PHYSIOLOGY: MAMMALS
BATS
Kelm D. H., Popa-Lisseanu A. G., Dehnhard M., Ibáñez C. (2016): Non-invasive monitoring of stress hormones in the bat Eptesicus isabellinus – Do fecal glucocorticoid metabolite concentrations correlate with survival?. General and Comparative Endocrinology 226: 27-35.
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Chronic stress may negatively impact fitness and survival in wildlife. Stress hormone analysis from feces is a non-invasive tool for identifying stressors and deducing about individual and population level fitness. Although many bat populations are endangered, fecal stress hormone analysis has not been established in bats as a method for focusing conservation efforts. The isabelline serotine bat, Eptesicus isabellinus, is exposed to human disturbance as its roosts are mostly found in anthropogenic structures. Moreover, this bat is host to various diseases and survival rates between colonies may vary significantly. To validate the analysis of fecal glucocorticoid metabolites, we applied an adrenocorticotropic hormone (ACTH) challenge and tested four different enzyme immunoassays (EIA) for measuring glucocorticoid concentrations. Cortisol and its metabolites showed the highest increase in blood and feces after the ACTH challenge, but corticosterone and its metabolites also increased significantly. Baseline fecal cortisol metabolite (FCM) concentrations did not increase until 1.5 h after the animals were captured, which is a convenient time lag for sample collection from captured animals. We furthermore compared baseline FCM concentrations between five colonies of E. isabellinus in Andalusia, Spain, and tested for their correlation with survival rates. FCM concentrations did not vary between colonies, but FCM levels increased with the animals’ age. FCM analysis may prove a useful tool for identifying bat colonies that experience uncommon environmental stress. However, inter-individual variation in hormone secretion, due to factors such as age, may require additional information to properly interpret differences in hormone concentrations.
Ryu H., Kinoshita K., Joo S., Kim S. S. (2021): Urinary creatinine varies with microenvironment and sex in hibernating Greater Horseshoe bats (Rhinolophus ferrumequinum) in Korea. BMC Ecology and Evolution 21: 77.
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In temperate regions many small mammals including bats hibernate during winter. During hibernation these small mammals occasionally wake up (arouse) to restore electrolyte and water balance. However, field data on water stress and concentration of bodily fluids during hibernation is scarce. Urinary creatinine concentration has long been used to calibrate urinary hormone concentration due to its close correlation with urine concentration. Therefore, by investigating urinary creatinine concentration, we can estimate bodily fluid concentration. In this study, we investigated changes in urinary creatinine from greater horseshoe bats (Rhinolophus ferrumequinum) hibernating in abandoned mineshafts in two regions in South Korea. We collected 74 urine samples from hibernating greater horseshoe bats from 2018 to 2019. We found that urinary creatinine concentration was higher in February and March and then declined in April. There were also indications of a sex difference in the pattern of change in creatinine concentration over the three months. Bats in the warmer and less humid mineshaft had higher urinary creatinine concentrations than bats in the colder and more humid mineshaft. These results indicate that hibernating bats face water stress as urinary concentration increases during winter and that water stress may vary depending on the microenvironment. Sex differences in behaviour during hibernation may influence arousal frequency and result in sex differences in changes in urinary creatinine concentration as hibernation progresses. Although further behavioural and endocrinal investigations are needed, our study suggests that urinary creatinine concentration can be used as a proxy to estimate the hydration status of bats and the effect of sex and environmental factors on arousal patterns during hibernation.
Sandoval-Herrera N. I., Mastromonaco G. F., Becker D. J., Simmons N. B., Welch K. C. (2021): Inter-and intra-specific variation in hair cortisol concentrations of Neotropical bats. Conservation Physiology 9: coab053.
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Quantifying hair cortisol has become popular in wildlife ecology for its practical advantages for evaluating stress. Before hair cortisol levels can be reliably interpreted, however, it is key to first understand the intrinsic factors explaining intra- and inter-specific variation. Bats are an ecologically diverse group of mammals that allow studying such variation. Given that many bat species are threatened or have declining populations in parts of their range, minimally invasive tools for monitoring colony health and identifying cryptic stressors are needed to efficiently direct conservation efforts. Here we describe intra- and inter-specific sources of variation in hair cortisol levels in 18 Neotropical bat species from Belize and Mexico. We found that fecundity is an important ecological trait explaining inter-specific variation in bat hair cortisol. Other ecological variables such as colony size, roost durability and basal metabolic rate did not explain hair cortisol variation among species. At the individual level, females exhibited higher hair cortisol levels than males and the effect of body mass varied among species. Overall, our findings help validate and accurately apply hair cortisol as a monitoring tool in free-ranging bats.
Vanderwolf K. J., Kyle C. J., Faure P. A., McAlpine D. F., Davy C. M. (2021): Skin pH varies among bat species and seasons and between wild and captive bats. Conservation Physiology 9: coab088.
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Skin is a key aspect of the immune system in the defence against pathogens. Skin pH regulates the activity of enzymes produced both by hosts and by microbes on host skin, thus implicating pH in disease susceptibility. Skin pH varies inter- and intra-specifically and is influenced by a variety of intrinsic and extrinsic variables. Increased skin alkalinity is associated with a predisposition to cutaneous infections in humans and dogs, and inter-specific and inter-individual variation in skin pH is implicated in differential susceptibility to some skin diseases. The cutaneous pH of bats has not been characterized but is postulated to play a role in susceptibility to white-nose syndrome (WNS), a fungal infection that has decimated several Nearctic bat species. We used non-invasive probes to measure the pH of bat flight membranes in five species with differing susceptibility to WNS. Skin pH ranged from 4.67 to 8.59 and varied among bat species, geographic locations, body parts, age classes, sexes and seasons. Wild Eptesicus fuscus were consistently more acidic than wild Myotis lucifugus, Myotis leibii and Perimyotis subflavus. Juvenile bats had more acidic skin than adults during maternity season but did not differ during swarming. Male M. lucifugus were more acidic than females during maternity season, yet this trend reversed during swarming. Bat skin was more acidic in summer compared to winter, a pattern also reported in humans. Skin pH was more acidic in captive than wild E. fuscus, suggesting environmental impacts on skin pH. The pH of roosting substrates affects skin pH in captive bats and may partially explain seasonal patterns in wild bats that use different roost types across seasons. Future research on the influence of pH on microbial pathogenic factors and skin barrier function may provide valuable insights on new therapeutic targets for treating bat skin conditions.
Edwards M. J., Stanley C. R., Hosie C. A., Richdon S., Price E., Wormell D., Smith T. E. (2022): Social roles influence cortisol levels in captive Livingstone’s fruit bats (Pteropus livingstonii). Hormones and Behavior 144: 105228.
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A critical component of conserving and housing species ex situ is an explicit scientific understanding of the physiological underpinnings of their welfare. Cortisol has been repeatedly linked to stress, and therefore used as an indicator of welfare for many species. In order to measure cortisol in the Livingstone’s fruit bat (Pteropus livingstonii; a critically endangered keystone species) without disturbing the captive population, we have developed and validated a non-invasive, novel hormone extraction procedure and faecal glucocorticoid assay. A total of 92 faecal samples, 73 from the P. livingstonii breeding colony at Jersey Zoo, Channel Islands and 19 samples from P. livingstonii housed at Bristol Zoological Gardens, UK, have been collected and analyzed. Mixed-effect modelling of the influence of physiological state variables on cortisol concentration revealed that lactating females had higher cortisol levels than non-lactating females, indicating that our assay is measuring biologically relevant hormone concentrations. Males and older bats also had higher cortisol than non-lactating females and younger individuals. Further analysis applied social network methodology to compare the cortisol levels of bats with different social roles. We found that individuals that linked social groups possessed higher than average cortisol levels and conversely, individuals with high-quality, positive relationships had lower cortisol levels. These results demonstrate, for the first time in a bat species, social mediation of stress hormones. Lastly, the frequency of vocalisation was found to positively correlate with cortisol concentration in males, suggesting that this behaviour may be used by animal management as a visual indicator of a bat’s hormonal status. Hence, this research has provided unique insights and empirical scientific knowledge regarding the relationship between the physiology and social behaviour of P. livingstonii, therefore allowing for recommendations to be made to optimise bat welfare at the individual level.
CARNIVORES
Bonier F., Quigley H., Austad S. N. (2004): A technique for non‐invasively detecting stress response in cougars. Wildlife Society Bulletin 32: 711-717.
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The ability to non‐invasively monitor stress hormone levels in free‐ranging animals could significantly aid in conservation and management efforts. Our objective in this study was to demonstrate the effectiveness of assay of fecal corticoid metabolites in detecting a stress response in cougars (Puma concolor). Fecal samples were collected from 9 captive cougars before and after an artificial stressor. Steroid hormones were extracted from the samples. Adrenal corticoid metabolite concentrations of the resulting extracts were quantified using cortisol and corticosterone assays. Results indicated that fecal glucocorticoid metabolite levels increased significantly 24 hours after the stressor in 6 of the 9 individuals. Behavior correlated with the hormonal response; all cougars that displayed a flight response to the stressor also had elevated stress hormone measures. Elevated levels of stress hormones were not observed in individuals that did not attempt to flee. We have demonstrated with this study that measurement of fecal hormone metabolites is sufficiently sensitive to detect an adrenal response to stress in cougars and could be applied in the field to monitor stress levels in free‐ranging populations.
Hunt K. E., Trites A. W., Wasser S. K. (2004): Validation of a fecal glucocorticoid assay for Steller sea lions (Eumetopias jubatus). Physiology & Behavior 80: 595-601.
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The Steller sea lion (Eumetopias jubatus) is listed as endangered in parts of its range and is suspected of suffering from ecological stressors that may be reflected by fecal glucocorticoid hormones. We validated a fecal glucocorticoid assay for this species with an adrenocorticotropic hormone (ACTH) challenge. Feces were collected from captive Steller sea lions (two males and two females) for 2 days before injection with ACTH, and for 4 or more days postinjection. Feces were freeze-dried, extracted with a methanol vortex method, and assayed for glucocorticoids. The assay demonstrated good parallelism and accuracy. All animals showed the expected peak of fecal glucocorticoid excretion after ACTH injection. However, the two males had higher baselines, higher peaks, and more delayed peaks than the females. Peak glucocorticoid excretion occurred at 5 and 28 h postinjection for the two females, and at 71 and 98 h for the two males. Correction for recoveries by the addition of tritiated hormones produced ACTH profiles that were virtually identical in pattern to uncorrected data, but with higher within-sample coefficients of variation. Based on these results, we conclude that this fecal glucocorticoid assay accurately reflects endogenous adrenal activity of Steller sea lions, and that recovery corrections are not necessary for this species when using the methanol vortex extraction method. More research is needed to address possible sex differences and other possible influences on fecal glucocorticoid concentrations.
Young K. M., Walker S. L., Lanthier C., Waddell W. T., Monfort S. L., Brown J. L. (2004): Noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analyses. General and Comparative Endocrinology 137: 148-165.
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Measurement of glucocorticoid metabolites in feces has become an accepted method for the noninvasive evaluation of adrenocortical activity. The objective of this study was to determine if a simple cortisol enzyme immunoassay (EIA) was suitable for monitoring adrenocortical activity in a variety of carnivore species. Performance of the cortisol EIA was gauged by comparison to a corticosterone radioimmunoassay (RIA) that has been used for measuring glucocorticoid metabolites in feces of numerous species. Tests for parallelism and extraction efficiency were used to compare the cortisol EIA and corticosterone RIA across eight species of carnivores (Himalayan black bear, sloth bear, domestic cat, cheetah, clouded leopard, black-footed ferret, slender-tailed meerkat, and red wolf). The biological relevance of immunoreactive glucocorticoid metabolites in feces was established for at least one species of each Carnivora family studied with an adrenocorticotropic hormone (ACTH) challenge. High performance liquid chromatography (HPLC) analysis of fecal extracts for each species revealed (1) the presence of multiple immunoreactive glucocorticoid metabolites in feces, but (2) the two immunoassays measured different metabolites, and (3) there were differences across species in the number and polarities of metabolites identified between assay systems. ACTH challenge studies revealed increases in fecal metabolite concentrations measured by the cortisol EIA and corticosterone RIA of ∼228–1145% and ∼231–4150% above pre-treatment baseline, respectively, within 1–2 days of injection. Concentrations of fecal glucocorticoid metabolites measured by the cortisol EIA and corticosterone RIA during longitudinal evaluation (i.e., >50 days) of several species were significantly correlated (P<0.0025, correlation coefficient range 0.383–0.975). Adrenocortical responses to physical and psychological stressors during longitudinal evaluations varied with the type of stimulus, between episodes of the same stimulus, and among species. Significant elevations of glucocorticoid metabolites were observed following some potentially stressful situations [anesthesia (2 of 3 subjects), restraint and saline injection (2 of 2 subjects), restraint and blood sampling (2 of 6 episodes), medical treatment (1 of 1 subject)], but not in all cases [e.g., gonadotropin injection (n=4), physical restraint only (n=1), mate introduction/breeding (n=1), social tension (n=1), construction (n=2) or relocation (n=1)]. Results reinforced the importance of an adequate baseline period of fecal sampling and frequent collections to assess adrenocortical status. The corticosterone RIA detected greater adrenocortical responses to exogenous ACTH and stressful exogenous stimuli in the Himalayan black bear, domestic cat (female), cheetah, clouded leopard, slender-tailed meerkat, and red wolf, whereas the cortisol EIA proved superior to resolving adrenocortical responses in the black-footed ferret and domestic cat (male). Overall results suggest the cortisol EIA tested in this study offers a practical method for laboratories restricted in the usage of radioisotopes (e.g., zoological institutions and field facilities) to integrate noninvasive monitoring of adrenocortical activity into studies of carnivore behavior and physiology.
Bechshøft T. Ø., Sonne C., Dietz R., Born E. W., Novak M. A., Henchey E., Meyer J. S. (2011): Cortisol levels in hair of East Greenland polar bears. Science of the Total Environment 409: 831-834.
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To demonstrate the ability to assess long-term hypothalamic–pituitary–adrenocortical (HPA) axis activity in polar bears (Ursus maritimus), a pilot study was conducted in which cortisol concentrations was analyzed in hair from 7 female (3–19 years) and 10 male (6–19 years) East Greenland polar bears sampled in 1994–2006. The hair was chosen as matrix as it is non-invasive, seasonally harmonized, and has been validated as an index of long-term changes in cortisol levels. The samples were categorized according to contamination: eight were clean (2 females, 6 males), 5 had been contaminated with bear blood (2 F, 3 M), and 4 with bear fat (3 F, 1 M). There was no significant difference in cortisol concentration between the three categories after external contamination was removed. However, contaminated hair samples should be cleaned before cortisol determination. Average hair cortisol concentration was 8.90 pg/mg (range: 5.5 to 16.4 pg/mg). There was no significant correlation between cortisol concentration and age (p = 0.81) or sampling year (p = 0.11). However, females had higher mean cortisol concentration than males (females mean: 11.0 pg/mg, males: 7.3 pg/mg; p = 0.01). The study showed that polar bear hair contains measurable amounts of cortisol and that cortisol in hair may be used in studies of long-term stress in polar bears.
Yang L. L., Zhang Q., Ge X. F., Jin X. L., Hu D. F., Zhao P. P., Yu X. J. (2011): Non-invasive determination of fecal steroid hormones relating to conservation practice in giant panda (Ailuropoda melanoleuca). Animal Biology 61: 335-347.
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The endangered giant panda is the flagship species of wildlife conservation, a status that results in heightened attention to their well-being. However, one factor that hinders the ability to monitor this species in the wild is the difficulty to measure relevant physiological parameters which quantify the survival status in conservation research. In this study, we employed a non-invasive sampling method and immunochemical assays to determine if measurement of fecal steroid hormones is a viable option in monitoring the sex ratios and stress levels of a giant panda population. The results indicate that 1) the yearly concentration of fecal testosterone is a possible biomarker for distinguishing gender, and 2) invasive methods of artificial insemination and semen collection as well as parturition, cause an increase in fecal cortisol concentration. This study suggests that the noninvasive sampling of feces can be a practical tool to monitor physiological stress in free-ranging and fenced giant panda populations, and could be useful in the forthcoming giant panda census in determining sex ratios in the wild.
Barja I., Escribano-Ávila G., Lara-Romero C., Virgós E., Benito J., Rafart E. (2012): Non-invasive monitoring of adrenocortical activity in European badgers (Meles meles) and effects of sample collection and storage on faecal cortisol metabolite concentrations. Animal Biology 62: 419-432.
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Due to the increasing demand for methods to quantify adrenal activity in response to stressors in wild animals, we evaluated whether stimulation with adrenocorticopic hormone (ACTH) increases faecal cortisol metabolite levels in European badgers. We also conducted several experiments to evaluate the effect of storage time and collection methods on cortisol metabolite levels of faecal samples. Faecal samples were collected from five captive European badgers (Meles meles) and cortisol metabolites were quantified by an enzyme immunoassay. Faecal cortisol metabolite levels increased 1-2 days after ACTH injection and dropped to pre-treatment levels 3 days after ACTH stimulation. We found that cortisol metabolite levels were not affected by the 8-hour time lapse from collection in the field until freezing of faecal samples, environmental exposure (temperature 25.66 ± 2.55°C; range 21.20-32.20) or mean relative humidity 62.77 ± 9.90% (range 42.00-79.00). Cortisol metabolites were not homogenously distributed in the scats. Cortisol metabolite levels were significantly lower when faecal extracts and samples were frozen for long periods of time (14 weeks or more). Thus, the measurement of faecal cortisol metabolites is a suitable method for the non-invasive evaluation of adrenocortical activity in European badgers. However, some aspects related to the collection and storage of faecal samples should be considered in future studies conducted with this species.
Ganswindt A., Muilwijk C., Engelkes M., Muenscher S., Bertschinger H., Paris M., Palme R., Cameron E. Z., Bennett N. C., Dalerum F. (2012): 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. Physiological and Biochemical Zoology 85: 194-199.
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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.
Bryan H. M., Adams A. G., Invik R. M., Wynne-Edwards K. E., Smits J. E. (2013): Hair as a meaningful measure of baseline cortisol levels over time in dogs. Journal of the American Association for Laboratory Animal Science 52: 189-196.
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Cortisol measurements of hair are becoming a valuable tool in monitoring chronic stress. To further validate this approach in domestic dogs, we compared the variability of cortisol immunoreactivity in hair with that in saliva and feces of dogs housed under constant social and physical conditions. Fecal (n = 268), and hair (n = 21) samples were collected over 3 mo from 7 dogs housed in a kennel and kept for training veterinary students in minimally invasive procedures. Salivary samples (n = 181) were collected 3 times daily twice weekly during the last month of the study. Hair and salivary samples were analyzed by enzyme immunoassay and feces by radioimmunoassay. HPLC coupled with tandem mass spectrometry was used to confirm the presence of cortisol in 3 hair samples. Variability of cortisol was compared across sample types by using repeated-measures ANOVA followed by paired t tests. Within dogs, cortisol immunoreactivity was less variable in hair than in saliva or feces. Averaged over time, the variability of fecal samples approached that of hair when feces were collected at least 4 times monthly. As predicted, the stable social and environmental condition of the dogs maintained repeatability over time and supported the hypothesis that data from hair samples reflect baseline cortisol levels. These findings indicate that determining cortisol immunoreactivity in hair is a more practical approach than is using samples of saliva or feces in monitoring the effects of long-term stressors such as social or physical environments and disease progression.
Bryan H. M., Darimont C. T., Paquet P. C., Wynne-Edwards K. E., Smits J. E. (2014): Stress and reproductive hormones reflect inter-specific social and nutritional conditions mediated by resource availability in a bear–salmon system. Conservation Physiology 2: cou010.
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Food availability can influence the nutritional and social dynamics within and among species. Our investigation focused on grizzly and black bears in coastal British Columbia, Canada, where recent and dramatic declines in their primary prey (salmon) raise concerns about potentially negative effects on bear physiology. We examined how salmon availability relates to stress and reproductive hormones in coastal grizzly (n = 69) and black bears (n = 68) using cortisol and testosterone. In hair samples from genotyped individuals, we quantified salmon consumption using stable isotope analysis and hormone levels by enzyme immunoassay. To estimate the salmon biomass available to each bear, we developed a spatially explicit approach based on typical bear home-range sizes. Next, we compared the relative importance of salmon consumption and salmon availability on hormone levels in male bears using an information theoretical approach. Cortisol in grizzly bears was higher in individuals that consumed less salmon, possibly reflecting nutritional stress. In black bears, cortisol was better predicted by salmon availability than salmon consumption; specifically, individuals in areas and years with low salmon availability showed higher cortisol levels. This indicates that cortisol in black bears is more strongly influenced by the socially competitive environment mediated by salmon availability than by nutritional requirements. In both species, testosterone generally decreased with increasing salmon availability, possibly reflecting a less competitive environment when salmon were abundant. Differences between species could relate to different nutritional requirements, social densities and competitive behaviour and/or habitat use. We present a conceptual model to inform further investigations in this and other systems. Our approach, which combines data on multiple hormones with dietary and spatial information corresponding to the year of hair growth, provides a promising tool for evaluating the responses of a broad spectrum of wildlife to changes in food availability or other environmental conditions.
