) is a small parrot native to Australia that is commonly held in zoos, laboratories, and private homes. Assessment of budgerigar stress levels would aid welfare monitoring and improve our understanding of their biology. Analyzing fecal glucocorticoid metabolites provides a noninvasive method to measure stress levels in birds. For this method to be reliable, the antibody to be used in an immunoassay must be carefully selected for each species, and validation must be performed. A common limitation in many existing assays is the inability to accurately detect variable fecal glucocorticoid metabolites in minute quantities of feces, requiring small samples to be combined. We have developed a double antibody radioimmunoassay protocol based on a commercially available ¹²⁵I‐corticosterone radioimmunoassay kit for use in detecting fecal glucocorticoid metabolites in small quantities (<20 mg) of budgerigar droppings. The assay was validated pharmacologically with an adrenocorticotropic hormone challenge and with oral administration of corticosterone. Our validation has demonstrated our assay is both sensitive and a reliable approach to noninvasive monitoring of stress in budgerigars. Zoo Biol. 32:112‐116, 2013. © 2012 Wiley Periodicals, Inc.     

Methodological Considerations in the Analysis of Fecal Glucocorticoid Metabolites in Tufted Capuchins (Cebus apella)

Analysis of fecal glucocorticoid (GC) metabolites has recently become the standard method to monitor adrenocortical activity in primates noninvasively. However, given variation in the production, metabolism, and excretion of GCs across species and even between sexes, there are no standard methods that are universally applicable. In particular, it is important to validate assays intended to measure GC production, test extraction and storage procedures, and consider the time course of GC metabolite excretion relative to the production and circulation of the native hormones. This study examines these four methodological aspects of fecal GC metabolite analysis in tufted capuchins (Cebus apella). Specifically, we conducted an adrenocorticotrophic hormone (ACTH) challenge on one male and one female capuchin to test the validity of four GC enzyme immunoassays (EIAs) and document the time course characterizing GC metabolite excretion in this species. In addition, we compare a common field-friendly technique for extracting fecal GC metabolites to an established laboratory extraction methodology and test for effects of storing “field extracts” for up to 1 yr. Results suggest that a corticosterone EIA is most sensitive to changes in GC production, provides reliable measures when extracted according to the field method, and measures GC metabolites which remain highly stable after even 12 mo of storage. Further, the time course of GC metabolite excretion is shorter than that described yet for any primate taxa. These results provide guidelines for studies of GCs in tufted capuchins, and underscore the importance of validating methods for fecal hormone analysis for each species of interest.     

Noninvasive analysis of fecal reproductive hormone metabolites in female veiled chameleons (Chamaeleo calyptratus) by enzyme immunoassay

The noninvasive technique of gonadal steroid metabolite measurement in feces for evaluation of reproductive activity has proven an effective and important tool for population management in various captive species, but has not yet been validated and used in reptile species. In this study, enzyme immunoassays (EIAs) were validated for the analysis of fecal samples from female veiled chameleons (Chamaeleo calyptratus) for estrogen (E2), testosterone (T), and progesterone (P) and their metabolites. High performance liquid chromatography and physiological methods (GnRH stimulation) were used for the validation of the assays. Biological events, such as skin color changes indicative of ovarian activity and oviposition, correlated with the cyclical pattern of E2, T and P metabolites in feces over a period of two reproductive cycles. This is the first study to report frequent longitudinal measurements of fecal hormone levels by EIA in a reptile species.     

Stability of Avian Fecal Corticosterone Metabolite Levels in Frozen Avian Feces

ABSTRACT Fecal corticosterone metabolites are commonly used in avian ecology as a measure of response to stress. Recent research on mammals suggested that the manner in which samples are stored could be critical to alleviating any storage handling bias. Cross-reacting metabolites can increase glucocorticoid metabolites even after samples are frozen and, thus, result in an overestimation of hormone levels as the time increases between when samples were collected and when levels are measured. We examined effects of sample storage time on fecal corticosterone metabolites for 2 avian species across 165 days. We observed no change in fecal corticosterone metabolites across the sampling periods in either fulvous whistling-ducks (Dendrocygna bicolor) or white ibis (Eudocimus albus). Results suggest that avian fecal corticosterone metabolite levels do not change when samples are frozen for long periods of time and that there were no differences in the response between the 2 species we compared. This study demonstrated that avian fecal corticosterone samples are accurate even after freezing and, thus, studies that seek to address conservation questions may rely on these data. Studies of additional bird species are needed to generalize our findings to other avian taxa.     

Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group-specific enzyme immunoassays for 20-oxo-pregnanes

The metabolism and excretion of progesterone in different animal species results in several fecal 5-reduced progesterone metabolites (pregnanes), which in recent studies were quantified using progesterone antibodies. To increase the accuracy of fecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immunoassays (EIA) using antibodies against 5α-pregnane-3β-ol-20-one 3HS:BSA (5α-20-one EIA) and 5β-pregnane-3α-ol-20-one 3HS:BSA (5β-20-one EIA) were developed. The assays showed high crossreactivities with pregnanes containing a 20-oxo group and are referred to as group-specific; results of these assays were compared with an EIA using an antibody against 6HS-progesterone (4-ene-20-one EIA). Fecal samples of both subspecies of the black rhinoceros (Diceros bicornis michaeli, n = 5, and Diceros bicornis minor, n = 1) during pregnancy were collected 1–3 times/week. HPLC separation showed three major immunoreactive fecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivities in the three EIAs provided strong evidence that these peaks are 5α-pregnane-3, 20-dione, 5α-pregnane-3α-ol-20-one, and 5α-pregnane-3β-ol-20-one. Pregnane values in the pregnant animals continuously increased between months 3–7 and were significantly (P < 0.01) elevated above the levels of nonpregnant animals (0.2 μg/g) by week 11. During months 6–13 concentrations in the 5α-20-one and in the 5β-20-one EIA (5–11 μg/g) were significantly (P < 0.01) higher than in the 4-ene-20-one EIA (1.5–3 μg/g). In conclusion, the immunoreactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determined more accurately with antibodies against pregnane-20-one-C3 conjugates, as compared with a progesterone antibody. © 1996 Wiley-Liss, Inc.     

Seasonal dynamics of agonistic behavior and hormones in an ex situ all‐male colony of large flying foxes

Large flying foxes (Pteropus vampyrus) are a socially complex species. In situ colonies typically comprise thousands of individuals in small harems of one male to many females. In ex situ environments, all‐male colonies are becoming more common due to a surplus of males in the population. There is limited information describing the hormonal and behavioral patterns of all‐male colonies during the breeding season. We assessed seasonal changes in hormones and behavior in an all‐male colony of 12 large flying foxes at Disney's Animal Kingdom^®. We validated hormone assays using morning urine and fecal samples to assess seasonal changes in excreted immunoreactive testosterone and glucocorticoid metabolites. We collected behavior data using an all‐occurrence method, recording agonistic behaviors related to territorial defense (hooking, biting, wing flexing, vocalizing, and wrestling), and sexual behavior (mounting and frontal grabbing). Results indicated that (i) we could reliably measure testosterone and glucocorticoid metabolites concentrations from fecal and urine samples collected from individual bats; (ii) there were distinct relationships between changes in levels of agonism and hormone concentrations throughout the year; and (iii) three agonistic behaviors (chasing, wrestling, and open‐mouth threat) peaked prior to the increase in testosterone and glucocorticoid hormones measured during the breeding season. These three behaviors could potentially be used as early indicators to signal the onset of the breeding season and allow time to implement ex situ management changes to reduce the incidence of agonism between individuals.     