Deng H., Jin X., Hu D., Liu D. (2014): Fecal cortisol content of wild giant pandas (Ailuropoda melanoleuca) to monitor human disturbance level in natural habitats. Animal Biology 64: 75-86.
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Wild animals are affected by growing human interference in their habitats and inevitably react internally to such stimuli. This study explores inherent physiological parameters to assess the effect of human interference on giant pandas in their wild habitat. Ninety-one fecal samples were collected within four nature reserves on Qinling Mountain. Fecal cortisol determinations were performed by radioimmunoassay. We categorized the intensity of local human disturbance and tested for correlations between the intensity of human disturbance and fecal cortisol metabolite levels. The results show a significant positive correlation between giant panda fecal cortisol metabolite levels and the degree of disturbance in their habitat. This study is the first to use a non-invasive monitoring method to analyze wild giant panda habitat disturbance, and demonstrates that cortisol metabolite levels in panda dung can objectively reflect the degree of panda habitat disturbance. The results provide a relatively objective means and method with which to evaluate the quality of wild giant panda habitat. This study highlights the need to monitor the effects of human disturbance on wildlife and to implement new policies in the management of nature reserves.
Gundlach N. H., Piechotta M., Siebert U. (2014): Is lachrymal fluid a potential method for cortisol measurement in wild harbor seals?: A pilot study. International Journal of Veterinary Medicine: Research & Reports 2016: 967043.
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Despite stable population size in harbor seals anthropogenic interferences in the marine environment still represent a highly discussed matter within research. In order to quantify and evaluate assumed stressful events in the marine environment, there is a permanent search for new methods and materials for the evaluation of stress. Previous studies are often based on venipuncture and determination of the classic “stress-related hormones” in blood, showing the change of activity of the hypothalamic pituitary adrenocortical (HPA) axis as well as the sympathetic adrenomedullary system. Though blood sampling represents the gold-standard, it is influenced by disturbance of sampling, representing an invasive stressful method itself. Therefore, new non—invasive approaches have to be developed to determine HPA modifications as well as other matrices to reduce stress caused by handling and trapping. In addition to saliva and blood, the present study tested cortisol measurement in lachrymal fluid as a possible method for rapid stress evaluation. Therefore, 17 harbor seals (13 male, four female) were examined during a health monitoring program in April 2013. Lachrymal fluid was collected using a swab which was carefully inserted into the conjunctival sack. For comparison, serum and saliva were also sampled afterwards. Cortisol was detected in lachrymal fluid, saliva and blood of all investigated animals. Evaluation of regression analysis showed a good correlation (R2 = 0.82) between serum cortisol and “free” cortisol in lachrymal fluid. This study showed for the first time the possibility of lachrymal fluid sampling as a non-invasive method to asses stress in harbor seals.
Lafferty D. J., Laudenslager M. L., Mowat G., Heard D., Belant J. L. (2015): Sex, diet, and the social environment: factors influencing hair cortisol concentration in free-ranging black bears (Ursus americanus). Plos One 10: e0141489.
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Increasingly, measures of glucocorticoid levels (e.g., cortisol), key components of the neuroendocrine stress axis, are being used to measure past hypothalamic-pituitary-adrenal (HPA) activity to index psychological and physiological stress exhibited by wildlife for assessing individual and population-level well-being. However, many intrinsic and extrinsic factors affect HPA activity in animals. Using American black bears (Ursus americanus; n = 116) as an ecological model and hair cortisol concentration (HCC) as an integrative measure of past HPA activity, we evaluated the influence of diet, sex and the social environment on black bear HCC in a free-ranging population that spanned adjoining ecoregions with differing densities of potential conspecific and heterospecific competitors. HCC varied by sex, with female HCC ranging from 0.6 to 10.7 pg/mg (median = 4.5 ± 1.2 mean absolute deviation [MAD]) and male HCC ranging from 0.5 to 35.1 pg/mg (median = 6.2 ± 2.6 MAD). We also observed a three-way interaction among sex, δ14C and ecoregion, which may indicate that some differences in HCC between female and male black bears results from variability in the nutritional needs of larger-bodied males relative to smaller-bodied females, slight differences in food resources use between ecoregions as well as sex-based differences regarding the social environment. Once we understand what drives sex-specific differences in HCC, HCC may aid our understanding of the physiological responses by bears and other wildlife to diverse environmental challenges.
Mesa-Cruz J. B., Brown J. L., Waits L. P., Kelly M. J. (2016): Non-invasive genetic sampling reveals diet shifts, but little difference in endoparasite richness and faecal glucocorticoids. Journal of Tropical Ecology 32: 226-239.
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Many Neotropical felids are threatened with extinction due to direct effects of habitat destruction and/or human persecution. However, indirect and synergistic effects of human-felid conflict remain under-studied and potentially include increased stress and diet shifts that may negatively impact felid health. We hypothesized that faecal glucocorticoid metabolites (FGM) and endoparasite species richness (ESR) would be higher, and diet would shift, for felids outside protected areas where conflict occurs. In north-western Belize, a scat-detector dog located 336 faecal samples, identified to species and individual using DNA analyses. DNA amplification success was substantially higher within protected areas than outside. We detected jaguar, puma, ocelot, jaguarundi and domestic cat. FGMs were higher in puma and jaguarundi than in other felids, while ESR was similar across felids with domestic cats exhibiting the highest number of genera. Diet partitioning occurred among felids, but domestic cats may compete with ocelot and jaguarundi for small prey. Outside of protected areas, large cats shifted their diet to smaller prey and livestock remains were not found. Contrary to our hypotheses, FGM and ESR did not differ inside versus outside protected areas, but sample sizes were low in human-modified areas. We provide a baseline on wild felid adrenal activity, endoparasites and diet and suggest improvements to increase sample sizes outside protected areas. Our research provides a template for expanding non-invasive sampling approaches more widely across the range of Neotropical felids.
Khonmee J., Rojanasthien S., Thitaram C., Sumretprasong J., Aunsusin A., Chaisongkram C., Songsasen N. (2017): Non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in dholes (Cuon alpinus). Conservation Physiology 5: cox001.
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To date, there is no information on reproductive endocrinology of dholes (Cuon alpinus). The objectives of the present study were as follows: (i) to characterize longitudinal profiles of gonadal steroids; and (ii) to examine the relationship between gonadal hormones and sexual behaviours in dholes. Three breeding pairs and two bachelor males were included in the study. Among these, four animals (2 males and 2 females; 4 years old) were imported from The Netherlands to Thailand 3 months before the study onset; the remaining individuals (3 males and 1 female; 5–7 years old) were native born. Faecal samples were collected 3–7 days/week for 12 months, extracted and assessed for gonadal hormone metabolites using a validated enzyme immunoassay. Observations of behaviour were conducted in 30 min sessions, 3–5 days/week. For the three breeding males, testosterone was elevated (P < 0.05) from October to January in the two imported males, whereas the concentration of steroid metabolites was high from April to June and from September to November in the native male. However, there was no clear seasonal pattern of reproductive hormone in the bachelor group. Oestrogen metabolite level of imported females was elevated for 9–12 days in January, followed by a rise in progestagen concentration. For native females, oestrogen metabolites were above the basal values in April and September, each of which was followed by a rise in progestagen concentration that remained elevated for 77 and 112 days, respectively. Sexual behaviours, including solicitation, mounting and copulations, were observed during the oestrogen peak in all females. Our findings indicate that reproductive seasonality of dholes may depend on the animals’ origin and social group.
Schell C. J., Young J. K., Lonsdorf E. V., Mateo J. M., Santymire R. M. (2017): Investigation of techniques to measure cortisol and testosterone concentrations in coyote hair. Zoo Biology 36: 220-225.
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Long‐term noninvasive sampling for endangered or elusive species is particularly difficult due to the challenge of collecting fecal samples before hormone metabolite desiccation, as well as the difficulty in collecting a large enough sample size from all individuals. Hair samples may provide an environmentally stable alternative that provides a long‐term assessment of stress and reproductive hormone profiles for captive, zoo, and wild mammals. Here, we extracted and analyzed both cortisol and testosterone in coyote (Canis latrans) hair for the first time. We collected samples from 5‐week old coyote pups (six female, six male) housed at the USDA‐NWRC Predator Research Facility in Millville, UT. Each individual pup was shaved in six different locations to assess variation in concentrations by body region. We found that pup hair cortisol (F 5,57.1 = 0.47, p = 0.80) and testosterone concentrations (F 5,60 = 1.03, p = 0.41) did not differ as a function of body region. Male pups generally had higher cortisol concentrations than females (males = 17.71 ± 0.85 ng/g, females = 15.48 ± 0.24 ng/g; F 1,57.0 = 5.06, p = 0.028). Comparatively, we did not find any differences between male and female testosterone concentrations (males = 2.86 ± 0.17 ng/g, females = 3.12 ± 0.21 ng/g; F 1,60 = 1.42, p = 0.24). These techniques represent an attractive method in describing long‐term stress and reproductive profiles of captive, zoo‐housed, and wild mammal populations.
Cattet M., Stenhouse G. B., Boulanger J., Janz D. M., Kapronczai L., Swenson J. E., Zedrosser A. (2018): Can concentrations of steroid hormones in brown bear hair reveal age class?. Conservation Physiology 6: coy001.
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Although combining genetic and endocrine data from non-invasively collected hair samples has potential to improve the conservation of threatened mammals, few studies have evaluated this opportunity. In this study, we determined if steroid hormone (testosterone, progesterone, estradiol and cortisol) concentration profiles in 169 hair samples collected from free-ranging brown bears (Ursus arctos) could be used to accurately discriminate between immature and adult bears within each sex. Because hair samples were acquired opportunistically, we also needed to establish if interactions between hormones and several non-hormone factors (ordinal day, year, contact method, study area) were associated with age class. For each sex, we first compared a suite of candidate models by Akaike Information Criteria model selection, using different adult-age thresholds (3, 4 and 5 years), to determine the most supported adult age. Because hair hormone levels better reflect the endocrine state at an earlier time, possibly during the previous year, then at the time of sampling, we re-analysed the data, excluding the records for bears at the adult-age threshold, to establish if classification accuracy improved. For both sexes, candidate models were most supported based on a 3-year-old adult-age threshold. Classification accuracy did not improve with the 3-year-old bear data excluded. Male age class was predicted with a high degree of accuracy (88.4%) based on the concomitant concentrations of all four hormones. Female age class was predicted with less accuracy (77.1%) based only on testosterone and cortisol. Accuracy was reduced for females, primarily because we had poor success in correctly classifying immature bears (60%) whereas classification success for adult females was similar to that for males (84.5%). Given the small and unbalanced sample used in this study, our findings should be viewed as preliminary, but they should also provide a basis for more comprehensive future studies.
Weiß B. M., Marcillo A., Manser M., Holland R., Birkemeyer C., Widdig A. (2018): A non‐invasive method for sampling the body odour of mammals. Methods in Ecology and Evolution 9: 420-429.
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Olfaction is a central aspect of mammalian communication, providing information about individual attributes such as identity, sex, group membership or genetic quality. Yet, the chemical underpinnings of olfactory cues remain little understood, one of the reasons being the difficulty in obtaining high quality samples for chemical analysis. In this study, we adjusted and evaluated the use of thermal desorption (TD) tubes, commonly used in plant metabolomic and environmental studies, for non-invasive sampling of mammalian body odour. We obtained chemical profiles of meerkat (Suricata suricatta) body odour samples, using TD tubes analysed with gas chromatography–mass spectrometry. TD tubes captured a wide range of volatile and semi-volatile organic compounds, including compounds likely originating from the target animals. Adjustment of sampling parameters (distance, volume, flow rate, interruption of sampling) to increase the feasibility for a non-invasive application yielded samples of adequate quality. However, to minimize the variability between samples, sampling parameters should be kept constant and samples should be collected when no conspecifics are close-by. The method was sensitive enough to pick up population differences in the chemical profiles of two captive groups of meerkats, demonstrating its applicability to biological questions. With sufficiently habituated animals, the method is applicable non-invasively, allowing short- and long-term studies on a wide range of questions, including e.g. chemical signatures of kinship, diet, individual health or reproductive state.
Calamari C. V., Furtado P. V., Nichi M., Martins G. S., Sobral G., Dias J. H. M., de Oliveira C. A. (2020): Hair as an alternative noninvasive matrix: sources of variation in testosterone levels. Domestic Animal Endocrinology 72: 106477.
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Hair is one widely used alternative matrix for endocrine studies. Not only can it maintain hormone content during storage for long periods of time, but its collection also induces little to no stress. Noninvasive techniques have broadened the opportunities for endocrine research, particularly regarding wild animals. Despite its advantages, many sources of variation may affect the steroid concentration found in hair, such as body location harvested, fur color, reproductive status, and sex. Thus, domestic species, such as the dog, are an excellent and approachable model for understanding this variability. For such, we addressed diverse sources of variation in testosterone concentrations from 24 domestic dogs (Canis lupus familiaris) of the Poodle breed of various colors and neuter status, and from both sexes. The variation comprised the comparison between 2 different matrices (blood vs hair); 2 different extraction storage methods (refrigerator vs freezer); 3 body regions (head, torso, and limbs); 3 coat colors (black, brown, and white); different neuter status (intact vs castrated males) and, finally, sex. Our results showed no correlation between blood and hair testosterone concentrations. Additionally, we did not find differences related to the storage method, body region, or coat color. There were differences in concentration between males and females, but not between females and castrated males. We discuss hair testosterone levels exhibited reasonable stability, and we present practical applications for both domestic and wildlife animals.
Candeias Í. Z. D., da Motta Lima C. F., Lemos F. G., Spercoski K. M., de Oliveira C. A., Songsasen N., de Barros Vaz Guimarães M. A. (2020): First assessment of hoary fox (Lycalopex vetulus) seasonal ovarian cyclicity by non-invasive hormonal monitoring technique. Conservation Physiology 8: coaa039.
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Reproduction is key to species survival, and reproductive physiology represents a high priority investigative area for conservation biology, as it provides a basic understanding of critical life-history traits, information that is helpful for the establishment of management strategies. Here, we generated knowledge about the reproductive endocrinology of the hoary fox (Lycalopex vetulus), a small canid (2.5–4 kg) endemic to open areas of the Brazilian Cerrado and listed in the Brazilian National List of Endangered species. Specifically, we utilized non-invasive hormone monitoring methods to assess oestrogen and progestagen metabolites from eight female hoary foxes housed in five zoological institutions in the state of São Paulo—Brazil. We observed the elevations of oestrogen and progestagen metabolites between July and September in six of the eight females. No significant evidence of ovarian activity was observed during other months. Two females, who shared the same enclosure, did not show a pattern of reproductive cyclicity. Based on these characteristics, we concluded that captive hoary foxes are seasonal monoestric, with the beginning of the oestrus cycle occurring mainly in July followed by 2 months of the luteal phase when conception does not occur. We suggest the dosage of faecal metabolites of estradiol and progesterone could be used to differentiate the reproductive period from a non-reproductive period in Lycalopex vetulus females, providing relevant information about their reproductive biology that may contribute to species conservation and management strategies, such as increased ex situ reproductive success.
Karpovich S. A., Horstmann L. A., Polasek L. K. (2020): Validation of a novel method to create temporal records of hormone concentrations from the claws of ringed and bearded seals. Conservation Physiology 8: coaa073.
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Ringed (Pusa hispida) and bearded seals (Erignathus barbatus) inhabit vast and often remote areas in the Arctic, making it difficult to obtain long-term physiological information concerning health and reproduction. These seals are experiencing climate-driven changes in their habitat that could result in physiological stress. Chronic physiological stress can lead to immunosuppression, decreased reproduction and decreased growth. Recently, keratin has become a popular matrix to measure steroid hormones, such as stress-related cortisol and reproduction-related progesterone. We developed and validated methods to extract cortisol and progesterone from the claws of adult female ringed (n = 20) and bearded (n = 3) seals using enzyme immunosorbent assays. As ringed and bearded seal claws grow, a pair of dark- and light-colored bands of keratin is deposited annually providing a guide for sampling. Two processing methods were evaluated, removal of claw material with a grinding bit or grinding followed by mechanical pulverization (102 paired samples from six claws, two each from three seals). Adding the mechanical pulverization step resulted in a 1.5-fold increase in hormone extraction. Progesterone from the proximal claw band was evaluated to biologically validate claw material as a measure of pregnancy in ringed seals (n = 14). Claws from pregnant seals had significantly higher claw progesterone concentrations than from non-pregnant seals. This suggests that the elevated progesterone associated with gestation was reflected in the claws, and that the most proximal claw band was indicative of pregnancy status at time of death. Thus, although the sample size was low and the collection dates unbalanced, this study demonstrates the potential to use claws to examine an extended time series (up to 12 yrs) of cortisol and progesterone concentrations in ringed and bearded seal claws.
Christianson D., Coleman T. H., Doan Q., Haroldson M. A. (2021): Physiological consequences of consuming low-energy foods: herbivory coincides with a stress response in Yellowstone bears. Conservation Physiology 9: coab029.
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Meat, fruit, seeds and other high-energy bear foods are often highly localized and briefly available and understanding which factors influence bear consumption of these foods is a common focus of bear conservation and ecology. However, the most common bear foods, graminoids and forbs, are more widespread but of lower quality. We poorly understand how herbage consumption impacts bear physiology, such as endocrine system function that regulates homeostasis and stress responses. Here, we described bear diets with a novel approach, measuring the concentration of chlorophyll in bear scats (faecal chlorophyll) to index the proportion of the recent diet that was composed of leaves from graminoids and forbs. We measured faecal chlorophyll and faecal cortisol in 351 grizzly (Ursus arctos, n = 255) and black bear (Ursus americanus, n = 96) scats from Yellowstone National Park in 2008–2009. We compared models of faecal chlorophyll and faecal cortisol concentrations considering the effects of spatial, dietary, scat and bear-specific factors including species. Faecal chlorophyll levels were the strongest predictor of faecal cortisol in a manner that suggested an endocrine response to a low-energy diet. Both compounds were highest during the spring and early summer months, overlapping the breeding season when higher energy foods were less available. Effects of scat composition, scat weathering, bear age, bear sex, species and other factors that have previously been shown to influence faecal cortisol in bears were not important unless faecal chlorophyll was excluded from models. The top models of faecal chlorophyll suggested grazing was primarily influenced by spatial attributes, with greater grazing closer to recreational trails, implying that elevated cortisol with grazing could be a response to anthropogenic activity. Our results confirm that higher stress hormone concentrations correspond with lower quality diets in bears, particularly grazing, and that faecal chlorophyll shows promise as a metric for studying grazing behaviour and its consequences.
Jepsen E. M., Scheun J., Dehnhard M., Kumar V., Umapathy G., Ganswindt A. (2021): Non-invasive monitoring of glucocorticoid metabolite concentrations in native Indian, as well as captive and re-wilded tigers in South Africa. General and Comparative Endocrinology 308: 113783.
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Over the last century, wild tiger (Panthera tigris) numbers have declined from over 100 000 individuals to fewer than 4 000, with animals now confined to less than 5% of their historic range due to habitat loss, persecution, inadequate management, and poaching. In contrast, 15 000–20 000 tigers are estimated to be housed in captivity, experiencing conditions vastly different than their wild counterparts. A total of 280 tigers are currently held at 44 different facilities within South Africa, including zoos, semi-captive ‘re-wilded’ populations, and pets; these animals provide a unique opportunity to measure the impact of extrinsic factors, found in exotic habitats, on the adrenocortical activity of tigers. By monitoring and comparing stress-related faecal glucocorticoid metabolite (fGCM) concentrations of tigers housed at different locations, and free ranging tigers in natural tiger reserves, this project aimed to get a better understanding of the impact of extrinsic factors on adrenocortical function as a measure of stress. The results of this study showed no significant difference in fGCM concentrations between captive, re-wilded, and free-ranging tigers with the exception of one site. Furthermore, factors such as sex and season were not significant drivers of fGCM concentrations. One study group had elevated fGCM concentrations, showing population variation in the stress response. This indicates that populations are able to cope with exotic environments, however, as population-specific differences in the stress response exist, we suggest management protocols be created for each population. This study offered the unique opportunity to see how well tigers are faring outside of their native range and if having re-wilded tigers in exotic locations is a potential welfare-acceptable management option for tiger conservation globally.
Keogh M. J., Charapata P., Fadely B. S., Zeppelin T., Rea L., Waite J. N., Burkanov V., Marshall C., Jones A., Sprowls C., Wooller M. J. (2021): Whiskers as a novel tissue for tracking reproductive and stress-related hormones in North Pacific otariid pinnipeds. Conservation Physiology 9: coaa134.
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Keratinized tissues, including whiskers, are ideal for acquiring a record of physiological parameters. Most tissues provide a snapshot of physiological status; however, whiskers may support longitudinal sampling for reproductive and stress-related hormones, if hormones are incorporated as whiskers grow and concentrations change with physiological state. Whiskers from female Steller sea lions (Eumetopias jubatus) and northern fur seals (Callorhinus ursinus) were serially sectioned and pulverized and steroid hormones were extracted. Standard methods were used to validate enzyme immunoassay kits for cortisol, progesterone, 17β-estradiol and testosterone. All hormones were measurable in whisker segments from both species with progesterone concentrations showing cyclical patterns, which appear to signify previous pregnancies or luteal phases. Yearly progesterone concentrations were greater in years a pup was produced compared with years when no pup was observed. Free-ranging female Steller sea lions had reproductive rates between 0 and 1.0 (0.53 ± 0.33, n = 12) using a yearly progesterone concentration of 30 pg/mg or greater to classify a reproductive year as producing a pup and below 30 pg/mg as non-reproductive. Cortisol concentrations were greater near the root and rapidly declined, lacking any obvious patterns, throughout the rest of the whisker. Progesterone and testosterone concentrations were able to help determine sex of unknown individuals. Immunohistochemistry revealed that steroid hormones most likely do not leach out of whiskers based on the deposition patterns of progesterone and cortisol being present throughout the whisker length. Overall, measuring steroid hormones in whiskers can reveal individual reproductive histories over multiple years in sea lions and fur seals. Cyclical patterns of δ15N were useful for identifying periods of up to ~10 years of growth within whiskers, and measuring both stable isotopes and hormones may be useful for differentiating periods of active gestation from diapause and potentially track multi-year reproductive histories of female otariids.
Kinoshita K. (2021): A field‐friendly method of measuring faecal glucocorticoid metabolite concentration as a simple stress checker in snow leopards. Methods in Ecology and Evolution 12: 1734-1746.
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The hormonal analysis of excrement has been employed to non-invasively assess the physiological conditions of domestic, experimental, zoo and wild animals to. However, conventional hormone analysis techniques require frozen and refrigerated reagents, and laboratory equipment; therefore, it is almost impossible to obtain the results on-site. This study attempted to establish a method for the simple and rapid quantitative on-site analysis of faecal glucocorticoid metabolite (fGM) concentrations; this method involved using hand-shaking for faecal hormone extraction, and immunochromatography using test strips and a smartphone application. This study focused specifically on snow leopards and developed a new, simple fGM measurement method to evaluate their stress levels on-site. First, the effects of ultraviolet exposure and bacterial activity on fGM concentration, including sample age, were evaluated using faeces from a captive snow leopard. Second, a field-friendly hormone extraction method was evaluated; this method does not require storage of faeces in the field because the hormones are extracted by hand-shaking of faeces in ethanol at the time of collection. In addition, to measure hormone concentrations on-site, a simple immunochromatography measurement kit was used. The concentrations were measured by quantifying the colour reactions using a smartphone application. There was no consistent increase or decrease in fGM concentration with ultraviolet irradiation time. In addition, a high correlation was observed between concentrations extracted using the conventional method (using a vortex mixer and methanol extraction) and those from the field-friendly method (r = 0.812, p < 0.05). Changes in fGM concentration measured using immunochromatography were also highly consistent with those measured using the conventional method (enzyme immunoassay; r = 0.825, p < 0.05). The analytical validation of immunochromatography also showed high accuracy, that is parallelism (ANCOVA: F = 0.597, df = 1, p = 0.445) and recovery tests (98.2% ± 5.1% and 107.5% ± 18.7%). Further, for biological validation, the change in fGM concentration was examined before and after the transportation of the snow leopards to another zoo. This field-friendly hormone analysis method is expected to contribute as a simple and rapid stress evaluation tool to manage conservation and animal welfare by monitoring the stress levels.
Sugianto N. A., Dehnhard M., Newman C., Macdonald D. W., Buesching C. D. (2021): A non-invasive method to assess the reproductive status of the European badger (Meles meles) from urinary sex-steroid metabolites. General and Comparative Endocrinology 301: 113655.
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Due to their unique reproductive physiology and behaviour, European badgers (Meles meles) are often used as a model to study mammalian reproduction. For reproductive endocrinology, circulating hormone levels are conventionally measured directly from blood samples. However, routine blood sampling is often not practical for wild animals and may induce stress affecting measurement accuracy. Non-invasive alternatives are thus of interest. Circulating hormones are metabolized through different routes, either by the kidneys, to be excreted through urine, or by the liver, to be excreted through faeces. These metabolites can thus be used as a proxy of hormone measurements, provided the species-specific metabolic characteristics are known. Here we tested the suitability of measuring urinary metabolites of circulating plasma sex-steroid hormones (testosterone in males and oestrogen in females) with enzyme immunoassays to assess the reproductive status of the European badger (Meles meles). Biological validation evidenced that urinary testosterone metabolite (UTM) and urinary total oestrogen metabolite (UEM) excretion patterns both corresponded with seasonal badger reproductive patterns on a population level, signaling correlation over a broad time frame. On an individual level, concurrent sampling of urine and plasma showed that male plasma testosterone and UTM levels correlated significantly across seasons, but no short term correlation was evident for total oestrogen and UEM in females. Thus, in badgers, urinary sex-steroid metabolites can be used reliably in the short term to assess male reproductive status at the individual level, but only at the broader population level for females.
Montgomery T. M., Greenberg J. R., Gunson J. L., John K., Laubach Z. M., Nonnamaker E., Person E. S., Rogers H., Ronis E. M., Smale L., Steinfield K. R., Strong R., Holekamp K. E., Beehner J. C. (2022): Measuring salivary cortisol in wild carnivores. Hormones and Behavior 137: 105082.
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Salivary hormone analyses provide a useful alternative to fecal and urinary hormone analyses in non-invasive studies of behavioral endocrinology. Here, we use saliva to assess cortisol levels in a wild population of spotted hyenas (Crocuta crocuta), a gregarious carnivore living in complex social groups. We first describe a novel, non-invasive method of collecting saliva from juvenile hyenas and validate a salivary cortisol assay for use in this species. We then analyze over 260 saliva samples collected from nearly 70 juveniles to investigate the relationships between cortisol and temporal and social variables in these animals. We obtain some evidence of a bimodal daily rhythm with salivary cortisol concentrations dropping around dawn and dusk, times at which cub activity levels are changing substantially. We also find that dominant littermates have lower cortisol than singleton juveniles, but that cortisol does not vary with age, sex, or maternal social rank. Finally, we examine how social behaviors such as aggression or play affect salivary cortisol concentrations. We find that inflicting aggression on others was associated with lower cortisol concentrations. We hope that the detailed description of our methods provides wildlife researchers with the tools to measure salivary cortisol in other wild carnivores.
Naidenko S. V., Alekseeva G. S., Klyuchnikova P. S., Erofeeva M. N. (2022): Application of felid hair for non-invasive tracking of animal reproductive status and adrenal activity. Animals 12: 2792.
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Hair can be a useful matrix to examine the hormonal status of an animal, although it is difficult to correlate the results to a specific time point. The aim of this study was to evaluate seasonal changes in cortisol and testosterone levels in the hair of four feline species (lynx, Lynx lynx, n = 8; Amur wildcat, Prionailurus bengalensis euptilurus, n = 8; caracal, Caracal caracal, n = 6 and domestic cat, Felis catus, n = 17) with different breeding strategies. Animals of both sexes were sampled over the year, once per season (every three months), and the concentrations of hair testosterone and cortisol were measured by EIA. Both hormones showed annual dynamic changes, which coincided with the reproductive seasonality of the studied species. Sexual differences in testosterone level were found only during the mating season (spring for lynx and Amur wildcats, spring–summer for domestic cats), when testosterone levels were higher in males than in females. Cortisol levels were higher in males than in females in domestic cats and Amur wildcat, but also only during the mating season. Seasonal increases in testosterone were observed in three seasonal breeders (lynx, Amur wildcat and domestic cat) but not in caracal, which had high testosterone levels over the whole year. In lynx and Amur wildcat, it decreased sharply in the summer. Cortisol levels increased during the mating period in domestic cat males and lynx of both sexes; in caracal, an increase in cortisol was related to the transfer of animals to smaller winter cages. Measurements of steroids in hair can provide a reliable method for evaluating the reproductive status and the activity of the hypothalamus–pituitary–adrenal axis in several felid species.
Roffler G. H., Karpovich S., Charapata P., Keogh M. J. (2022): Validation and measurement of physiological stress and reproductive hormones in wolf hair and claws. Wildlife Society Bulletin 46: e1330.
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The use of keratinized tissues (e.g., hair, claws) to investigate physiological effects of environmental and anthropogenic stressors in free-ranging wildlife populations has increased because these tissues retain steroid hormones during growth and are relatively easy to collect and store in the field. We measured reproductive and stress-related steroid hormones in wolves (Canis lupus ligoni; n = 31) captured on Prince of Wales Island, Alaska, USA, during 1993–1994 and 2012–2014, representing periods of time when both wolf harvest and densities ranged from high to moderate. We validated enzyme immunoassay kits to measure steroid hormone concentrations in wolf guard hair, undercoat hair, and claw tip samples. Progesterone, testosterone, and cortisol were extracted and measured in the 3 keratinous tissues from wolves of different age class, sex, residency status, and collection periods. Within each tissue type, progesterone and testosterone were positively correlated (guard hair, r = 0.59, P = 0.003; undercoat hair, r = 0.55, P = 0.011; claws, r = 0.62, P ≤ 0.001) and cortisol concentrations were not related to either reproductive hormone. We were able to measure hormone concentrations in archived keratinous tissues collected up to 25 years earlier to assess stress and reproductive activity in historical samples. Our study validates a method for measuring steroid hormones in hair, and for the first time, continuously growing claws in wolves. Measurement of hormone concentrations in keratinous tissues may aid in the assessment of reproductive activity and physiological stress responses in wolf populations over long-term time periods (i.e., decades) to enhance conservation efforts of an important apex predator.
Fokidis H. B., Brock T., Newman C., Macdonald D. W., Buesching C. D. (2023): Assessing chronic stress in wild mammals using claw-derived cortisol: a validation using European badgers (Meles meles). Conservation Physiology 11: coad024.
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Measuring stress experienced by wild mammals is increasingly important in the context of human-induced rapid environmental change and initiatives to mitigate human-wildlife conflicts. Glucocorticoids (GC), such as cortisol, mediate responses by promoting physiological adjustments during environmental perturbations. Measuring cortisol is a popular technique; however, this often reveals only recent short-term stress such as that incurred by restraining the animal to sample blood, corrupting the veracity of this approach. Here we present a protocol using claw cortisol, compared with hair cortisol, as a long-term stress bio-indicator, which circumvents this constraint, where claw tissue archives the individual’s GC concentration over preceding weeks. We then correlate our findings against detailed knowledge of European badger life history stressors. Based on a solid-phase extraction method, we assessed how claw cortisol concentrations related to season and badger sex, age and body-condition using a combination of generalized linear mixed models (GLMM) (n = 668 samples from 273 unique individuals) followed by finer scale mixed models for repeated measures (MMRM) (n = 152 re-captured individuals). Claw and hair cortisol assays achieved high accuracy, precision and repeatability, with similar sensitivity. The top GLMM model for claw cortisol included age, sex, season and the sex*season interaction. Overall, claw cortisol levels were significantly higher among males than females, but strongly influenced by season, where females had higher levels than males in autumn. The top fine scale MMRM model included sex, age and body condition, with claw cortisol significantly higher in males, older and thinner individuals. Hair cortisol was more variable than claw; nevertheless, there was a positive correlation after removing 34 outliers. We discuss strong support for these stress-related claw cortisol patterns from previous studies of badger biology. Given the potential of this technique, we conclude that it has broad application in conservation biology.
CETACEANS
Hunt K. E., Rolland R. M., Kraus S. D., Wasser S. K. (2006): Analysis of fecal glucocorticoids in the North Atlantic right whale (Eubalaena glacialis). General and Comparative Endocrinology 148: 260-272.
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Very little is known about the endocrinology of the baleen whales. The highly endangered North Atlantic right whale (NARW; Eubalaena glacialis) is a good model species, because most NARW individuals are photo-identified with known histories. We used an 125I corticosterone assay, shown to reliably measure cortisol metabolites, to determine glucocorticoid metabolite concentrations in 177 NARW fecal samples collected between 1999–2004 in the Bay of Fundy, Canada. Fecal glucocorticoid metabolite concentrations varied significantly with sex and reproductive category, being highest in pregnant females (mean ±SE: 238.14 ± 74.37 ng/g) and mature males (71.6 ± 11.36), intermediate in lactating females (39.33 ± 5.82), and lower in non-reproducing females (23.11 ± 4.25) and immature males (34.33 ± 5.01) and females (14.0 ± 0.41). One case also suggests that glucocorticoids rise markedly in response to severe entanglement in fishing lines. Whales with fecal glucocorticoid content over 100 ng/g (termed “high-cort” samples) were rare, and included most pregnant females, some mature males, a fatally entangled whale, and several very young animals. Glucocorticoid concentrations were highly correlated with androgen concentrations in males and pregnant females. We analyzed the elution profiles of glucocorticoid and androgen metabolites in 13 samples with high-performance liquid chromatography (HPLC) to determine the extent to which androgen metabolites cross-react with our glucocorticoid assay. Males, pregnant females, non-pregnant females, and “high-cort” whales each had distinctly different immunoreactive HPLC profiles of glucocorticoid and androgen metabolites. A major glucocorticoid metabolite was prominent in all “high-cort” whales including the fatally entangled whale. The major fecal androgen was not testosterone but was instead a more nonpolar steroid (possibly dihydrotestosterone), which may be diagnostic of males. Androgen metabolites showed only minor cross-reactivity to our glucocorticoid assay, having a slight influence on glucocorticoid results in particular individuals. We conclude that fecal glucocorticoid analysis appears to be a useful measure of adrenal activity and reproductive condition for NARW.
Hogg C. J., Rogers T. L., Shorter A., Barton K., Miller P. J. O., Nowacek D. (2009): Determination of steroid hormones in whale blow: it is possible. Marine Mammal Science 25: 605-618.
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The feasibility of using analysis of hormone content of whale blow samples to assess reproductive function is addressed. A suitable collection method and analytical technique using liquid chromatography-mass spectrometry (LC-MS) has been developed. Blow samples were collected opportunistically from free-ranging humpback whales (Megaptera novaeangliae) (n= 35) and North Atlantic right whales (Eubalaena glacialis) (n= 18) using a 13-m carbon fiber pole with a collection device. Samples were analyzed for the presence of testosterone and progesterone using a 55% isocratic gradient with LC-MS. Testosterone was detected in four humpback whale samples and eight northern right whale samples. Progesterone was detected in seven humpback whale samples and eight northern right whale samples. This is the first documented use of lung mucosa to determine the presence of reproductive hormones in any free-swimming cetacean and may provide a novel non-invasive technique to quantify the hormonal condition of free-swimming animals that spend brief periods of time at the water’s surface.
Ayres K. L., Booth R. K., Hempelmann J. A., Koski K. L., Emmons C. K., Baird R. W., Balcomb-Bartok K., Hanson M. B., Ford M. J., Wasser S. K. (2012): Distinguishing the impacts of inadequate prey and vessel traffic on an endangered killer whale (Orcinus orca) population. Plos One 7: e36842.
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Managing endangered species often involves evaluating the relative impacts of multiple anthropogenic and ecological pressures. This challenge is particularly formidable for cetaceans, which spend the majority of their time underwater. Noninvasive physiological approaches can be especially informative in this regard. We used a combination of fecal thyroid (T3) and glucocorticoid (GC) hormone measures to assess two threats influencing the endangered southern resident killer whales (SRKW; Orcinus orca) that frequent the inland waters of British Columbia, Canada and Washington, U.S.A. Glucocorticoids increase in response to nutritional and psychological stress, whereas thyroid hormone declines in response to nutritional stress but is unaffected by psychological stress. The inadequate prey hypothesis argues that the killer whales have become prey limited due to reductions of their dominant prey, Chinook salmon (Oncorhynchus tshawytscha). The vessel impact hypothesis argues that high numbers of vessels in close proximity to the whales cause disturbance via psychological stress and/or impaired foraging ability. The GC and T3 measures supported the inadequate prey hypothesis. In particular, GC concentrations were negatively correlated with short-term changes in prey availability. Whereas, T3 concentrations varied by date and year in a manner that corresponded with more long-term prey availability. Physiological correlations with prey overshadowed any impacts of vessels since GCs were lowest during the peak in vessel abundance, which also coincided with the peak in salmon availability. Our results suggest that identification and recovery of strategic salmon populations in the SRKW diet are important to effectively promote SRKW recovery.
Rolland R. M., Parks S. E., Hunt K. E., Castellote M., Corkeron P. J., Nowacek D. P., Wasser S. K., Kraus S. D. (2012): Evidence that ship noise increases stress in right whales. Proceedings of the Royal Society B: Biological Sciences 279: 2363-2368.
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Baleen whales (Mysticeti) communicate using low-frequency acoustic signals. These long-wavelength sounds can be detected over hundreds of kilometres, potentially allowing contact over large distances. Low-frequency noise from large ships (20–200 Hz) overlaps acoustic signals used by baleen whales, and increased levels of underwater noise have been documented in areas with high shipping traffic. Reported responses of whales to increased noise include: habitat displacement, behavioural changes and alterations in the intensity, frequency and intervals of calls. However, it has been unclear whether exposure to noise results in physiological responses that may lead to significant consequences for individuals or populations. Here, we show that reduced ship traffic in the Bay of Fundy, Canada, following the events of 11 September 2001, resulted in a 6 dB decrease in underwater noise with a significant reduction below 150 Hz. This noise reduction was associated with decreased baseline levels of stress-related faecal hormone metabolites (glucocorticoids) in North Atlantic right whales (Eubalaena glacialis). This is the first evidence that exposure to low-frequency ship noise may be associated with chronic stress in whales, and has implications for all baleen whales in heavy ship traffic areas, and for recovery of this endangered right whale population.
Thompson L. A., Spoon T. R., Goertz C. E. C., Hobbs R. C., Romano T. A. (2014): Blow collection as a non-invasive method for measuring cortisol in the beluga (Delphinapterus leucas). Plos One 9: e114062.
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Non-invasive sampling techniques are increasingly being used to monitor glucocorticoids, such as cortisol, as indicators of stressor load and fitness in zoo and wildlife conservation, research and medicine. For cetaceans, exhaled breath condensate (blow) provides a unique sampling matrix for such purposes. The purpose of this work was to develop an appropriate collection methodology and validate the use of a commercially available EIA for measuring cortisol in blow samples collected from belugas (Delphinapterus leucas). Nitex membrane stretched over a petri dish provided the optimal method for collecting blow. A commercially available cortisol EIA for measuring human cortisol (detection limit 35 pg ml−1) was adapted and validated for beluga cortisol using tests of parallelism, accuracy and recovery. Blow samples were collected from aquarium belugas during monthly health checks and during out of water examination, as well as from wild belugas. Two aquarium belugas showed increased blow cortisol between baseline samples and 30 minutes out of water (Baseline, 0.21 and 0.04 µg dl−1; 30 minutes, 0.95 and 0.14 µg dl−1). Six wild belugas also showed increases in blow cortisol between pre and post 1.5 hour examination (Pre 0.03, 0.23, 0.13, 0.19, 0.13, 0.04 µg dl−1, Post 0.60, 0.31, 0.36, 0.24, 0.14, 0.16 µg dl−1). Though this methodology needs further investigation, this study suggests that blow sampling is a good candidate for non-invasive monitoring of cortisol in belugas. It can be collected from both wild and aquarium animals efficiently for the purposes of health monitoring and research, and may ultimately be useful in obtaining data on wild populations, including endangered species, which are difficult to handle directly.