Use of group-specific antibodies to detect fecal progesterone metabolites during the estrous cycle of cows

Progesterone is metabolized to pregnanediones and hydroxylated pregnanes prior to its fecal excretion. Therefore, use of progesterone antibodies underestimates the actual amount of fecal metabolites. To improve the methodology of noninvasive fecal progesterone metabolite analysis, enzymeimmunoassays (EIA) using group-specific antibodies against 5-reduced 20-oxo-pregnane-C3-conjugates were developed. Fecal and milk samples were collected at 1- to 2-d intervals during the morning and evening milking throughout 1 estrous cycle in dairy cows (n = 12). Six immunoreactive metabolites were detected in the feces with high performance liquid chromatography (HPLC), eluting as 5α- and 5β-reduced pregnanes containing a 20-oxo-group (20-oxo-pregnanes). Fecal samples of 3 cows were analyzed by 3 EIAs using antibodies against 4-pregnene-6α-ol-3,20-dione 6HS:BSA (6HS-progesterone), 5α-pregnane-3β-ol-20-one 3HS:BSA and 5β-pregnane-3β-ol-20-one 3HS:BSA, respectively. The follicular and luteal phases were identifiable with each EIA. Luteal phase values and the differences between mean follicular (Days 0 to 2 and 19 to 21) and luteal phase (Days 10 to 16) values obtained with the 5-pregnane EIAs were 3- to 4-fold higher than with the 6HS-progesterone EIA. Since results with the former 2 EIAs were almost identical, the remaining samples were only analyzed by the EIA for 5β-pregnane-3α-ol-20-one. Fecal 20-oxo-pregnane concentrations were parallel to milk progesterone values, but had a lag time of about 0.5 d; the coefficient of correlation (P < 0.001) was 0.76 (y = 155.2 × + 37.2). Fecal 20-oxo-pregnane concentrations during the follicular and luteal phase were 39.5 ± 2.2 and 341 ± 15.2 ng/g feces, respectively. In conclusion, fecal 20-oxo-pregnanes are significantly correlated to milk progesterone concentrations. They consist of several metabolites and compared to a 6HS-progesterone antibody, their evaluation was improved using antibodies against 5-reduced pregnanes.     

Non-invasive monitoring of glucocorticoid metabolites in brown hyaena (Hyaena brunnea) feces

The brown hyaena (Hyaena brunnea) is the least known of the large predators of southern Africa. The current IUCN status of the brown hyaena is “Near Threatened”, and there are conservation concerns related to a general lack of biological knowledge of the species. For instance, a better knowledge of the responses to environmental and social stressors would improve our abilities to sustainably manage brown hyaena populations in both captive and free‐ranging environments. We conducted adrenocorticotrophic hormone (ACTH) challenges in one female and one male adult brown hyaena at Lion Park Zoo, South Africa, to validate measurements of glucocorticoid metabolites (GCM) in brown hyaena feces via an enzyme immunoassay (EIA). We also measured gastrointestinal transit times (GIT times) and the GCM degradation in feces left in ambient temperature for up to 32 hr to more reliably assess the use of this assay as a tool for non‐invasive glucocorticoid measurements. Intramuscular injections of synthetic ACTH yielded GCM levels of 388% (female) and 2,682% (male) above baseline with peak increases occurring 25‐ to 40‐hr after injection. The time delay of fecal GCM excretion approximately corresponded with food transit time in the brown hyaenas. Fecal GCM levels declined significantly over time since defecation. Our results provided a good validation that fecal GCMs accurately reflect circulating glucocorticoid stress hormones in brown hyaenas, but we highlight that samples have to be frozen immediately after defecation to avoid bias in the measurements as a result of bacterial degredation. Zoo Biol 30:451–458, 2011. © 2010 Wiley‐Liss, Inc.     

Biological Validations of Fecal Glucocorticoid,Testosterone, and Progesterone Metabolite Measurements in Captive Stumptail Macaques (<Emphasis Type="Italic">Macaca arctoides</Emphasis>)

Measuring hormone metabolites in fecal samples allows the noninvasive assessment of some steroid hormones in primates. However, noninvasive hormone assays need analytical and biological validation owing to variation in hormone metabolism and excretion between the sexes and across species. We aimed to validate the measurement of fecal glucocorticoid (fGC), testosterone (fT), and progesterone (fP) metabolites in 15 captive stumptail macaques (Macaca arctoides). We collected fecal samples before and after we induced a stress response by restraining and injecting the subjects with saline solution. We then measured hormone metabolites using a methanol extraction technique and ¹²⁵I radioimmunoassay kits. We analyzed the change in glucocorticoid production before and after the stressor, as well as sexual and social rank differences. For fT metabolite levels we investigated variation with sex, age, and social rank, and for fP metabolite levels, we tested for sexual and cycle phase differences. We found a significant increase in fGC metabolite levels 22–25 h poststressor in both sexes. The increase was greater in high-ranking than in low-ranking individuals. Levels of fT metabolites were higher in males than in females, correlated positively with rank only in males, and correlated negatively with age in both sexes. fP metabolite levels were higher in females than in males, and were higher during the luteal phase than in the follicular phase. These findings indicate that our assays reliably detected hormonal changes related to stress (fGC) and detected differences between social and sexual categories (fT, fP) in stumptail macaques.     