Corkeron P., Rolland R. M., Hunt K. E., Kraus S. D. (2017): A right whale pootree: classification trees of faecal hormones identify reproductive states in North Atlantic right whales (Eubalaena glacialis). Conservation Physiology 5: cox006.
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Immunoassay of hormone metabolites extracted from faecal samples of free-ranging large whales can provide biologically relevant information on reproductive state and stress responses. North Atlantic right whales (Eubalaena glacialis Müller 1776) are an ideal model for testing the conservation value of faecal metabolites. Almost all North Atlantic right whales are individually identified, most of the population is sighted each year, and systematic survey effort extends back to 1986. North Atlantic right whales number <500 individuals and are subject to anthropogenic mortality, morbidity and other stressors, and scientific data to inform conservation planning are recognized as important. Here, we describe the use of classification trees as an alternative method of analysing multiple-hormone data sets, building on univariate models that have previously been used to describe hormone profiles of individual North Atlantic right whales of known reproductive state. Our tree correctly classified the age class, sex and reproductive state of 83% of 112 faecal samples from known individual whales. Pregnant females, lactating females and both mature and immature males were classified reliably using our model. Non-reproductive [i.e. ‘resting’ (not pregnant and not lactating) and immature] females proved the most unreliable to distinguish. There were three individual males that, given their age, would traditionally be considered immature but that our tree classed as mature males, possibly calling for a re-evaluation of their reproductive status. Our analysis reiterates the importance of considering the reproductive state of whales when assessing the relationship between cortisol concentrations and stress. Overall, these results confirm findings from previous univariate statistical analyses, but with a more robust multivariate approach that may prove useful for the multiple-analyte data sets that are increasingly used by conservation physiologists.
Richard J. T., Robeck T. R., Osborn S. D., Naples L., McDermott A., LaForge R., Romano T. A., Sartini B. L. (2017): Testosterone and progesterone concentrations in blow samples are biologically relevant in belugas (Delphinapterus leucas). General and Comparative Endocrinology 246: 183-193.
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Steroid hormone analysis in blow (respiratory vapor) may provide a minimally invasive way to assess the reproductive status of wild cetaceans. Biological validation of the method is needed to allow for the interpretation of hormone measurements in blow samples. Utilizing samples collected from trained belugas (Delphinapterus leucas, n = 20), enzyme immunoassays for testosterone and progesterone were validated for use with beluga blow samples. Testosterone concentrations in 40 matched blood and blow samples collected from 4 male belugas demonstrated a positive correlation (R2 = 0.52, p < 0.0001). Progesterone concentrations in 64 matching blood and blow samples from 11 females were also positively correlated (R2 = 0.60, p < 0.0001). Testosterone concentrations (mean ± SD) in blow samples collected from adult males (119.3 ± 14.2 pg/ml) were higher (p < 0.01) than that of a juvenile male (<8 years) (59.4 ± 6.5 pg/ml) or female belugas (54.1 ± 25.7 pg/ml). Among adult males, testosterone concentrations in blow demonstrated a seasonal pattern of secretion, with peak secretion occurring during the breeding season (February–April, 136.95 ± 33.8 pg/ml). Progesterone concentrations in blow varied by reproductive status; pregnant females (410.6 ± 87.8 pg/ml) and females in the luteal phase of the estrous cycle (339.5 ± 51.0 pg/ml) had higher (p < 0.0001) blow progesterone concentrations than non-pregnant females without a corpus luteum (242.5 ± 27.3 pg/ml). Results indicate that blow sample analysis can be used to detect variation in reproductive states associated with large differences in circulating testosterone or progesterone in belugas.
Burgess E. A., Hunt K. E., Kraus S. D., Rolland R. M. (2018): Quantifying hormones in exhaled breath for physiological assessment of large whales at sea. Scientific Reports 8: 10031.
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Exhaled breath analysis is a non-invasive assessment tool that has shown promise in human diagnostics, and could greatly benefit research, management, and conservation of large whales. However, hormone assessment of whale respiratory vapor (blow) has been challenged by variable water content and unknown total volume of collected samples. To advance this technique, we investigated urea (a compound present in narrow range in circulation) as a normalizing factor to correct for blow sample concentration. Normalized progesterone, testosterone, and cortisol concentrations of 100 blow samples from 46 photo-identified North Atlantic right whales (Eubalaena glacialis) were more biologically relevant compared to absolute estimates, varying by sex, age class, or individual. Progesterone was elevated in adult females compared with other cohorts and highest in one independently confirmed pregnant female. For both sexes, testosterone was two-fold higher in reproductively mature whales but studied adult females showed the widest variation. Cortisol was present in relatively low concentrations in blow and demonstrated variation between individual whales, suggesting potential for studies of individual differences in adrenal activity. Incorporation of methodologies that normalize sample concentration are essential for blow hormone analysis of free-swimming whales, and measurement of urea could be used to optimize non-invasive physiological assessment of whales.
Valenzuela-Molina M., Atkinson S., Mashburn K., Gendron D., Brownell Jr R. L. (2018): Fecal steroid hormones reveal reproductive state in female blue whales sampled in the Gulf of California, Mexico. General and Comparative Endocrinology 261: 127-135.
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Steroid hormone assessment using non-invasive sample collection techniques can reveal the reproductive status of aquatic mammals and the physiological mechanisms by which they respond to changes in their environment. A portion of the eastern North Pacific blue whale (Balaenoptera musculus) population that seasonally visits the Gulf of California, Mexico has been monitored using photo-identified individuals for over 30 years. The whales use the area in winter-early spring for nursing their calves and feeding and it therefore is well suited for fecal sample collection. Using radioimmunoassays in 25 fecal samples collected between 2009 and 2012 to determine reproductive state and stress, we validated three steroid hormones (progesterone, corticosterone and cortisol) in adult female blue whales. Females that were categorized as pregnant had higher mean fecal progesterone metabolite concentrations (1292.6 415.6 ng) than resting and lactating females (14.0 3.7 ng; 23.0 5.4 ng, respectively). Females classified as pregnant also had higher concentrations of corticosterone metabolites (37.5 9.9 ng) than resting and lactating females (17.4 2.0 ng; 16.8 2.8 ng, respectively). In contrast, cortisol metabolite concentrations showed high variability between groups and no significant relationship to reproductive state. We successfully determined preliminary baseline parameters of key steroid hormones by reproductive state in adult female blue whales. The presence of pregnant or with luteal activity and known lactating females confirms that the Gulf of California is an important winter-spring area for the reproductive phase of these blue whales. The baseline corticosterone levels we are developing will be useful for assessing the impact of the increasing coastal development and whale-watching activities on the whales in the Gulf of California.
Hunt K. E., Robbins J., Buck C. L., Bérubé M., Rolland R. M. (2019): Evaluation of fecal hormones for noninvasive research on reproduction and stress in humpback whales (Megaptera novaeangliae). General and Comparative Endocrinology 280: 24-34.
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Fecal hormone analysis shows high potential for noninvasive assessment of population-level patterns in stress and reproduction of marine mammals. However, the marine environment presents unique challenges for fecal sample collection. Data are still lacking on collection methodology and assay validations for most species, particularly for those mysticete whales that have variable diets. In this study we tested collection techniques for fecal samples of free-swimming humpback whales (Megaptera novaeangliae), and validated immunoassays for five steroid and thyroid hormones. Resulting data were used for preliminary physiological validations, i.e., comparisons to independently confirmed sex and reproductive state. Pregnant females had significantly higher fecal progestins and glucocorticoids than did other demographic categories of whales. Two possible cases of previously undetected pregnancies were noted. Males had significantly higher fecal testosterone metabolites than nonpregnant females. Fecal glucocorticoids were significantly elevated in pregnant females and mature males compared to nonpregnant females. Calf fecal samples had elevated concentrations of all fecal hormones. Fecal thyroid hormones showed a significant seasonal decline from spring to summer. Though sample sizes were small, and sampling was necessarily opportunistic, these patterns indicate that noninvasive fecal hormone analysis may facilitate studies of reproduction, stress and potentially energetics in humpback whales.
Lemos L. S., Olsen A., Smith A., Chandler T. E., Larson S., Hunt K., Torres L. G. (2020): Assessment of fecal steroid and thyroid hormone metabolites in eastern North Pacific gray whales. Conservation Physiology 8: coaa110.
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Baleen whale fecal samples have high potential for endocrine monitoring, which can be used as a non-invasive tool to identify the physiological response to disturbance events and describe population health and vital rates. In this study, we used commercial enzyme-linked immunosorbent assays to validate and quantify fecal steroid (progestins, androgens and glucocorticoids) and thyroid hormone metabolite concentrations in eastern North Pacific gray whales (Eschrichtius robustus) along the Oregon coast, USA, from May to October of 2016–2018. Higher mean progestin metabolite concentrations were observed in postweaning females, followed by pregnant females. Mean androgen, glucocorticoid and thyroid metabolites were higher in mature males. Progestin, glucocorticoids and thyroid fecal metabolites varied significantly by year, with positive correlations between progestin and androgen, and between glucocorticoid and thyroid metabolites. We also present two case studies of a documented injured whale and a mature male displaying reproductive competitive behavior, which provide reference points for physiologically stressed individuals and adult breeding males, respectively. Our methods and findings advance the knowledge of baleen whale physiology, can help guide future research on whale physiology and can inform population management and conservation efforts regarding minimizing the impact of anthropogenic stressors on whales.
Hudson J. M., Anderson W. G., Marcoux M. (2021): Measurement of cortisol in blow samples collected from free‐swimming beluga whales (Delphinapterus leucas). Marine Mammal Science 37: 888-900.
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Blow or respiratory vapor collection is a noninvasive technique for monitoring the physiology of cetaceans and although this technique shows promise, a major challenge of blow collection is difficulty quantifying samples due to variable amounts of seawater contamination. Here, we aimed to (1) determine whether blow samples could be collected from free-swimming belugas, (2) assess whether urea could be used as a dilution marker to normalize blow samples, and (3) determine which factors influenced cortisol and urea concentrations. We collected a total of 252 blow samples from free-swimming belugas in the Churchill River in Manitoba, Canada. Cortisol, an indicator of individual stress and health, was detected in 65.87% of samples with concentrations ranging from 6.73 to 963.17 pg/ml of extract volume, while urea was detected in 90.48% of samples with concentrations ranging from 0.21 to 20.30 mg/L. We were unable to verify the efficacy of urea as a dilution marker to normalize blow samples from free-swimming belugas; however, absolute cortisol concentrations varied based on sample device and quantity rating. Although we demonstrated that blow can be successfully collected from free-swimming belugas, further refinement of this technique is needed before it can be used as a reliable method for physiological assessments.
Reckendorf A., Schmicke M., Bunskoek P., Anderson Hansen K., Thybo M., Strube C., Siebert U. (2021): Is harbor porpoise (Phocoena phocoena) exhaled breath sampling suitable for hormonal assessments? Animals 11: 907.
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Over the last decades, exhaled breath sampling has been established for laboratory analysis in various cetacean species. Due to their small size, the usability of respiratory vapor for hormone assessments was questionable in harbor porpoises (Phocoena phocoena). This pilot study compared three different blow collection devices for their suitability in the field and during laboratory processing: a sterile petri dish covered by a Nitex membrane, as well as sterile 50 mL centrifuge tubes with or without manganese(II) chloride as a stabilizer. Collected exhales varied between three, five or ten, depending on feasibility. Hormones were extracted through an ether mix, followed by centrifugal evaporation and cortisol analysis using an immunoassay. Although close to the lower end of the assay’s dynamic range, the ELISA produced results (n = 110, 0.102–0.937 ng/mL). Hence, a simple 50 mL centrifuge tube was determined as the best suited blow collection device, while three consecutive exhales proved sufficient to yield results. These findings are promising regarding the suitability of exhaled breath as a matrix for future endocrine and immune system-related studies in harbor porpoises. If further advanced, blow sampling can become an important, non-invasive tool for studying and monitoring health, stress levels and diseases in harbor porpoises.
Pirotta E., Fernandez Ajó A., Bierlich K. C., Bird C. N., Buck C. L., Haver S. M., Haxel J. H., Hildebrand L., Hunt K. E., Lemos L. S., New L., Torres L. G. (2023): Assessing variation in faecal glucocorticoid concentrations in gray whales exposed to anthropogenic stressors. Conservation Physiology 11: coad082.
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Understanding how individual animals respond to stressors behaviourally and physiologically is a critical step towards quantifying long-term population consequences and informing management efforts. Glucocorticoid (GC) metabolite accumulation in various matrices provides an integrated measure of adrenal activation in baleen whales and could thus be used to investigate physiological changes following exposure to stressors. In this study, we measured GC concentrations in faecal samples of Pacific Coast Feeding Group (PCFG) gray whales (Eschrichtius robustus) collected over seven consecutive years to assess the association between GC content and metrics of exposure to sound levels and vessel traffic at different temporal scales, while controlling for contextual variables such as sex, reproductive status, age, body condition, year, time of year and location. We develop a Bayesian Generalized Additive Modelling approach that accommodates the many complexities of these data, including non-linear variation in hormone concentrations, missing covariate values, repeated samples, sampling variability and some hormone concentrations below the limit of detection. Estimated relationships showed large variability, but emerging patterns indicate a strong context-dependency of physiological variation, depending on sex, body condition and proximity to a port. Our results highlight the need to control for baseline hormone variation related to context, which otherwise can obscure the functional relationship between faecal GCs and stressor exposure. Therefore, extensive data collection to determine sources of baseline variation in well-studied populations, such as PCFG gray whales, could shed light on cetacean stress physiology and be used to extend applicability to less-well-studied taxa. GC analyses may offer greatest utility when employed as part of a suite of markers that, in aggregate, provide a multivariate measure of physiological status, better informing estimates of individuals’ health and ultimately the consequences of anthropogenic stressors on populations.
Ross A. R., Liao X., Brown T. M. (2023): Simultaneous determination of steroid hormones and pharmaceuticals in killer whale (Orcinus orca) faecal samples by liquid chromatography tandem mass spectrometry. Conservation Physiology 11: coad081.
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We describe a non-invasive method for profiling selected hormones, pharmaceuticals and personal care products (PPCPs) in killer whales (Orcinus orca) based on analysis of faecal samples by liquid chromatography tandem mass spectrometry (LC–MS/MS). The method targets 21 compounds of interest including glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, selective serotonin uptake inhibitors and an antibacterial/antifungal agent. This method is suitable for routine simultaneous determination of target compounds in killer whale faecal samples as well as validation of immunoassays for the detection and measurement of steroid hormones in faeces. The optimized method involves extraction of freeze-dried faecal material with reagent alcohol and water followed by isolation of the analytes using solid phase extraction with hydrophilic–lipophilic balance cartridges and liquid–liquid extraction with methyl tertiary-butyl ether. Reconstituted extracts were analysed by LC–MS/MS using an electrospray ionization interface. Method limit of quantification ranged from 0.06 to 45.2 ng/g in freeze-dried faecal samples. Except for sertraline, triclosan and estradiol (which was not recovered at the lowest spiked concentration), average intra- and inter-day precisions were within 10%, and average recoveries were between 89.3% and 129.3%, for faecal samples spiked with 5.3, 26.7 or 133 ng/g of each analyte. The method was applied successfully to the analysis of hormones and PPCPs in whale faeces during which 17α-hydroxyprogesterone, a common intermediate in steroid biosynthesis that cross-reacts with precursors and sulphated conjugates in immunoassays, was identified and quantified in all samples.
ELEPHANTS
Munshi‐South J., Tchignoumba L., Brown J., Abbondanza N., Maldonado J. E., Henderson A., Alonso A. (2008): Physiological indicators of stress in African forest elephants (Loxodonta africana cyclotis) in relation to petroleum operations in Gabon, Central Africa. Diversity and Distributions 14: 995-1003.
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Human activities are major determinants of forest elephant (Loxodonta africana cyclotis) distribution in Gabon, but the types and intensity of disturbance that elephants can tolerate are not known. We conducted dung surveys within the Gamba Complex of Protected Areas in SW Gabon to examine (1) the feasibility of noninvasive faecal analyses for monitoring stress physiology, and (2) the influence of petroleum operations on stress levels in forest elephants. We identified multiple dung piles from the same individual by matching their eight‐locus microsatellite genotypes, and measured faecal concentrations of glucocorticoid metabolites as an indicator of stress in areas subject to different levels of disturbance: (1) Loango National Park (2) an ‘industrial corridor’ dominated by oil fields, and (3) a nearby area of human settlements. We obtained unique microsatellite genotypes and faecal glucocorticoid metabolite (FGM) concentrations for 150 forest elephant individuals, which is the largest hormonal data set for wild African forest elephants to date. Adults exhibited higher mean FGM concentrations than juveniles, and in contradiction of our expectations of chronic stress around oil fields, elephants in Loango National Park exhibited significantly higher FGM concentrations than elephants in the industrial corridor. We argue that forest elephants in the industrial corridor of the Gamba Complex have become acclimated to oil fields, resulting in part from oil company regulations that minimize stressful interactions between elephants and petroleum operations. Our findings for a flagship species with substantial ecological requirements bode well for other taxa, but additional studies are needed to determine whether oil operations are compatible over their life span with rain forest ecosystems in Central Africa.
Ahlering M. A., Millspaugh J. J., Woods R. J., Western D., Eggert L. S. (2011): Elevated levels of stress hormones in crop‐raiding male elephants. Animal Conservation 14: 124-130.
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Crop raiding is one of the most common forms of human–elephant conflict. Deterring elephants from raiding crops requires an understanding of the factors influencing the behavior of the individuals involved. We collected fecal samples from five group ranches in southern Kenya where crop‐raiding incidents had occurred (n =10) and two protected areas, Amboseli National Park (n =24) and Maasai Mara National Reserve (n =20). We used molecular sexing to sex the individuals and radioimmunoassay kits to determine the level of glucocorticoid metabolites (i.e. stress hormones) in their dung. All crop‐raiding individuals were male and had a significantly elevated concentration of glucocorticoid metabolites as compared with the Amboseli elephants (W =12, P =0.0005). We detected no significant difference between Maasai Mara elephants and either Amboseli or the crop‐raiding elephants when just males were compared. Our results suggest that crop raiding may be related to stress in elephants.
Ahlering M. A., Maldonado J. E., Eggert L. S., Fleischer R. C., Western D., Brown J. L. (2013): Conservation outside protected areas and the effect of human‐dominated landscapes on stress hormones in savannah elephants. Conservation Biology 27: 569-575.
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Biodiversity conservation strategies are increasingly focused on regions outside national protected areas, where animals face numerous anthropogenic threats and must coexist with human settlements, livestock, and agriculture. The effects of these potential threats are not always clear, but they could have profound implications for population viability. We used savannah elephants (Loxodonta africana) as a case study to assess the physiological stress associated with living in a human‐livestock‐dominated landscape. We collected samples over two 3‐month periods in 2007 and 2008. We used fecal DNA to identify 96 individual elephants in a community conservation area (CCA) and measured fecal glucocorticoid metabolite (FGM) concentrations as a proxy for stress. The CCA is community Maasai land managed for livestock and wildlife. We compared the FGM concentrations from the CCA to FGM concentrations of 40 elephants in Amboseli National Park and 32 elephants in the Maasai Mara National Reserve, where human settlements and intense livestock grazing were absent. In the CCA, we found no significant individual differences in FGM concentrations among the elephants in 2007 (p = 0.312) or 2008 (p = 0.412) and no difference between years (p = 0.616). The elephants in the CCA had similar FGM concentrations to the Maasai Mara population, but Amboseli elephants had significantly lower FGM concentrations than those in either Maasai Mara or the CCA (Tukey pairwise test, p < 0.001), due primarily to females excreting significantly lower FGM relative to males (p = 0.025). In the CCA, there was no relation among female group size, average pairwise group relatedness, and average group FGM concentration. We found no clear evidence of chronic stress in elephants living on CCA communal land, which is encouraging for conservation strategies promoting the protection of animals living outside protected areas.
Hunninck L., Ringstad I. H., Jackson C. R., May R., Fossøy F., Uiseb K., Killian W., Palme R., Røskaft E. (2017): Being stressed outside the park – conservation of African elephants (Loxodonta africana) in Namibia. Conservation Physiology 5: cox067.