A non-invasive method for measuring glucocorticoid metabolites (GCM) in Mountain hares (<Emphasis Type="Italic">Lepus timidus</Emphasis>)

In this study, we evaluated a non-invasive method for measuring glucocorticoid metabolites (GCM) in the Mountain hare (Lepus timidus). An adrenocorticotrophic hormone challenge test was performed in order to select an appropriate enzyme immunoassay (EIA) to measure faecal GCM. Finally, an 11-oxoaetiocholanolone EIA and a 5α-pregnane-3β,11β,21-triol-20-one EIA were chosen. Both assays showed small fluctuations in baseline values and a clear response after stimulation of the adrenocortical activity. To test the stability of faecal metabolites under field conditions, the effects of different storage conditions and periods on GCM concentrations were examined. The assays revealed low fluctuations in metabolite concentrations within the storage period of 12, 24, 48 and 72 h, both at ambient temperatures of 10°C and 25°C, respectively. A washing-out effect of water was found for both assays, which must be taken into account in field studies. The results indicate that this non-invasive method can be used to evaluate glucocorticoid levels of free-ranging Mountain hares.     

Characterization of reproductive cycles and adrenal activity in the black‐footed ferret (Mustela nigripes) by fecal hormone analysis

The reproductive cycle of the black‐footed ferret (Mustela nigripes) was characterized by enzyme immunoassay (EIA) analysis of ovarian fecal steroids (estradiol, progestins) in 29 females over two consecutive breeding seasons. Estrous status was determined by measuring the vulva size and examining the percentage of superficial cells in vaginal lavages. Mean fecal estradiol concentrations were correlated with vulval area (r = 0.370, P < 0.0001) and the percentage of superficial cells (r = 0.380, P < 0.0001). Ovulation resulted in a rise in fecal progestin concentrations 5 days after breeding that differed (P < 0.05) between pregnant (n = 14) and pseudopregnant (n = 12) females during the late luteal phase (days 12–40), with concentrations remaining higher in pregnant animals. Gestation length was 41.3 ± 0.7 days with 3.6 ± 0.4 kits produced per female. Litter size correlated significantly (P < 0.05) with fecal estradiol, but not progestins during the 12 to 40 days after breeding. Females failing to breed (n = 3) remained in estrus for 31 ± 6.2 days before ovulation induction with human chorionic gonadotropin. Adrenal activity in male (n = 4) and female (n = 6) black‐footed ferrets was monitored by quantifying fecal corticoid metabolites after a series of manipulations (physical restraint, intramuscular saline, intramuscular gel adrenocorticotropic hormone (ACTH), intramuscular liquid ACTH). A significant (P < 0.0001) increase in fecal corticoids above the pre‐treatment baseline occurred 20 to 44 hours after restraint (five of 10 animals), saline (six of nine), gel ACTH (seven of 10), and liquid ACTH (nine of 10) treatments. Immunoreactivity of high‐performance liquid chromatography–separated fecal elutes was compared using antibodies against cortisol and corticosterone. The cortisol EIA demonstrated immunoreactivity that co‐eluted with ³H‐cortisol, whereas a corticosterone radioimmunoassay detected a metabolite peak that co‐eluted with ³H‐corticosterone in addition to a slightly less polar and one considerably more polar peak. Despite recognizing different metabolites, both assays produced similar temporal profiles of corticoid excretion after manipulation. This study provides new information on the black‐footed ferret regarding differences in fecal steroid excretion patterns between pregnancy and pseudopregnancy and the potential application of fecal corticoid metabolite monitoring for evaluating responses to stressors associated with practices used in breeding management. Zoo Biol 20:517–536, 2001. © 2002 Wiley‐Liss, Inc.     

Noninvasive monitoring of fecal cortisol metabolites in the eastern chipmunk (Tamias striatus): validation and comparison of two enzyme immunoassays

Monitoring fecal glucocorticoid metabolites in wild animals, using enzyme immunoassays, enables the study of endocrinological patterns relevant to ecology and evolution. While some researchers use antibodies against the parent hormone (which is typically absent from fecal samples), others advocate the use of antibodies designed to detect glucocorticoid metabolites. We validated two assays to monitor fecal cortisol metabolites in the eastern chipmunk (Tamias striatus). We compared an antibody produced against cortisol and one produced against 5α-pregnane-3β, 11β, 21-triol-20-one using a radiometabolism study and an injection with adrenocorticotropic hormone (ACTH). Most cortisol metabolites were excreted in the urine (～83%). Peak excretion in the feces occurred 8 h after injection. Both assays detected an increase in fecal cortisol metabolite levels after injection of ACTH. Males, but not females, exhibited a circadian variation in metabolite levels. The sexes did not exhibit any difference over the time course and route of excretion or the relative increase in fecal cortisol metabolite levels after ACTH injection. The cortisol assay displayed higher reactivity to ACTH injection relative to baseline than did the metabolite assay. While both antibodies gave comparable results, the cortisol antibody was more sensitive to changes in plasma cortisol levels in eastern chipmunks.     

Excretion of corticosteroids in urine and faeces of hares (Lepus europaeus)

Increased production of glucocorticoids by the adrenal cortex is found in mammals under stress. As cortisol itself is absent in the faeces, an enzyme immunoassay (11-oxoaetiocholanolone) measuring 11,17-dioxoandrostanes has already been established to measure faecal cortisol metabolites in ruminants for non-invasive monitoring of adrenocortical activity. The aim of this study was to establish route and delay of excretion of glucocorticoids in hares and to determine whether a cortisol-, corticosterone- or this new enzyme immunoassay is best suited to detect faecal glucocorticoid metabolites. In the first experiment radioactive-labelled glucocorticoids (¹⁴C-cortisol and ³H-corticosterone) were administered intravenously to two groups of three hares in metabolic cages. All voided urine and faecal samples were collected for 4 days. Metabolites of both steroids were found predominantly in the urine (91 ± 4%). Peak concentrations were observed in the first urinary sample following infusion (13 ± 6 h) and in the faeces with a delay of about 1 day (23 ± 7 h). Most of the radioactivity was not extractable with diethylether, indicating that the metabolites excreted in urine and faeces are mainly conjugated or polar unconjugated ones. This was confirmed by reverse-phase high-performance liquid chromatography separations of the metabolites, which also revealed marked differences concerning the metabolism of the two glucocorticoids injected. Compared with the cortisol and the corticosterone enzyme immunoassay, only the group-specific enzyme immunoassay for 11,17-dioxoandrostanes detected high quantities of immunoreactive metabolites. In a second experiment hares (n=20) were stressed by rousing them three times (5 min, 10 min and another 5 min) with a 20-min break in-between. Faecal samples were collected 2 days before until 4 days after stress and analysed using the 11-oxoaetiocholanolone enzyme immunoassay. After stress significantly (P < 0.001) increased 11,17-dioxoandrostane concentrations were found. Based on these results, measuring 11,17-dioxoandrostanes in faeces enables non-invasive monitoring of disturbances in hares and thus provides a tool for field investigations elucidating the role of stress in hare populations. Accepted: 24 November 1999     

Validation of noninvasive monitoring of adrenocortical endocrine activity in ground-feeding aardwolves (Proteles cristata): exemplifying the influence of consumption of inorganic material for fecal steroid analysis