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The conservation of the African savanna elephant (Loxodonta africana) is of prime importance for many African countries. Interactions between elephants and humans are known to induce stress and thereby have the potential to affect elephants’ fitness. In Namibia, anthropogenic disturbances are increasing due to increasing human population size and development, particularly near protected areas, such as national parks. In this study, we investigated elephant stress levels in relation to their land use, specifically their protection status, comparing elephants within Etosha National Park in Namibia with elephants residing outside the park. We noninvasively collected dung samples of 91 elephants and determined the concentration of faecal glucocorticoid metabolites (fGCM), an indicator of physiological stress. Elephants outside the park (N = 35) had significantly higher concentrations of fGCM than those inside ENP (N = 56), suggesting that, despite including community-based conservancies, unprotected areas are more stressful for elephants than protected areas, most likely due to increased interactions with humans. We also found that males had lower fGCM concentrations than females, but no significant effect of age, body size or group size was detected. Additionally, herd sizes were significantly smaller and calf recruitment was potentially lower in unprotected areas. These findings underpin the importance of protected areas such as ENP, while encouraging decision-makers to continue reducing and mitigating potential human-induced disturbances.
Pokharel S. S., Singh B., Seshagiri P. B., Sukumar R. (2019): Lower levels of glucocorticoids in crop‐raiders: diet quality as a potential ‘pacifier’ against stress in free‐ranging Asian elephants in a human‐production habitat. Animal Conservation 22: 177-188.
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Overlapping habitats and sharing of resources between elephants and people has led to intense elephant-human conflicts, especially crop depredation by elephants, across elephant-range countries. While raiding agricultural crops, elephants face numerous threats from people through chase, injury and the risk of death which could enhance the associated energetic costs, ultimately elevating their stress levels. We hypothesized that crop-raiders (in the human-production habitat) would show higher faecal glucocorticoid metabolite (fGCM) levels, a proxy of stress-response, as compared to nonraiders (in protected forests). To study this, 208 faecal samples were collected from crop-raiding elephants in a human-production habitat and 394 samples from nonraiding elephants in protected forests during 2013 and 2015. Contrary to our expectation, fGCM levels were significantly higher in nonraiding than in crop-raiding elephants of both sexes. As one of the possible factors for lower fGCM in elephants inhabiting the human-production habitat, the influence of benefits obtained from foraging here was assessed. For this, the difference in vegetation greenness (standing biomass) between the human-production habitat and the protected forests was analysed from remotely-sensed Normalized Differential Vegetation Index (NDVI), and further confirmed by measuring dietary quality (faecal nitrogen (N) content and C:N ratio as proxies for crude protein). Interestingly, higher NDVI values (greater biomass availability), higher N content and lower faecal C:N ratio (indicating higher protein content in the diet) of elephants in the human-production habitat suggested enhanced nutritional levels here as compared to protected forests. Further, there were significant correlations between faecal C:N ratio (positive) or N content (negative) and fGCM levels. These findings suggest that crop-raiding comes with the benefits of a superior quality diet which may help in reducing human-induced stress-response in elephants inhabiting or foraging within human-production habitats.
Szott I. D., Pretorius Y., Ganswindt A., Koyama N. F. (2019): Physiological stress response of African elephants to wildlife tourism in Madikwe Game Reserve, South Africa. Wildlife Research 47: 34-43.
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Wildlife tourism has been shown to increase stress in a variety of species and can negatively affect survival, reproduction, welfare, and behaviour of individuals. In African elephants, Loxodonta africana, increased physiological stress has been linked to use of refugia, rapid movement through corridors, and heightened aggression towards humans. However, we are unaware of any studies assessing the impact of tourism pressure (tourist numbers) on physiological stress in elephants. We used faecal glucocorticoid metabolite (fGCM) concentrations to investigate whether tourist numbers in Madikwe Game Reserve, South Africa, were related to changes in physiological stress in elephants. We repeatedly collected dung samples (n = 43) from 13 individually identified elephants over 15 months. Using a generalised linear mixed model and a Kenward–Roger approximation, we assessed the impact of monthly tourist numbers, season, age, and sex on elephant fGCM concentrations. High tourist numbers were significantly related to elevated fGCM concentrations. Overall, fGCM concentrations increased by 112% (from 0.26 to 0.55 µg g−1 dry weight) in the months with the highest tourist pressure, compared to months with the lowest tourist pressure. Managers of fenced reserves should consider providing potential alleviation measures for elephants during high tourist pressure, for example, by ensuring that refuge areas are available. This may be of even higher importance if elephant populations have had traumatic experiences with humans in the past, such as poaching or translocation. Such management action will improve elephant welfare and increase tourist safety. Although tourism can generate substantial revenue to support conservation action, careful monitoring of its impact on wildlife is required to manage potential negative effects.
Carlin E., Teren G., Ganswindt A. (2020): Non-invasive assessment of body condition and stress-related fecal glucocorticoid metabolite concentrations in African elephants (Loxodonta africana) roaming in Fynbos vegetation. Animals 10: 814.
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Fynbos is a unique endemic vegetation type belonging to the Cape Floral Kingdom in the Western Cape Province of South Africa, representing the smallest of the six floral kingdoms in the world. Nowadays, only a few game reserves in this region support populations of African elephants (Loxodonta africana), and thus, little information exists regarding the suitability of the nutritionally poor Fynbos vegetation for these megaherbivores. Using already established non-invasive methods, the monitoring of individual body conditions and fecal glucocorticoid metabolite (fGCM) concentrations, as a measure of physiological stress, was performed to examine a herd of 13 elephants in a Western Cape Province Private Game Reserve, during two monitoring periods (April and June 2018), following a severe drought. The results indicate that overall median body condition scores (April and June: 3.0, range 2.0–3.0) and fGCM concentrations (April: 0.46 µg/g dry weight (DW), range 0.35–0.66 µg/g DW; June: 0.61 µg/g DW, range 0.22–1.06 µg/g DW) were comparable to those of other elephant populations previously studied utilizing the same techniques. These findings indicate that the individuals obtain sufficient nutrients from the surrounding Fynbos vegetation during the months monitored. However, a frequent assessment of body conditions and stress-associated fGCM concentrations in these animals would assist conservation management authorities and animal welfare practitioners in determining ways to manage this species in environments with comparably poorer nutritional vegetation.
Plangsangmas T., Brown J. L., Thitaram C., Silva-Fletcher A., Edwards K. L., Punyapornwithaya V., Towiboon P., Somgird C. (2020): Circadian rhythm of salivary immunoglobulin A and associations with cortisol as a stress biomarker in captive Asian elephants (Elephas maximus). Animals 10: 157.
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Salivary immunoglobulin A (sIgA) has been proposed as a potential indicator of welfare for various species, including Asian elephants, and may be related to adrenal cortisol responses. This study aimed to distinguish circadian rhythm effects on sIgA in male and female Asian elephants and compare patterns to those of salivary cortisol, information that could potentially have welfare implications. Subjects were captive elephants at an elephant camp in Chiang Mai province, Thailand (n = 5 males, 5 females). Salivette® kits were used to collect saliva from each elephant every 4 h from 06:00 to 22:00 h for 3 consecutive days (n = 15 samples/elephant). Enzyme immunoassays were used to quantify concentrations of IgA and cortisol in unextracted saliva. Circadian rhythm patterns were determined using a generalized least-squares method. Both sIgA and cortisol followed a circadian rhythm, although the patterns differed. sIgA displayed a daily quartic trend, whereas cortisol concentrations demonstrated a decreasing linear trend in concentrations throughout the day. There was no clear relationship between patterns of sIgA and salivary cortisol, implying that mechanisms of control and secretion differ. Results demonstrate for the first time that circadian rhythms affect sIgA, and concentrations follow a daily quartic pattern in Asian elephants, so standardizing time of collection is necessary.
Szott I. D., Pretorius Y., Ganswindt A., Koyama N. F. (2020): Normalized difference vegetation index, temperature and age affect faecal thyroid hormone concentrations in free-ranging African elephants. Conservation Physiology 8: coaa010.
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Conservation biologists can use hormone measurements to assess animals’ welfare, reproductive state, susceptibility to stressors, as well as energy expenditure. Quantifying hormone concentrations from faecal samples is particularly advantageous as samples can be collected without disturbing animals’ behaviour. In order for an endocrine marker to be useful for wildlife managers, we need to understand how extrinsic and intrinsic factors affect hormone concentrations in free-ranging animal populations. Thyroid hormones are linked to basal metabolic rate and energy expenditure. Previous research demonstrated that triiodothyronine (T3) can be measured successfully in faecal matter of African elephants, Loxodonta africana. However, to our knowledge, research into factors affecting changes in elephant T3 levels has only been carried out in captive elephants so far. Thus, we present the first study of faecal T3 metabolite (mT3) concentrations of a large population of free-ranging African elephants. Over 15 months, we collected faecal samples from identified (n = 43 samples) and unidentified (n = 145 samples) individuals in Madikwe Game Reserve, South Africa. We investigated whether vegetative productivity [normalized difference vegetation index (NDVI)] in interaction with mean monthly temperature, age and sex affected mT3 concentrations. We found a significant negative interaction effect of NDVI and temperature. Increasing NDVI was related to higher concentrations of mT3, but increasing temperature was related to a decrease in mT3 concentrations in individually identified and unidentified elephants. In unidentified individuals, juvenile elephants had significantly higher mT3 concentrations compared to adult elephants. Faecal T3 can successfully be quantified in samples from free-ranging elephant populations and thus provides insight into energy expenditure in large herbivores.
de Andrés P. J., Cáceres S., Crespo B., Silván G., Illera J. C. (2021): Non-invasive determination of annual fecal cortisol, androstenedione, and testosterone variations in a herd of male Asian elephants (Elephas maximus) and their relation to some climatic variables. Animals 11: 2723.
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The measurement of stress and reproductive hormones in wild animal species by non-invasive methods is of special interest. To assess whether the adrenal and gonadal hormones show annual variations in male Asian elephants (Elephas maximus) and to evaluate whether there is any influence of climatic variables on hormonal secretion, fecal samples were taken from a herd of 7 Asian elephants over a 14-month period to subsequently determine the concentrations of testosterone (T), androstenedione (A4), and cortisol (C) by a validated immunoassay technique. Data referring to three climatic variables in the place and period of study were collected, namely monthly mean values of temperature, humidity and rainfall. Levels of T and A4 showed two major increases in July (T: 1088.35 ± 131.04 ng/g; A4: 480.40 ± 50.86 ng/g) and October (T: 825.09 ± 31.60 ng/g; A4: 319.96 ± 32.69 ng/g) (p < 0.05). Our results show a secretion of fecal androgens dependent on temperature (T and A4), and humidity (T). Male musth was detected during the initial increases of T and A4 levels. The highest concentrations of C were observed in September (156.67 ± 60.89 ng/g) (p < 0.05), probably due to the stressful fights that occurred during the musth period. The observed results of the fecal levels of T, A4 and C were similar to those obtained by invasive methods. In conclusion, fecal secretion of the three hormones in these captive male Asian elephants showed variations related in some cases to different weather factors.
Pokharel S. S., Yoneda H., Yanagi M., Sukumar R., Kinoshita K. (2021): The tail-tale of stress: an exploratory analysis of cortisol levels in the tail-hair of captive Asian elephants. PeerJ 9: e10445.
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Assessment of physiological states by measuring biomarkers, such as cortisol, has significantly contributed to the monitoring of health, welfare and management of animals. Immunoreactive cortisol in hair (hC) has been used widely for deciphering ‘stressful’ past-events in various wild and captive animals. However, no such studies have been done in long-lived mammals. In this first exploratory study in elephants, we assessed (i) tail-hair growth rate (TGR) and (ii) hC levels in tail-hair samples from six captive Asian elephants from two zoos in Japan for comparing hC levels with zoo-keepers’ records of distinct biological events over a c.0.5–2.0-year period. Tail-hair samples were cut into segments (based on monthly growth rate), pulverized or minced and a validated cortisol enzyme-immunoassay employed to measure hC levels. When the hC levels of all individuals were compared with the keepers’ records, a posteriori, most of the high hC levels were found to be associated with ‘stressful’ or distinct behavioural events such as pathological (anaemia, colic infection, skin infection, oral sores), psychosocial (reluctance in entering the enclosure, presence of a calf) and husbandry practice-related (contact trials/ space sharing) conditions, indicating that tail-hair indeed can be a potential ‘retrospective’ calendar of physiological health of an animal. Our observations open up the possibility of using the tail-hair as an alternative matrix to reconstruct the physiological history of elephants.
Seltmann M. W., Jackson J., Lynch E., Brown J. L., Htut W., Lahdenperä M., Lummaa V. (2022): Sex-specific links between the social landscape and faecal glucocorticoid metabolites in semi-captive Asian elephants. General and Comparative Endocrinology 319: 113990.
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Although social behaviour is common in group-living mammals, our understanding of its mechanisms in long-lived animals is largely based on studies in human and non-human primates. There are health and fitness benefits associated with strong social ties, including increased life span, reproductive success, and lower disease risk, which are attributed to the proximate effects of lowered circulating glucocorticoid hormones. However, to deepen our understanding of health-social dynamics, we must explore species beyond the primate order. Here, using Asian elephants as a model species, we combine social data generated from semi-captive timber elephants in Myanmar with measurements of faecal glucocorticoid metabolite (FGM) concentrations. These data enable a “natural experiment” because individuals live in work groups with different demographic compositions. We examine sex-specific FGM concentrations for four different aspects of an individuals’ social world: general sociality, work group size, sex ratio and the presence of immatures (<5 years) within the work group. Males experienced lower FGM concentrations when engaged in more social behaviours and residing in female-biased work groups. Surprisingly, females only exhibited lower FGM concentrations when residing with calves. Together, our findings highlight the importance of sociality on individual physiological function among elephants, which may have broad implications for the benefits of social interactions among mammals.
Troup G., Heinsohn R., King L. E., Edwards K. L. (2022): Exploring seasonal variation in the faecal glucocorticoid concentrations of African elephants (Loxodonta africana) living in a drought-prone, anthropogenic landscape. Wildlife Research 49: 415-427.
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The wide-ranging movement of African elephants (Loxodonta africana) is largely driven by the spatio-temporal distribution of water and forage, and often leads to their travelling outside of formally protected areas. With an increase in drier periods predicted across Africa due to climate change, it is critically important to understand how elephants physiologically respond to alterations in the availability and distribution of resources. We assessed variation in the adrenal activity of elephants living in Kenya’s Tsavo East National Park between wet and dry seasons, as well as among individuals found in Tsavo East National Park and privately protected Rukinga Wildlife Sanctuary (part of the Kasigau REDD+ wildlife corridor) in the dry season, when the area experiences an influx of elephants in search of alternative resources. We opportunistically collected fresh elephant faecal samples across the two seasons and locations for analysis of faecal glucocorticoid metabolite (fGCM, a proxy for stress) and nitrogen (Nf, an indirect measure of diet quality) concentrations. The Normalised Difference Vegetation Index (NDVI) was employed as an additional indicator of habitat quality. In Tsavo East N.P. Nf and NDVI were both significantly lower during the dry season, indicating poorer habitat quality compared with the wet season. Although elephant fGCM concentrations tended to be higher in the dry season than the wet, the differences were not significant. There was no difference between elephant fGCMs measured in Tsavo East N.P. and Rukinga W.S. during the dry season, nor in habitat quality between the two locations. Elephants living in Tsavo may be physiologically unaffected by (or adapt to) typical seasonal changes in habitat quality that could lead to nutritional stress; however, whether this is the case during extended periods of severe drought requires further investigation. Rukinga W.S. provides a safe haven of sufficient habitat quality for elephants searching for alternative resources during this period. Extended dry periods are likely to become increasingly common in semiarid savannahs, and implications for wildlife must be closely monitored. Privately protected land outside formally protected areas plays an important role in conservation efforts, which should be considered when making land management plans.
Lacomme L., Guerbois C., Fritz H., Ganswindt A., Rey B. (2023): Validation of a field-friendly faeces drying and storage method for quantifying faecal glucocorticoid metabolites in African elephants (Loxodonta africana) opens up new perspectives for conservationists. Conservation Physiology 11: coad053.
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Faecal glucocorticoid metabolites (fGCMs) are a relevant means of non-invasively assessing adrenocortical activity and thus, a key physiological stress response in wildlife populations. However, the widespread use of fGCMs as a stress-related biomarker in conservation biology is often hampered by the logistical challenge of storing collected faecal material frozen until it reaches the laboratory for analysis. Although alternative approaches to minimize potential alteration of fGCM composition post-defecation have been recently identified, there is to our knowledge, no satisfactory alternative method established for the preservation of elephant dung. In this study, we validated a field-friendly protocol for dehydrating African elephant faeces samples using a food dehydrator with desiccant and investigated the stability of fGCM concentrations in the dehydrated faeces when stored at ambient temperature. We collected 40 faecal samples from African elephants and compared fGCM concentrations of freeze-dried and dehydrated sample sub-sets. Samples dried in the field showed a slight but significant overall −6% reduction in fGCM concentration compared with frozen control samples. However, fGCM concentrations following field dehydration protocol match those of control samples with high accuracy, as evidenced by the low bias and strong coefficient of determination between the two approaches (R2 = 0.88). In addition, over nearly 2 months, storage time at ambient temperature of the dehydrated samples had no effect on the fGCM concentrations compared with those measured in the control samples (F-statistic = 1.82; P = 0.18). Dehydrating the samples in the field thus provides an easy and cost-effective alternative to freezing, especially when working in remote areas with unstable electrical supply. Our results encourage the widespread use of fGCMs by conservationists as non-invasive means of steroid monitoring of African elephants in the current context of a general increase in wildlife welfare research. Future studies are needed to extend the use of this protocol to other species and to other steroid classes.
HEDGEHOGS, SHREWS, AND DESMANS
Medger K., Prins A., Lutermann H., Ganswindt A., Ganswindt S. B., Bennett N.C. (2021): Repeatability of daily profiles of baseline glucocorticoid metabolites measured in the urine and faeces of eastern rock sengis (Elephantulus myurus). General and Comparative Endocrinology 312: 113857.
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Repeatability of hormone concentrations is of great interest for studies investigating the evolution of hormonal traits. Particularly the repeatability of glucocorticoids (GCs) in response to a stressor is frequently investigated, but often only point (initial and/or response value), or single measures are used. A new method takes into account the entire individual hormone profile and generates an individual profile repeatability (PR) score. The method was developed for response profiles, but it may also be valuable for baseline values in species with diurnal changes in hormone concentrations. GCs are determined in a variety of matrices, and repeatability can vary considerably depending on the matrix. We investigated the repeatability of baseline GC metabolite (GCM) concentrations measured in urine (uGCM) and faeces (fGCM) of captive eastern rock sengis (Elephantulus myurus) using the more traditional linear mixed model approach and the PR method. GCMs were assessed over 24 h and measurements were repeated twice with two weeks between replicates. A diurnal rhythm in GCM concentrations associated with the activity period of the sengis was found in urine, but not in the faeces. Urinary GCM concentrations exhibited a moderate repeatability, whereas the repeatability of fGCM concentrations was low. Urinary GCM concentrations and their repeatability differed between the sexes; with higher concentrations and lower PR scores in females. No such sex differences were apparent for fGCM concentrations and the PR score was not able to characterise repeatability of fGCM concentrations, which were lacking a distinct profile. The PR score enabled a successful quantification of the repeatability of the diurnal uGCM profiles. Hormone profile, sex and sample matrix can affect hormonal traits considerably and the results may be obscured if these factors are not carefully considered.
Rasmussen S. L., Kalliokoski O., Dabelsteen T., Abelson K. (2021): An exploratory investigation of glucocorticoids, personality and survival rates in wild and rehabilitated hedgehogs (Erinaceus europaeus) in Denmark. BMC Ecology and Evolution 21: 96.
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The European population of hedgehogs (Erinaceus europaeus) is declining. It is therefore essential to optimise conservation initiatives such as the rehabilitation of sick, injured and orphaned hedgehogs. Wild animals placed in captivity may be prone to chronic stress, potentially causing negative health effects. Therefore, the effects of these rehabilitation efforts should consequently be evaluated. Furthermore, hand-raising orphaned hedgehogs is a laborious and costly task, and it is therefore relevant to document whether they have equal post release survival rates compared to their wild conspecifics. The objectives of this research were therefore to conduct an exploratory study of glucocorticoid levels in hedgehogs from different backgrounds and compare the post release survival of translocated, rehabilitated and wild, juvenile hedgehogs as well as the possible effect on survival of differences in shy or bold behaviour (personality) exhibited by individuals. We measured glucocorticoid levels in 43 wild-caught (n = 18) and rehabilitated (n = 25) hedgehogs and compared the post release survival and spatial behaviour of 18 translocated juvenile hedgehogs (eight hand-raised and ten wild) until hibernation. The possible effect on survival of differences in shy or bold behaviour (personality) exhibited by 17 juvenile individuals (seven hand-raised and ten wild) was also examined. Rehabilitated individuals and females had higher levels of faecal corticosterone metabolites compared to wild individuals and males, respectively. Rehabilitated individuals showed higher levels of saliva corticosterone than wild. The personality tests labelled 13 individuals as shy and 11 as bold. Post release survival was 57% for rehabilitated and 50% for wild individuals. Neither background nor personality affected post release survival. Home range measures were 3.54 and 4.85 ha. Mean dispersal length from the release sites was 217 ± 100 m. The higher levels of corticosterone observed in rehabilitated compared to wild hedgehogs calls for consideration of the duration of admission to wildlife rehabilitation centres to reduce stress levels in the patients. Hand-raised juveniles appear to have the same prospects as wild, and personality does not seem to affect post release survival in hedgehogs, indicating that hand-raising of orphaned juvenile hedgehogs is a relevant contribution to the conservation of this species.