Biologically inert material in feces may confound interpretations of noninvasive fecal endocrine data, because it may induce variance related to differences in foraging behavior rather than to differences in endocrine activity. We evaluated two different enzyme immunoassays (EIAs) for the noninvasive evaluation of adrenocortical activity in ground-feeding aardwolves (Proteles cristata) and tested the influence of soil content in aardwolf feces on the interpretation of fecal glucocorticoid metabolite data. Using adrenocorticotropic hormone (ACTH) challenges for validation, we successfully identified a cortisol EIA suitable for assessing adrenocortical activity in aardwolves. An alternatively tested 11-oxoetiocholanolone EIA failed to detect a biologically relevant signal after ACTH administration. Although the proportion of inorganic content in aardwolf feces did not alter qualitative conclusions from the endocrine data, the data related to mass of organic content had a larger amount of variance attributed to relevant biological contrasts and a lower amount of variance attributed to individual variation, compared with data related to total dry mass of extracted material. Compared with data expressed as dry mass of extracted material, data expressed as mass of organic content may provide a more refined and statistically powerful measure of endocrine activity in species that ingest large amounts of indigestible material.     

Individual‐based analysis of hair corticosterone reveals factors influencing chronic stress in the American pika

Glucocorticoids are often measured in wildlife to assess physiological responses to environmental or ecological stress. Hair, blood, saliva, or fecal samples are generally used depending on the timescale of the stress response being investigated and species‐specific considerations. Here, we report the first use of hair samples to measure long‐term corticosterone levels in the climate‐sensitive American pika (Ochotona princeps). We validated an immunoassay‐based measurement of corticosterone extracted from hair samples and compared corticosterone estimates obtained from plasma, hair, and fecal samples of nine pikas. To demonstrate an ecological application of this technique, we characterized physiological stress in 49 pikas sampled and released at eight sites along two elevational transects. Microclimate variation was measured at each site using both ambient and subsurface temperature sensors. We used an information theoretic approach to compare support for linear, mixed‐effects models relating corticosterone estimates to microclimate, body size, and sex. Corticosterone was measured accurately in pika hair samples after correcting for the influence of sample mass on corticosterone extraction efficiency. Hair‐ and plasma‐based estimates of corticosterone were weakly correlated. The best‐supported model suggested that corticosterone was lower in larger, male pikas, and at locations with higher ambient temperatures in summer. Our results are consistent with a general negative relationship between body mass and glucocorticoid concentration observed across mammalian species, attributed to the higher mass‐specific metabolic rates of smaller bodied animals. The higher corticosterone levels in female pikas likely reflected the physiological demands of reproduction, as observed in a wide array of mammalian species. Additionally, we establish the first direct physiological evidence for thermal stress in the American pika through nonlethal sampling of corticosterone. Interestingly, our data suggest evidence for cold stress likely induced during the summer molting period. This technique should provide a useful tool to researchers wishing to assess chronic stress in climate‐sensitive mammals.     

Influence of Radiotransmitters on Fecal Glucocorticoid Levels of Free-Ranging Male American Kestrels

ABSTRACT Although radiotelemetry is considered a valuable technique for ornithological field studies, several assumptions have been made about the impact that transmitters may have on the estimation of behavioral, ecological, and reproductive parameters. To assess the potential effects of backpack radiotransmitters, we captured and assigned 8 male American kestrels (Falco sparverius) into 2 groups: radiotagged (n = 6) and control individuals (leg-banded, n = 2). Thereafter, we collected feces approximately 2 hours after capture (day −1), and subsequently during days 0 (releasing day), 4, 7, 15, 30, 40, and 55. Prior to fecal analysis, we validated the corticosterone enzyme immunoassay using standard procedures (e.g., parallelism, dose-response curve), and we confirmed physiological significance of fecal glucocorticoid metabolites through adrenocorticotropin challenge, which induced an increase of 4-fold (446.10 ± 60.73 ng/g) above baseline (114.27 ± 15.23 ng/g) within 4 hours (P < 0.001). Both groups exhibited a significant increase in fecal glucocorticoids during day 0 (P < 0.001), but concentrations returned to preattachment values within 4 days. Fecal glucocorticoid concentrations did not differ between samples of radiotagged and leg-banded kestrels (P > 0.05). In spite of the small number of monitored subjects, these findings suggested that radiotransmitters did not affect adrenocortical activity in these male American kestrels.