MARSUPIALS
McKenzie S., Deane E. M. (2005): Faecal corticosteroid levels as an indicator of well-being in the tammar wallaby, Macropus eugenii. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 140: 81-87.
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Faecal corticosteroid levels were measured in five female tammar wallabies, Macropus eugenii, at Macquarie University, NSW, Australia, to assess their reliability as indicators of well-being in this species. Animals were challenged with a change in conditions over the course of approximately 1 week, comprising movement from group yards to isolation in individual yards, in order to impose a disturbance to homeostasis (“stress”). Faecal samples were collected in 24-h intervals during the study period and analysed for corticosteroid concentration. The use of enzyme immunoassay for the measurement of corticosteroids in marsupial faecal pellets was validated. We observed a significant increase in faecal corticosteroids upon isolation and movement. Faecal corticosteroids remained above initial levels in all five animals throughout the study period, suggesting that faecal corticosteroid concentrations may be a useful indicator of a change in animal well-being. Faecal corticosteroid levels did not correlate with serum cortisol levels, implying that the use of noninvasive methods in a representative marsupial, the tammar wallaby, has the potential to provide information that is not readily apparent using blood-based protocols. Faecal corticosteroid analysis therefore has the potential for application in monitoring the well-being of captive and managed marsupial populations, as part of an integrated system of measures of animal health and well-being.
Davies N. A., Gramotnev G., McAlpine C., Seabrook L., Baxter G., Lunney D., Rhodes J. R., Bradley A. (2013): Physiological stress in koala populations near the arid edge of their distribution. Plos One 8: e79136.
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Recent research has shown that the ecology of stress has hitherto been neglected, but it is in fact an important influence on the distribution and numbers of wild vertebrates. Environmental changes have the potential to cause physiological stress that can affect population dynamics. Detailed information on the influence of environmental variables on glucocorticoid levels (a measure of stress) at the trailing edge of a species’ distribution can highlight stressors that potentially threaten species and thereby help explain how environmental challenges, such as climate change, will affect the survival of these populations. Rainfall determines leaf moisture and/or nutritional content, which in turn impacts on cortisol concentrations. We show that higher faecal cortisol metabolite (FCM) levels in koala populations at the trailing arid edge of their range in southwestern Queensland are associated with lower rainfall levels (especially rainfall from the previous two months), indicating an increase in physiological stress when moisture levels are low. These results show that koalas at the semi-arid, inland edge of their geographic range, will fail to cope with increasing aridity from climate change. The results demonstrate the importance of integrating physiological assessments into ecological studies to identify stressors that have the potential to compromise the long-term survival of threatened species. This finding points to the need for research to link these stressors to demographic decline to ensure a more comprehensive understanding of species’ responses to climate change.
Dowle M., Webster K. N., Deane E. (2013): Faecal glucocorticoid metabolite concentrations in the free-ranging bandicoots (Perameles nasuta and Isoodon obesulus) of northern Sydney. Australian Mammalogy 35: 1-7.
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Knowledge of how animals cope with their environment is fundamental to the management of free-ranging populations. Urban animals face increased competition for resources, habitat fragmentation and predation. These pressures may impact an individual’s welfare by releasing glucocorticoid hormones in the blood through a response from the hypothalamic–pituitary–adrenal axis, resulting in altered energy storage and utilisation. This study aimed to determine the applicability of measuring faecal glucocorticoid metabolites in free-ranging bandicoots by using a simple enzyme immunoassay. We used long-nosed and southern brown bandicoots in northern Sydney to investigate whether environmental and demographic variables can influence faecal glucocorticoid metabolites. Long-nosed bandicoots showed similar faecal glucocorticoid metabolite concentrations between suburban backyards and National Park populations. Higher faecal glucocorticoid metabolites were recorded in female southern brown bandicoots than in males, whilst female and male long-nosed bandicoots had similar glucocorticoid metabolite levels. Ectoparasite load, body condition and season did not influence faecal glucocorticoid metabolites. This non-invasive method has a broad application and can be used to provide biological information to guide management of populations within a conservation context.
Narayan E. J., Webster K., Nicolson V., Mucci A., Hero J. M. (2013): Non-invasive evaluation of physiological stress in an iconic Australian marsupial: The Koala (Phascolarctos cinereus). General and Comparative Endocrinology 187: 39-47.
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Koalas (Phascolarctos cinereus) are the only extant representatives of Australia’s unique marsupial family Phascolarctidae and were listed as nationally Vulnerable in 2012. Causes of mortality are diverse, although the disease chlamydiosis, dog attacks, collisions with cars, and loss of habitat represent the principal reasons for the continued species decline. Koala breeding facilities in Queensland and New South Wales, Australia have been established for conservation and tourism. Non-invasive monitoring of physiological stress is important for determining the sub-lethal effects of environmental stressors on the well-being, reproduction and survival of Koalas in Zoos and also in the wild. In this study, we developed a faecal cortisol metabolite (FCM) enzyme-immunoassay (EIA) for monitoring physiological stress in Koalas from two established Zoos in Australia and also within a free-living sub-population from Queensland. Biological validation of the FCM EIA was done using an adrenocorticotropic hormone (ACTH) challenge. We discovered excretory lag-times of FCM of 24 h in females (n = 2) and 48 h in male (n = 2) Koalas in response to the ACTH challenge. FCM levels showed an episodic and delayed peak response lasting up to 9 days post ACTH challenge. This finding should be taken into consideration when designing future experiments to study the impacts of short-term (acute) and chronic stressors on the Koalas. Laboratory validations were done using parallelism and recovery checks (extraction efficiency) of the cortisol standard against pooled Koala faecal extracts. Greater than 99% recovery of the cortisol standard was obtained as well as a parallel displacement curve against Koala faecal extracts. FCM levels of the captive Koalas (n = 10 males and 13 females) significantly differed by sex, reproductive condition (lactating versus non-lactating Koalas) and the handling groups. Handled male Koalas had 200% higher FCM levels than their non-handled counterparts, while females were not affected by handling as long they were not undergoing lactation. There was no significant difference in FCM levels between the captive and wild Koalas (n = 9 males and 7 females). Overall, these results provide foundation knowledge on non-invasive FCM analysis in this iconic Australian marsupial. Non-invasive stress endocrinology opens up opportunities for evaluating the sub-lethal physiological effects of management activities (including caging, translocation) on the nutritional status, reproductive behaviors and disease status of captive and managed in situ Koala populations.
Narayan E. J., Evans N., Hero J. M. (2014): Monitoring physiological stress in semi-free ranging populations of an endangered Australian marsupial, the Greater Bilby (Macrotis lagotis). European Journal of Wildlife Research 60: 727-735.
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Rapid and reliable physiological evaluation of stress is necessary for understanding the potential impacts of environmental changes on managed populations of threatened mammals. In situ populations of Australia’s iconic marsupial, the greater bilby (Macrotis lagotis), are nearing extinction due to the impacts of competition and predation by feral animals and unpredictable climatic events (summer heat waves). In this study, we focussed our aim to identify a non-invasive method to measure adrenal activity in the species and also to identify potential factors that should be considered when comparing physiological stress in semi-free ranging populations of the species. We validated an enzyme immunoassay (EIA) for detecting fecal cortisol metabolites (FCM) from fresh fecal pellets taken from bilbies within four captive sites and two semi-free ranging populations around Queensland and New South Wales, Australia. Our FCM EIA successfully detected the ‘raise and fall’ pattern of FCM levels within 3 days of exogenous adrenocorticotropic hormone (ACTH) challenge. Mean FCM levels differed significantly between the captive sites and between sexes. All male bilbies grouped outdoor in captivity expressed the highest mean FCM level in comparison to all captive males that were housed individually or as groups indoors. Also, semi-free ranging bilbies expressed higher mean FCM levels than the captive bilbies. Overall, our study successfully validated a non-invasive tool for monitoring physiological stress in the greater bilby. In the future, it will be worthwhile to consider factors such as housing conditions, sex and location when comparing the adrenal sensitivity to environmental changes, to help evaluate the success of management interventions (such as predator free enclosures) and support the survival of the species.
Hing S., Narayan E. J., Thompson R. A., Godfrey S. S. (2017): Identifying factors that influence stress physiology of the woylie, a critically endangered marsupial. Journal of Zoology 302: 49-56.
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Faecal glucocorticoid metabolites are minimally invasive stress physiology indices that can be used to understand how animals respond to physical and/or psychological challenges (stressors) and inform how to optimize conservation management in view of these stressors. We investigated contextual biological, environmental and parasitological factors influencing variation in baseline faecal cortisol metabolite (FCM) concentration in a critically endangered marsupial, the woylie (syn. brush-tailed bettong, Bettongia penicillata). Woylies have undergone a rapid and significant population decline, with environmental stressors exacerbating disease suggested to contribute to these ongoing declines. We conducted a longitudinal field study of 15 adult woylies (9 females, 6 males) in a captive, naturalistic facility. FCM concentration in faecal samples (n = 269) collected monthly over 20 months was quantified by enzyme immunoassay in parallel with measures of body condition, sex, season, female reproductive status and the presence of endoparasites and ectoparasites. Linear mixed effect modelling revealed a significant effect of season, sex, body condition index and nematode parasite status on FCM. Overall, mean FCM was lowest in summer and highest in autumn and winter, and females had higher mean FCM than males. There was a significant but weak negative association between body condition and FCM. When woylies were shedding oxyurid nematode eggs they had higher mean FCM compared to when they were not shedding. In future, knowledge of factors that influence FCM fluctuations in woylies may be considered when carrying out potentially stressful conservation interventions that may influence the future survival of this unique and threatened species.
Jensen M. A., Moseby K. E., Paton D. C., Fanson K. V. (2019): Non-invasive monitoring of adrenocortical physiology in a threatened Australian marsupial, the western quoll (Dasyurus geoffroii). Conservation Physiology 7: coz069.
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Reintroduction has become an increasingly important conservation tool in Australia, yet the effects of stress on species during reintroduction programs have received little attention. The use of enzyme immunoassays to measure faecal glucocorticoid metabolites (FGM) is a useful non-invasive technique to monitor adrenal activity but requires validation before they can be reliably used. As part of a large reintroduction project, the goals of this study were to 1) monitor FGM in 53 western quolls (Dasyurus geoffroii) following capture from the wild and transfer to a holding facility and use this stressor to biologically validate an enzyme immunoassay; 2) determine if biological factors, such as sex, age, weight or source population affect baseline FGM levels; and 3) examine individual variation in the acute adrenal response of quolls to the capture and transfer associated with reintroductions. We successfully validated an assay that targets glucocorticoid metabolites with a 5α-3β,11β-diol structure and found that sex significantly influenced both baseline and peak FGM output in western quolls, whereas age, weight and source population did not. We also observed considerable variation among individuals in the magnitude and duration of their physiological response to capture and transfer. Using the methods described here, FGM analysis may provide further information about the adrenal activity of the western quoll and improve future conservation efforts for this threatened species.
Narayan E. (2019): Physiological stress levels in wild koala sub-populations facing anthropogenic induced environmental trauma and disease. Scientific Reports 9: 6031.
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Australian small mammals such as koalas must cope with immense pressure from anthropogenic induced stressors or trauma such as bushfires, vehicle collision impacts and habitat disturbance and land clearance. In addition, they must cope with diseases such as chlamydia. To date, there is no published literature on physiological stress levels in wild koala populations compared with identified environmental stressors. This study investigated physiological stress levels within sub-populations of wild koalas encountering environmental trauma and disease from New South Wales (NSW), Queensland (QLD) and South Australia (SA). Physiological stress was determined using a faecal glucocorticoid (or cortisol) metabolites (FGMs) enzyme-immunoassay (EIA) from 291 fresh faecal samples collected from wild koalas at the point of rescue. A healthy breeding sub-population from a forest reserve in QLD acted as a control group. Clearance of prime Eucalyptus habitat had the largest impact on FGMs, followed by bushfire related factors (e.g. flat demeanour, dehydration and burns injury). Koalas with other sources of physical injury (dog-attacks and vehicle collisions) and disease (chlamydia) also had higher FGMs compared to healthy wild koalas. Healthy wild koalas expressed the lowest median levels of FGMs. Overall, the results highlight that anthropogenic-induced stressors tend to increase physiological stress in wild koalas. Thus, the ultimate stressors such as habitat clearance and bush fire events could increase the incidences of proximate stressors such as dog attacks and vehicle collisions, and increase risks of foliage shortage, diseases and mortality. Therefore, there is need for ecological monitoring, conservation management actions and policy changes to curb the koala population crisis, especially within on-going and future land and road development programs.
Scheun J., Geiser F., Ganswindt A., Nowack J. (2019): Non-invasive evaluation of stress hormone responses in a captive population of sugar gliders (Petaurus breviceps). Australian Mammalogy 42: 176-184.
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Faecal hormone monitoring offers a robust tool to non-invasively determine the physiological stress experienced by an individual when faced with natural or human-driven stressors. Although already quantified for several species, the method needs to be validated for each new species to ensure reliable quantification of the respective glucocorticoids. Here we investigated whether measurement of faecal glucocorticoid metabolite (fGCM) provides a feasible and non-invasive way to assess the physiological state of sugar gliders (Petaurus breviceps), an arboreal marsupial native to Australia, by using both a biological and physiological validation. Our analysis confirmed that the cortisol enzyme immunoassay (EIA) was the most appropriate assay for monitoring fGCM concentrations in sugar gliders. Comparing the fGCM response to the physiological and the biological validation, we found that while the administration of ACTH led to a significant increase in fGCM concentration in all individuals, only six of eight individuals showed a considerable fGCM response following the biological validation. Our study identified the most appropriate immunoassay for monitoring fGCM concentrations as an indicator of physiological stress in sugar gliders, but also supports recent suggestions that, if possible, both biological and physiological stressors should be used when testing the suitability of an EIA for a species.
Brunton E. A., Clemente C. J., Burnett S. E. (2020): Not all urban landscapes are the same: interactions between urban land use and stress in a large herbivorous mammal. Ecological Applications 30: e02055.
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Urbanization significantly impacts the health and viability of wildlife populations yet it is not well understood how urban landscapes differ from non-urban landscapes with regard to their effects on wildlife. This study investigated the physiological response of eastern grey kangaroos (Macropus giganteus) to land use at a landscape scale. Using fecal glucocorticoid metabolites (FGM) we compared stress levels of kangaroo populations in urban and non-urban environments. We modeled FGM concentrations from 24 kangaroo populations against land use (urban or non-urban) and other anthropogenic and environmental factors, using a linear modeling approach. We found that land use was a significant predictor of FGM concentrations in eastern grey kangaroos with significant differences in concentrations between urban and non-urban populations. However, the direction of the relationship differed between northern and southern regions of Australia. In the northern study sites, kangaroos in urban areas had significantly higher FGM levels than their non-urban counterparts. In contrast, in southern sites, where kangaroos occur in high densities in many urban areas, urban kangaroos had lower FGM concentrations than non-urban kangaroos. Rainfall and temperature were also significant predictors of FGM and the direction of the relationship was consistent across both regions. These results are consistent with the contrasting abundance and persistence of kangaroo populations within the urban matrix between the two study regions. In the northern region many populations have declined over the last two decades and are fragmented, also occurring at lower densities than in southern sites. Our study indicates that it is the characteristics of urban environments, rather than the urban environment per se, which determines the extent of impacts of urbanization on kangaroos. This research provides insights into how the design of urban landscapes can influence large mammal populations.
Cope H. R., Keeley T., Keong J., Smith D., Silva F. R., McArthur C., Webster K. N., Mella V. S., Herbert C. A. (2022): Validation of an enzyme immunoassay to measure faecal glucocorticoid metabolites in common brushtail possums (Trichosurus vulpecula) to evaluate responses to rehabilitation. Animals 12: 1627.
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Volunteer wildlife rehabilitators rescue and rehabilitate thousands of native animals every year in Australia. However, there is little known about how exposure to novel stimuli during rehabilitation could affect the physiology of wildlife. We investigated this question in a species that commonly enters rehabilitation, the common brushtail possum (Trichosurus vulpecula). We evaluated five enzyme immunoassays (EIA) to determine the most suitable for measuring faecal glucocorticoid metabolites (FGM) as a proxy for evaluating the response of brushtail possums to potential stressors during rehabilitation. An adrenocorticotrophin hormone (ACTH) challenge was conducted on wild-caught possums to determine the best-performing EIA based on the successful detection of FGM peaks in at least two of three possums. While a number of assays met these criteria, the 11-oxoaetiocholanolone (abbreviation: 72a) EIA was selected as it had the largest amplitude of change in response to the ACTH challenge. This assay was then used to measure FGM concentrations in 20 possums during rehabilitation. There was high variation in baseline FGM concentrations and response to captivity between possums. Significant changes in FGM levels were detected in most possums during captivity, but were not reliably associated with potentially stressful events that were identified by rehabilitators. The probability of an FGM peak occurring within five days of a potentially stressful event was about 50%, regardless of the type of event. Our study has demonstrated that injured and orphaned possums show changes in FGMs during captivity and rehabilitation and has identified events that can induce a physiological response in some individuals. We recommend that research now focus on the relationship between these responses during rehabilitation and pre- and post-release survival.
MONOTREMES
Braga A. F., Hunt K. E., Dillon D., Minicozzi M., Nicol S. C., Buck C. L. (2022): Can spines tell a story? Investigation of echidna spines as a novel sample type for hormone analysis in monotremes. General and Comparative Endocrinology 325: 114053.
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The short-beaked echidna (Tachyglossus aculeatus) is a monotreme endemic to Australia and New Guinea, and is the most widespread native mammal in Australia. Despite its abundance, there are considerable gaps in our understanding of echidna life history such as reproductive cycles in both sexes, patterns of stress physiology, and possible seasonal changes in metabolism. Slow-growing integumentary sample types comprised of keratin (hair, claw, etc.) have been used in other wildlife to assess these questions via analysis of longitudinal patterns in steroid and thyroid hormones that are deposited in these tissues as they grow. Hairs and spines comprise the pelage of echidnas, the spines being keratinized structures homologous to hair. Thus, echidna spines could be a viable sample type for hormone analysis contributing to a better understanding of the biology of echidnas. The aim of this work was to determine whether steroid hormones are detectable in echidna spines, to perform assay validations, and to establish a protocol for extracting and quantifying hormones in echidna spines using commercially available assay kits. We also inspected cross-sectioned spines using light and electron microscopy for any evidence of annual growth markers that might enable inferences about spine growth rate. Corticosterone, progesterone, estradiol, and testosterone were detectable in all samples, and echidna spine extract passed standard assay validations (parallelism and accuracy), indicating that commercially available assay kits can quantify hormones accurately in this sample type. No visible growth marks were identified in the spines and thus spine growth rate is currently unknown. Echidna spines show promise as a novel matrix from which hormones can be quantified; next steps should involve determination of spine annual growth rate, possible seasonal changes in growth rate, and persistence of spines over time in order to perform physiological validations, i.e., relationship between physiological status and hormone concentrations in spines.
Russell F. A., Johnston S. D., Hill A., Roser A., Meer H., Fenelon J. C., Renfree M. B., Keeley T. (2022): Validation of a non-invasive assessment technique for quantifying faecal glucocorticoid metabolite concentrations in the short-beaked echidna (Tachyglossus aculeatus). General and Comparative Endocrinology 327: 114092.
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The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic–pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response.
PRIMATES
Higham J. P., Vitale A. B., Rivera A. M., Ayala J. E., Maestripieri D. (2010): Measuring salivary analytes from free-ranging monkeys. Physiology & Behavior 101: 601-607.
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Studies of large free-ranging mammals have been revolutionized by non-invasive methods for assessing physiology, which usually involve the measurement of fecal or urinary biomarkers. However, such techniques are limited by numerous factors. To expand the range of physiological variables measurable non-invasively from free-ranging primates, we developed techniques for sampling monkey saliva by offering monkeys ropes with oral swabs sewn on the ends. We evaluated different attractants for encouraging individuals to offer samples, and proportions of individuals in different age/sex categories willing to give samples. We tested the saliva samples we obtained in three commercially available assays: cortisol, salivary alpha amylase, and secretory immunoglobulin A. We show that habituated free-ranging rhesus macaques will give saliva samples voluntarily without training, with 100% of infants, and over 50% of adults willing to chew on collection devices. Our field methods are robust even for analytes that show poor recovery from cotton, and/or that have concentrations dependent on salivary flow rate. We validated the cortisol and SAA assays for use in rhesus macaques by showing aspects of analytical validation, such as that samples dilute linearly and in parallel to assay standards. We also found that values measured correlated with biologically meaningful characteristics of sampled individuals (age and dominance rank). The SIgA assay tested did not react to samples. Given the wide range of analytes measurable in saliva but not in feces or urine, our methods considerably improve our ability to study physiological aspects of the behavior and ecology of free-ranging primates, and are also potentially adaptable to other mammalian taxa.
Heintz M. R., Santymire R. M., Parr L. A., Lonsdorf E. V. (2011): Validation of a cortisol enzyme immunoassay and characterization of salivary cortisol circadian rhythm in chimpanzees (Pan troglodytes). American Journal of Primatology 73: 903-908.
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Monitoring concentrations of stress hormones is an important tool for behavioral research and conservation for animals both in the wild and captivity. Glucocorticoids can be measured in mammals as an indicator of stress by analyzing blood, feces, urine, hair, feathers, or saliva. The advantages of using saliva for measuring cortisol concentrations are three‐fold: it is minimally invasive, multiple samples can be collected from the same individual in a short timeframe, and cortisol has a relatively short response time in saliva as compared with other materials. The purpose of this study was to: (1) conduct an adrenocorticotropic hormone (ACTH) challenge as a physiological validation for an enzyme immunoassay to measure salivary cortisol in chimpanzees and (2) characterize the circadian rhythm of salivary cortisol in chimpanzees. We determined that salivary cortisol concentrations peaked 45 min following the ACTH challenge, which is similar to humans. Also, salivary cortisol concentrations peaked early in the morning and decreased throughout the day. We recommend that saliva collection may be the most effective method of measuring stress reactivity and has the potential to complement behavioral, cognitive, physiological, and welfare studies.
Shutt K., Setchell J. M., Heistermann M. (2012): Non-invasive monitoring of physiological stress in the Western lowland gorilla (Gorilla gorilla gorilla): validation of a fecal glucocorticoid assay and methods for practical application in the field. General and Comparative Endocrinology 179: 167-177.
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Enzymeimmunoassays (EIAs) allow researchers to monitor stress hormone output via measurement of fecal glucocorticoid metabolites (FGCMs) in many vertebrates. They can be powerful tools which allow the acquisition of otherwise unobtainable physiological information from both captive animals and wild animals in remote forest habitats, such as great apes. However, methods for hormone measurement, extraction and preservation need to be adapted and validated for field settings. In preparation for a field study of Western lowland gorillas (Gorilla gorilla gorilla) in the Central African Republic we used samples from captive gorillas collected around opportunistic stressful situations to test whether four different glucocorticoid EIAs reflected adrenocortical activity reliably and to establish the lag-time from the stressor to peak excretion. We also validated a field extraction technique and established a simple, non-freezer-reliant method to preserve FGCMs in extracts long-term. We determined the rate of FGCM change over 28 days when samples cannot be extracted immediately and over 12 h when feces cannot be preserved immediately in alcohol. Finally, we used repeat samples from identified individuals to test for diurnal variation in FGCM output. Two group-specific assays measuring major cortisol metabolites detected the predicted FGCM response to the stressor reliably, whereas more specific cortisol and corticosterone assays were distinctly less responsive and thus less useful. We detected a lag time of 2–3 days from stressor to peak FGCM excretion. Our field extraction method performed as well as an established laboratory extraction method and FGCMs in dried extracts stored at ambient temperatures were as stable as those at −20 °C over 1 yr. Hormones in non-extracted feces in alcohol were stable up to 28 days at ambient temperatures. FGCMs in un-fixed gorilla feces deteriorated to almost 50% of the original values within 6 h under field conditions. We detected no diurnal variation in FGCMs in samples from wild gorillas. Our study highlights the importance of thorough biological and immunological validation of FGCM assays, and presents validated, practical methods for the application of non-invasive adrenocortical monitoring techniques to field conservation contexts where it is crucially needed.
Rimbach R., Link A., Heistermann M., Gómez-Posada C., Galvis N., Heymann E. W. (2013): Effects of logging, hunting, and forest fragment size on physiological stress levels of two sympatric ateline primates in Colombia. Conservation Physiology 1: cot031.
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Habitat fragmentation and anthropogenic disturbances are of major concern to the conservation of endangered species because of their potentially negative impact on animal populations. Both processes can impose physiological stress (i.e. increased glucocorticoid output) on animals, and chronically elevated stress levels can have detrimental effects on the long-term viability of animal populations. Here, we investigated the effect of fragment size and human impact (logging and hunting pressure) on glucocorticoid levels of two sympatric Neotropical primates, the red howler monkey (Alouatta seniculus) and the critically endangered brown spider monkey (Ateles hybridus). These two species have been reported to contrast strongly in their ability to cope with anthropogenic disturbances. We collected faecal samples from eight spider monkey groups and 31 howler monkey groups, living in seven and 10 different forest fragments in Colombia, respectively. We measured faecal glucocorticoid metabolite (FGCM) levels in both species using previously validated methods. Surprisingly, fragment size did not influence FGCM levels in either species. Spider monkeys showed elevated FGCMs in fragments with the highest level of human impact, whereas we did not find this effect in howler monkeys. This suggests that the two species differ in their physiological responsiveness to anthropogenic changes, further emphasizing why brown spider monkeys are at higher extinction risk than red howler monkeys. If these anthropogenic disturbances persist in the long term, elevated FGCM levels can potentially lead to a state of chronic stress, which might limit the future viability of populations. We propose that FGCM measurements should be used as a tool to monitor populations living in disturbed areas and to assess the success of conservation strategies, such as corridors connecting forest fragments.
Hämäläinen A., Heistermann M., Fenosoa Z. S. E., Kraus C. (2014): Evaluating capture stress in wild gray mouse lemurs via repeated fecal sampling: method validation and the influence of prior experience and handling protocols on stress responses. General and Comparative Endocrinology 195: 68-79.
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Reliable measurements of physiological stress are increasingly needed for eco-physiological research and for species conservation or management. Stress can be estimated by quantifying plasma glucocorticoid levels, but when this is not feasible, glucocorticoid metabolites are often measured from feces (FGCM). However, evidence is accumulating on the sensitivity of FGCM measurements to various nuisance factors. Careful species- and context-specific validations are therefore necessary to confirm the biological relevance and specificity of the method. The goals of this study were to: (1) establish and validate sampling methods and an enzyme immunoassay to measure FGCM in the gray mouse lemur (Microcebus murinus); (2) explore causes of variability in the FGCM measurements, and; (3) assess the consequences of capturing and handling for free-living individuals by quantifying their stress responses via repeated fecal sampling within capture sessions. We further assessed the influence of different handling protocols and the animals’ previous capture experience on the magnitude of the physiological response. Our validations identified the group-specific measurement of 11ß-hydroxyetiocholanolone as the most suitable assay for monitoring adrenocortical activity. The sample water content and the animal’s age were found to significantly influence baseline FGCM-levels. Most captured animals exhibited a post-capture FGCM-elevation but its magnitude was not related to the handling protocol or capture experience. We found no evidence for long-term consequences of routine capturing on the animals’ stress physiology. Hence the described methods can be employed to measure physiological stress in mouse lemurs in an effective and relatively non-invasive way.
Rangel-Negrín A., Coyohua-Fuentes A., Chavira R., Canales-Espinosa D., Dias P. A. D. (2014): Primates living outside protected habitats are more stressed: the case of black howler monkeys in the Yucatán Peninsula. Plos One 9: e112329.
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The non-invasive monitoring of glucocorticoid hormones allows for the assessment of the physiological effects of anthropogenic disturbances on wildlife. Variation in glucocorticoid levels of the same species between protected and unprotect areas seldom has been measured, and the available evidence suggests that this relationship may depend on species-specific habitat requirements and biology. In the present study we focused on black howler monkeys (Alouatta pigra), a canopy-dwelling primate species, as a case study to evaluate the physiological consequences of living in unprotected areas, and relate them with intragroup competition and competition with extragroup individuals. From February 2006 to September 2007 we collected 371 fecal samples from 21 adults belonging to five groups (two from protected and three from unprotected areas) in Campeche, Mexico. We recorded agonistic interactions within groups and encounters with other groups (1,200 h of behavioral observations), and determined fecal glucocorticoid metabolite (FGM) concentrations with radioimmunoassays. We used linear mixed models and Akaike’s information criterion to choose the best model explaining variation in FGM concentrations between protected and unprotected areas calculated from five categorical variables: habitat type (protected vs. unprotected), participation in agonistic interactions, intergroup encounters, sex and female reproductive state, and season. The best model included habitat type, the interaction between habitat type and agonism, and the interaction between habitat type and season. FGM concentrations were higher in unprotected habitats, particularly when individuals were involved in agonistic interactions; seasonal variation in FGM concentrations was only detected in protected habitats. High FGM concentrations in black howler monkeys living in unprotected habitats are associated with increased within-group food competition and probably associated with exposure to anthropogenic stressors and overall food scarcity. Because persistent high GC levels can be detrimental to health and fitness, populations living in disturbed unprotected areas may not be viable in the long-term.
Nugraha T. P., Heistermann M., Agil M., Purwantara B., Supriatna I., Gholib G., van Schaik C. P., Weingrill T. (2017): Validation of a field-friendly extraction and storage method to monitor fecal steroid metabolites in wild orangutans. Primates 58: 285-294.
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Measuring hormone metabolites from feces is the most often used method to assess hormonal status in wildlife. Although immediate freezing of fecal samples collected in the field is the best method to minimize the risk of degradation of hormones over time, this is often not possible in remote field sites. Therefore, alternative storage and preservation methods for fecal samples are required in these conditions. We conducted an experiment to investigate if fecal glucocorticoid (FGCM) and progesterone metabolite (pregnanediol-3-glucuronide; PdG) levels measured from samples that were extracted with a simple, field-friendly methodology correlate with those generated from frozen samples. We also evaluated whether storing fecal samples in alcohol is a suitable alternative to preserve FGCM and PdG concentrations long-term (i.e. over a 9-month period) at locations where fecal extraction is not feasible. Finally, we tested if the hormone concentrations in unpreserved fecal samples of orangutans change over 14 h when stored at ambient conditions, representing the maximum duration between sample collection and return to the camp. FGCM and PdG levels measured from samples that were extracted with the field-friendly method showed strong correlations with those generated from frozen samples, and mean levels did not differ significantly between these methods. FGCM concentrations showed no significant change compared to control samples when fecal samples were stored for up to 6 months in alcohol at ambient temperature and PdG concentrations even remained stable for up to 9 months of storage. FGCM concentrations of fecal samples kept at ambient temperature for up to 14 h post-defecation did not significantly differ compared to control samples frozen immediately after collection. These results provide the basis for the successful monitoring of the physiological status of orangutans living in remote natural settings, like those included in the Indonesian reintroduction programs.
Young C., Ganswindt A., McFarland R., De Villiers C., Van Heerden J., Ganswindt S., Barrett L., Henzi S. P. (2017): Faecal glucocorticoid metabolite monitoring as a measure of physiological stress in captive and wild vervet monkeys. General and Comparative Endocrinology 253: 53-59.
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The development of non-invasive techniques to analyse physiological stress in mammalian species has revolutionised field-based endocrinology. However, careful validation of the methods used to determine faecal glucocorticoid metabolite (fGCM) and other hormone concentrations are required on a species- and sex-specific basis. In this study, we performed an adrenocorticotropic hormone (ACTH) stimulation test on four (two male and two female) captive vervet monkeys (Chlorocebus pygerythrus) to determine the most appropriate enzyme immunoassay (EIA) from a suite of available EIAs. Furthermore, we took advantage of a potentially stressful event in our wild vervet population from Samara Private Game Reserve, South Africa, to examine if an alpha–beta female rank reversal increases the physiological stress of those individuals directly involved, as well as other group members. Both our physiological and biological validation studies revealed that a cortisol assay was the most appropriate EIA for monitoring fGCM alterations in vervet monkeys. In addition, we found that the observed rank-reversal had no significant effect on the physiological stress levels of uninvolved group members. Our study highlights that physiological validation is imperative and, where possible, should be conducted in parallel with a carefully considered biologically-relevant test under natural conditions. Overall, our results provide a necessary step for future studies to examine physiological stress of vervet monkeys via fGCM monitoring by validating a suitable EIA for this species. This paves the way for future research into the health and welfare of both captive and wild vervet monkeys, and will allow researchers to assess the behavioural, social and ecological correlates of physiological stress levels of this species.
Buti T. E. M., Kugelmeier T., Sobral G., Viau Furtado P., do Valle Dutra de Andrade Neves D., Alvarenga de Oliveira C. (2018): Fecal glucocorticoid metabolites and assay validation: Stress response evaluation in captive brown howler monkeys (Alouatta clamitans). Journal of Medical Primatology 47: 226-231.
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The advent of non‐invasive methods provides a powerful alternative to stress studies as the use of stressful handling techniques is no longer needed. However, many factors influence hormone metabolism such as sex, diet, and metabolic rate. Thus, validation should be species‐ and matrix‐specific. To assess stress response in brown howler monkeys Alouatta clamitans, we adopted an ACTH challenge test and parallelism to provide physiological and laboratorial validation. Radioimmunoassay was used to measure fecal levels of corticosterone. All challenged animals presented a peak in fecal glucocorticoids levels the day after the treatment, while control animals did not. There were no significant sex differences, but females with infants had higher levels of corticosterone. Corticosterone levels showed parallelism to the standard curve of the diagnostics kit. Collectively, the data suggest that the method was validated and is useful for monitoring stress, thereby helping in conservation programs both in captivity and in the wild. Transit time information may be coupled with travel distance in seed dispersal studies.
Broche N., Takeshita R. S., Mouri K., Bercovitch F. B., Huffman M. A. (2019): Salivary alpha-amylase enzyme is a non-invasive biomarker of acute stress in Japanese macaques (Macaca fuscata). Primates 60: 547-558.
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Salivary alpha-amylase (sAA) enzyme functions as a digestive enzyme in many species that consume starch in their diet. Human studies have also revealed that sAA enzyme activity levels are positively correlated with the release of the stress hormone norepinephrine, allowing sAA to act as a biomarker for sympathetic nervous system activity. Recent non-human primate studies have incorporated sAA as a physiological stress marker. However, no published reports have investigated the time course of sAA from a stressful event to return to baseline levels in non-human primates. Furthermore, no validation of sAA as a stress biomarker has been reported for Japanese macaques (Macaca fuscata). This study had two primary aims: (1) to develop a systematic method for non-invasive saliva collection and, (2) to investigate sAA as a biomarker of acute stress in M. fuscata in order to better understand its acute stress-related characteristics. We developed a non-invasive method for cooperative saliva collection using positive reinforcement training (PRT) and tracked individual progress over 595 trials in ten individually housed Japanese macaques. We detected sAA enzyme in M. fuscata via kinetic reaction assay, then performed 22 acute stress tests. Four tests met conditions for interpreting sAA in response to an acute stressor and these results show that on average sAA activity rapidly increased post-stressor (mean ± SD = 4.2 ± 0.9 min) and returned to baseline shortly thereafter (10.4 ± 0.6 min). Our report reveals for the first time the temporal dynamics of sAA when applying acute stress to Japanese macaques and could be a useful tool for assessing animal welfare.
Eckardt W., Stoinski T. S., Rosenbaum S., Santymire R. (2019): Social and ecological factors alter stress physiology of Virunga mountain gorillas (Gorilla beringei beringei). Ecology and Evolution 9: 5248-5259.
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Living in a rapidly changing environment can alter stress physiology at the population level, with negative impacts on health, reproductive rates, and mortality that may ultimately result in species decline. Small, isolated animal populations where genetic diversity is low are at particular risks, such as endangered Virunga mountain gorillas (Gorilla beringei beringei). Along with climate change‐associated environmental shifts that are affecting the entire population, subpopulations of the Virunga gorillas have recently experienced extreme changes in their social environment. As the growing population moves closer to the forest’s carrying capacity, the gorillas are coping with rising population density, increased frequencies of interactions between social units, and changing habitat use (e.g., more overlapping home ranges and routine ranging at higher elevations). Using noninvasive monitoring of fecal glucocorticoid metabolites (FGM) on 115 habituated Virunga gorillas, we investigated how social and ecological variation are related to baseline FGM levels, to better understand the adaptive capacity of mountain gorillas and monitor potential physiological indicators of population decline risks. Generalized linear mixed models revealed elevated mean monthly baseline FGM levels in months with higher rainfall and higher mean maximum and minimum temperature, suggesting that Virunga gorillas might be sensitive to predicted warming and rainfall trends involving longer, warmer dry seasons and more concentrated and extreme rainfall occurrences. Exclusive use of smaller home range areas was linked to elevated baseline FGM levels, which may reflect reduced feeding efficiency and increased travel efforts to actively avoid neighboring groups. The potential for additive effects of stress‐inducing factors could have short‐ and long‐term impacts on the reproduction, health, and ultimately survival of the Virunga gorilla population. The ongoing effects of environmental changes and population dynamics must be closely monitored and used to develop effective long‐term conservation strategies that can help address these risk factors.
Lavin S. R., Woodruff M. C., Atencia R., Cox D., Woodruff G. T., Setchell J. M., Wheaton C. J. (2019): Biochemical and biological validations of a faecal glucocorticoid metabolite assay in mandrills (Mandrillus sphinx). Conservation Physiology 7: coz032.
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Stress is a major factor in determining success when releasing endangered species into the wild but is often overlooked. Mandrills (Mandrills sphinx) are vulnerable to extinction due to habitat loss and demand for bush meat and the pet trade. To help bolster in situ populations, rehabilitated rescued mandrills recently were released into a protected area in the Republic of Congo. The goal of this study was to validate the use of faecal glucocorticoid metabolite enzyme immunoassays (EIAs) in mandrills and test field-friendly faecal hormone extraction techniques that can subsequently be used to monitor the stress physiology and welfare of mandrills throughout the release process. Using faecal samples collected from ex situ mandrills, we tested cortisol, corticosterone, 11β-hydroxyetiocholanolone (69a), and 11-oxoetiocholanolone EIAs. Absolute concentrations, hormone profiles following medical procedures or translocation, and high-performance liquid chromatography fraction immunoreactivity showed that the 69a assay was the best choice to monitor the stress response in this species. Samples with delayed extraction or drying times had 40–80% lower 69a concentrations than samples extracted immediately post-collection and frozen. The 69a EIA is an appropriate assay for monitoring welfare in this species in situ or ex situ, and results indicated that consistent extraction methods are important for accurate comparisons.
Thompson C. L., Bottenberg K. N., Lantz A. W., de Oliveira M. A., Melo L. C., Vinyard C. J. (2020): What smells? Developing in‐field methods to characterize the chemical composition of wild mammalian scent cues. Ecology and Evolution 10: 4691-4701.
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Olfactory cues play an important role in mammalian biology, but have been challenging to assess in the field. Current methods pose problematic issues with sample storage and transportation, limiting our ability to connect chemical variation in scents with relevant ecological and behavioral contexts. Real-time, in-field analysis via portable gas chromatography–mass spectrometry (GC-MS) has the potential to overcome these issues, but with trade-offs of reduced sensitivity and compound mass range. We field-tested the ability of portable GC-MS to support two representative applications of chemical ecology research with a wild arboreal primate, common marmoset monkeys (Callithrix jacchus). We developed methods to (a) evaluate the chemical composition of marmoset scent marks deposited at feeding sites and (b) characterize the scent profiles of exudates eaten by marmosets. We successfully collected marmoset scent marks across several canopy heights, with the portable GC-MS detecting known components of marmoset glandular secretions and differentiating these from in-field controls. Likewise, variation in the chemical profile of scent marks demonstrated a significant correlation with marmoset feeding behavior, indicating these scents’ biological relevance. The portable GC-MS also delineated species-specific olfactory signatures of exudates fed on by marmosets. Despite the trade-offs, portable GC-MS represents a viable option for characterizing olfactory compounds used by wild mammals, yielding biologically relevant data. While the decision to adopt portable GC-MS will likely depend on site- and project-specific needs, our ability to conduct two example applications under relatively challenging field conditions bodes well for the versatility of in-field GC-MS.
Long C., Tordiffe A., Sauther M., Cuozzo F., Millette J., Ganswindt A., Scheun J. (2021): Seasonal drivers of faecal glucocorticoid metabolite concentrations in an African strepsirrhine primate, the thick-tailed greater galago (Otolemur crassicaudatus). Conservation Physiology 9: coab081.
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As global non-human primate populations show dramatic declines due to climate change, land transformation and other anthropogenic stressors, it has become imperative to study physiological responses to environmental change in order to understand primate adaptability and enhance species conservation strategies. We examined the effects of seasonality on faecal glucocorticoid metabolite (fGCM) concentrations of free-ranging male and female thick-tailed greater galagos (Otolemur crassicaudatus) in an Afromontane habitat. To do so, we established an enzyme immunoassay (EIA) for monitoring fGCM concentrations in the species using a biological validation. Following this, faecal samples were collected each month over the course of a year from free-ranging males and females situated in the Soutpansberg Mountains, Limpopo, South Africa. Multivariate analyses revealed lactation period was a driver of fGCM levels, whereas sex and food availability mostly influenced seasonal fGCM concentrations in the total population. Thus far, the results of this study show that drivers of fGCM levels, an indication of increased adrenocortical activity, in O. crassicaudatus are numerous and complex within the natural environment. The species may be adapted to such conditions and an extreme change to any one component may result in elevated fGCM levels. This increases our understanding of strepsirrhine primate physiology and offers initial insights into species adaptability to a challenging environment.
Beehner J. C., Alfaro J., Allen C., Benítez M. E., Bergman T. J., Buehler M. S., Carrera S. C., Chester E. M., Deschner T., Fuentes A., Gault C. M., Godoy I., Jack K. M., Kim J. D., Kolinski L., Kulick N. K., Losch T., Ordonez J. C., Perry S. E., Pinto F., Reilly O. T., Johnson E. T., Wasserman M. D. (2022): Using an on-site laboratory for fecal steroid analysis in wild white-faced capuchins. General and Comparative Endocrinology 329: 114109.
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Hormone laboratories located “on-site” where field studies are being conducted have a number of advantages. On-site laboratories allow hormone analyses to proceed in near-real-time, minimize logistics of sample permits/shipping, contribute to in-country capacity-building, and (our focus here) facilitate cross-site collaboration through shared methods and a shared laboratory. Here we provide proof-of-concept that an on-site hormone laboratory (the Taboga Field Laboratory, located in the Taboga Forest Reserve, Costa Rica) can successfully run endocrine analyses in a remote location. Using fecal samples from wild white-faced capuchins (Cebus imitator) from three Costa Rican forests, we validate the extraction and analysis of four steroid hormones (glucocorticoids, testosterone, estradiol, progesterone) across six assays (DetectX® and ISWE, all from Arbor Assays). Additionally, as the first collaboration across three long-term, wild capuchin field sites (Lomas Barbudal, Santa Rosa, Taboga) involving local Costa Rican collaborators, this laboratory can serve as a future hub for collaborative exchange.
RABBITS, HARES, AND PIKAS
Monclús R., Rödel H. G., Palme R., Holst D. V., Miguel J. D. (2006): Non-invasive measurement of the physiological stress response of wild rabbits to the odour of a predator. Chemoecology 16: 25-29.
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Stress has been widely studied in different mammals, but the physiological stress reaction that the odour of a predator could induce in preys has not received much attention. Besides, not all the animals would respond to the same extent to a known stressor. We developed an experimental procedure with eleven naïve European rabbits (Oryctolagus cuniculus) in order to determine the individual physiological response to the olfactory detection of a potential predator. The rabbits were housed singly in small enclosures with a concrete burrow system and food and water were available ad libitum. The animals followed a control trial, without odour, and an experimental trial where we confronted the rabbits with fox (Vulpes vulpes) odour. Furthermore, another sample of eleven rabbits followed a control procedure subjected to the same housing and handling procedures but without facing the predator odour. In order to assess the physiological response we analysed the concentration of glucocorticoid metabolites in the faeces of the rabbits. Therefore, everyday faecal samples were collected and analysed with an enzyme immunoassay in order to measure the corticosterone metabolites (CM), particularly, those metabolites with a 5α-3β, 11β-diol structure. After validating the assay for wild rabbits, we found that the simulated presence of a predator (fox odour) in the enclosure resulted in an increase in faecal CM concentrations. However, the stressor did not affect all the animals in the same way. We found a general increase in the individual differences. In particular, males experienced a higher increase than females, though the overall response was similar for both sexes. To our knowledge this is one of the first attempts to analyse the assessment of the risk of predation by means of non-invasive methods.
Rehnus M., Hackländer K., Palme R. (2009): A non-invasive method for measuring glucocorticoid metabolites (GCM) in Mountain hares (Lepus timidus). European Journal of Wildlife Research 55: 615-620.
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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.
Sheriff M. J., Bosson C. O., Krebs C. J., Boonstra R. (2009): A non-invasive technique for analyzing fecal cortisol metabolites in snowshoe hares (Lepus americanus). Journal of Comparative Physiology B 179: 305-313.
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To develop non-invasive techniques for monitoring steroid stress hormones in the feces of free-living animals, extensive knowledge of their metabolism and excretion is essential. Here, we conducted four studies to validate the use of an enzyme immunoassay for monitoring fecal cortisol metabolites in snowshoe hares (Lepus americanus). First, we injected 11 hares with radioactive cortisol and collected all voided urine and feces for 4 days. Radioactive metabolites were recovered predominantly in the urine (59%), with only 8% recovered in the feces. Peak radioactivity was detected an average of 3.5 and 5.7 h after injection in the urine and feces, respectively. Second, we investigated diurnal rhythms in fecal cortisol metabolites by measuring recovered radioactivity 2 days after the radioactive cortisol injection. The total amount of radioactivity recovered showed a strong diurnal rhythm, but the amount of radioactivity excreted per gram of feces did not, remaining constant. Third, we injected hares with dexamethasone to suppress fecal cortisol metabolites and 2 days later with adrenocorticotropic hormone to increase fecal cortisol metabolites. Dexamethasone decreased fecal cortisol metabolites concentrations by 61% and adrenocorticotropic hormone increased them by 1,000%, 8–12 h after injection. Fourth, we exposed hares to a simulated predator (dog). This increased the fecal cortisol metabolites concentrations by 175% compared with baseline concentrations 8–12 h after exposure. Thus, this enzyme immunoassay provides a robust foundation for non-invasive field studies of stress in hares.
Wilkening J. L., Ray C., Sweazea K. L. (2013): Stress hormone concentration in Rocky Mountain populations of the American pika (Ochotona princeps). Conservation Physiology 1: cot027.
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The American pika (Ochotona princeps) is considered a sentinel species for detecting ecological effects of climate change. Pikas are declining within a large portion of their range, but previous studies have focused only on local pika extinction as a metric of change. We designed a procedure which can provide an earlier warning signal, based on non-invasive sampling and analysis of physiological stress in living pikas. Pikas were sampled at several locations in the Rocky Mountains for the measurement of glucocorticoid metabolites (GCMs) in faeces. Using a time series of faecal pellets from 12 individuals, we detected a significant increase in faecal GCM level in response to capture, thus biologically validating the use of a corticosterone enzyme immunoassay. We also established baseline, peak, and post-peak GCM concentrations for pikas in the Rocky Mountains, which varied according to gender and individual. This is the first study to measure stress hormone metabolites in any species of pika. The methods developed and validated in this study can be used to add non-invasive measurements of physiological stress to pika monitoring programmes and other research designed to assess pika vulnerability to predicted changes in climate. Pika monitoring programmes currently in place use a protocol that relates current site use by pikas with data on local habitat characteristics, such as elevation, to infer potential effects of climate change. Data generated by these monitoring studies can be used to identify the trends in site use by pikas in relationship to habitat covariates. However, this approach does not take into account the role of behavioural thermoregulation and the pika’s use of microhabitats to ameliorate variations in climate. Incorporating a stress metric, such as GCM concentration, will provide relatively direct evidence for or against the hypothesis that pikas can be stressed by climate regardless of behavioural adaptations.
Rehnus M., Wehrle M., Palme R. (2014): Mountain hares Lepus timidus and tourism: stress events and reactions. Journal of Applied Ecology 51: 6-12.
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Winter tourism in the European Alps has developed rapidly over the past few decades, leading to the expansion of ski resorts, growing numbers of visitors and a massive increase in snow sport activities such as free-ride skiing and snowboarding, backcountry skiing and snowshoeing. Wildlife is often disturbed by these largely unpredictable activities, and animals may have limited opportunities to adapt. Mountain hares Lepus timidus are affected by this increase in alpine tourism, but their physiological and behavioural reactions to tourist activity are still unknown. We measured the levels of faecal glucocorticoid metabolites (GCM) in wild mountain hares living in areas that had no, medium or high levels of tourist activity during winter in 2011. Furthermore, we compared the changes in GCM excretion, behaviour and food intake of six captive mountain hares following predator challenge experiments from early to mid-winter. Our field results showed that GCM excretion is positively correlated with increased tourism intensity. In the predator challenge experiments, hares spent less time resting and grooming (including re-ingesting faecal pellets) during and after the stress treatments. These stress events lead to higher energy demands due to flushing, increased GCM levels, and disrupted the energy intake that hares derive from faeces. We conclude that mountain hares living in areas with frequent human winter recreational activities show changes in physiology and behaviour that demand additional energy in winter, when access to food resources is limited by snow. To bring down the frequency of stress threats for mountain hares, we recommend that managers keep forests inhabited by mountain hares free of tourism infrastructure and retain undisturbed forest patches within skiing areas. Other species such as black grouse Tetrao tetrix and/or capercaillie Tetrao urogallus are also likely to benefit from such management activities because they share similar habitat requirements with mountain hares.
Wilkening J. L., Ray C., Varner J. (2015): Relating sub-surface ice features to physiological stress in a climate sensitive mammal, the American pika (Ochotona princeps). Plos One 10: e0119327.
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The American pika (Ochotona princeps) is considered a sentinel species for detecting ecological effects of climate change. Pikas are declining within a large portion of their range, and ongoing research suggests loss of sub-surface ice as a mechanism. However, no studies have demonstrated physiological responses of pikas to sub-surface ice features. Here we present the first analysis of physiological stress in pikas living in and adjacent to habitats underlain by ice. Fresh fecal samples were collected non-invasively from two adjacent sites in the Rocky Mountains (one with sub-surface ice and one without) and analyzed for glucocorticoid metabolites (GCM). We also measured sub-surface microclimates in each habitat. Results indicate lower GCM concentration in sites with sub-surface ice, suggesting that pikas are less stressed in favorable microclimates resulting from sub-surface ice features. GCM response was well predicted by habitat characteristics associated with sub-surface ice features, such as lower mean summer temperatures. These results suggest that pikas inhabiting areas without sub-surface ice features are experiencing higher levels of physiological stress and may be more susceptible to changing climates. Although post-deposition environmental effects can confound analyses based on fecal GCM, we found no evidence for such effects in this study. Sub-surface ice features are key to water cycling and storage and will likely represent an increasingly important component of water resources in a warming climate. Fecal samples collected from additional watersheds as part of current pika monitoring programs could be used to further characterize relationships between pika stress and sub-surface ice features.
Wilkening J. L., Ray C., Varner J. (2016): When can we measure stress noninvasively? Postdeposition effects on a fecal stress metric confound a multiregional assessment. Ecology and Evolution 6: 502-513.
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Measurement of stress hormone metabolites in fecal samples has become a common method to assess physiological stress in wildlife populations. Glucocorticoid metabolite (GCM) measurements can be collected noninvasively, and studies relating this stress metric to anthropogenic disturbance are increasing. However, environmental characteristics (e.g., temperature) can alter measured GCM concentration when fecal samples cannot be collected immediately after defecation. This effect can confound efforts to separate environmental factors causing predeposition physiological stress in an individual from those acting on a fecal sample postdeposition. We used fecal samples from American pikas (Ochotona princeps) to examine the influence of environmental conditions on GCM concentration by (1) comparing GCM concentration measured in freshly collected control samples to those placed in natural habitats for timed exposure, and (2) relating GCM concentration in samples collected noninvasively throughout the western United States to local environmental characteristics measured before and after deposition. Our timed‐exposure trials clarified the spatial scale at which exposure to environmental factors postdeposition influences GCM concentration in pika feces. Also, fecal samples collected from occupied pika habitats throughout the species’ range revealed significant relationships between GCM and metrics of climate during the postdeposition period (maximum temperature, minimum temperature, and precipitation during the month of sample collection). Conversely, we found no such relationships between GCM and metrics of climate during the predeposition period (prior to the month of sample collection). Together, these results indicate that noninvasive measurement of physiological stress in pikas across the western US may be confounded by climatic conditions in the postdeposition environment when samples cannot be collected immediately after defecation. Our results reiterate the importance of considering postdeposition environmental influences on this stress metric, especially in multiregional comparisons. However, measurements of fecal GCM concentration should prove useful for population monitoring within an eco‐region or when postdeposition exposure can be minimized.
Waterhouse M. D., Sjodin B., Ray C., Erb L., Wilkening J., Russello M. A. (2017): Individual‐based analysis of hair corticosterone reveals factors influencing chronic stress in the American pika. Ecology and Evolution 7: 4099-4108.
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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.
RODENTS
Pettitt B. A., Wheaton C. J., Waterman J. M. (2007): Effects of storage treatment on fecal steroid hormone concentrations of a rodent, the Cape ground squirrel (Xerus inauris). General and Comparative Endocrinology 150: 1-11.
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Fecal steroid analysis is an increasingly common non-invasive technique used in both captive and field studies to measure an animal’s approximate hormonal levels and corresponding physiological state. Fecal collection in the field necessitates storage and transportation methods that will prevent the degradation of hormonal metabolites by fecal bacteria. To determine the most stable and therefore preferred method of storage, 48 fecal samples were collected from six captive female Cape ground squirrels (Xerus inauris). Each sample was randomly divided into three sub-samples to be processed for storage through freezing, drying, or preservation in ethanol. Frozen samples were stored at −20 °C, dried-treated samples were desiccated in a conventional oven at 40 °C for 4 h, and alcohol-treated samples were preserved in 3 ml of 95% ethanol. Samples were stored for 330 days followed by enzyme immunoassay analysis (EIA) to determine their progestogen and estrone conjugate (E1C) concentrations. Validations were performed to establish that the progestogen and E1C assays accurately measure fecal progestogen and estrone conjugate concentrations and were sensitive enough to detect biologically meaningful differences in these steroid metabolite concentrations in female X. inauris. Validation results showed a significant difference in progestogen concentrations of gravid females compared to sub-adults and non-gravid females. There was also a significant difference in estrone conjugates between sub-adult and adult females. Duration of storage time did not affect progestogen or estrone metabolite concentrations after being frozen for 3 months. Storage treatment results showed no significant difference between frozen and dried samples, but a significant difference was found between frozen and ethanol samples in both progestogen and estrone conjugate concentrations demonstrating that drying feces provides a reliable method for long-term preservation of fecal steroid concentrations and is the better alternative when freezing is not a viable option.
Bosson C. O., Palme R., Boonstra R. (2009): Assessment of the stress response in Columbian ground squirrels: laboratory and field validation of an enzyme immunoassay for fecal cortisol metabolites. Physiological and Biochemical Zoology 82: 291-301.
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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 5α‐3β,11β‐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, livetrapped, 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.
Dantzer B., McAdam A. G., Palme R., Fletcher Q. E., Boutin S., Humphries M. M., Boonstra R. (2010): Fecal cortisol metabolite levels in free-ranging North American red squirrels: assay validation and the effects of reproductive condition. General and Comparative Endocrinology 167: 279-286.
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Patterns in stress hormone (glucocorticoid: GC) levels and their relationship to reproductive condition in natural populations are rarely investigated. In this study, we (1) validate an enzyme-immunoassay to measure fecal cortisol metabolite (FCM) levels in North American red squirrels (Tamiasciurus hudsonicus), and (2) examine relationships between FCM levels and reproductive condition in a free-ranging red squirrel population. Injected radiolabeled cortisol was entirely metabolized and excreted in both the urine (mean ± SE; 70.3 ± 0.02%) and feces (29.7 ± 0.02%), with a lag time to peak excretion in the feces of 10.9 ± 2.3 h. Our antibody reacted with several cortisol metabolites, and an adrenocorticotropic injection significantly increased FCM levels above baseline levels at 8 h post-injection. Relative to baseline levels, manipulation by handling also tended to increase FCM levels at 8 h post-manipulation, but this difference was not significant. FCM levels did not differ significantly between samples frozen immediately and 5 h after collection. Reproductive condition significantly affected FCM levels in free-ranging females (pregnant > lactating > post-lactating > non-breeding) but not males (scrotal testes vs. abdominal testes). Among females with known parturition dates, FCM levels increased during gestation, peaked at parturition, and declined during lactation. The difference between pregnant and lactating females was therefore dependent upon when the fecal samples were obtained during these periods, suggesting caution in categorizing reproductive stages. This study demonstrates the utility of fecal hormone metabolite assays to document patterns of glucocorticoid levels in free-ranging animals.
Blumstein D. T., Chi Y. Y. (2012): Scared and less noisy: glucocorticoids are associated with alarm call entropy. Biology Letters 8: 189-192.
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The nonlinearity and arousal hypothesis predicts that highly aroused mammals will produce nonlinear, noisy vocalizations. We tested this prediction by measuring faecal glucocorticoid metabolites (GCMs) in adult yellow-bellied marmots (Marmota flaviventris), and asking if variation in GCMs was positively correlated with Wiener entropy—a measure of noise. Contrary to our prediction, we found a significant negative relationship: marmots with more faecal GCMs produced calls with less noise than those with lower levels of GCMs. A previous study suggested that glucocorticoids modulate the probability that a marmot will emit a call. This study suggests that, like some other species, calls emitted from highly aroused individuals are less noisy. Glucocorticoids thus play an important, yet underappreciated role, in alarm call production.
Montiglio P. O., Pelletier F., Palme R., Garant D., Réale D., Boonstra R. (2012): Noninvasive monitoring of fecal cortisol metabolites in the eastern chipmunk (Tamias striatus): validation and comparison of two enzyme immunoassays. Physiological and Biochemical Zoology 85: 183-193.
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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.
Smith J. E., Monclús R., Wantuck D., Florant G. L., Blumstein D. T. (2012): Fecal glucocorticoid metabolites in wild yellow-bellied marmots: experimental validation, individual differences and ecological correlates. General and Comparative Endocrinology 178: 417-426.
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Natural selection is expected to shape phenotypic traits that permit organisms to respond appropriately to the environments in which they live. One important mechanism by which animals cope with changes in their environment is through physiological responses to stressors mediated by glucocorticoid hormones. Here we perform biological and physiological validations of a minimally-invasive technique for assessing fecal corticosterone metabolites (FCMs) in captive and wild groups of yellow-bellied marmots (Marmota flaviventris). Then we draw from ten years of data on these obligate hibernators at the Rocky Mountain Biological Laboratory in Colorado, USA to assess the extent to which seasonal and daily changes explain naturalistic variation in baseline levels of FCMs. Interestingly, beyond important population-level variation with respect to year, season, time of day, sex, age and reproductive state, we found repeatable inter-individual differences in FCMs, suggesting this hormonal trait might be a meaningful target of selection. FCM levels were 68% lower in captive than wild marmots, suggesting that the natural environment in which these animals occur is generally more challenging or less predictable than life in captivity. Most live-trapping events failed to represent stressors for wild marmots such that repeated measurements of traits were possible with minimal “stress” to subjects. We also document the natural ranges of annual and seasonal variation necessary for understanding the extent to which anthropogenic assaults represent stressors for wild mammals. Taken together, this study provides a foundation for understanding the evolution of hormonal traits and has important welfare and conservation implications for field biologists.
Bosson C. O., Palme R., Boonstra R. (2013): Assessing the impact of live-capture, confinement, and translocation on stress and fate in eastern gray squirrels. Journal of Mammalogy 94: 1401-1411.
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Live-capture and translocation are methods to deal with nuisance eastern gray squirrels in North America, but it is unknown how these methods affect squirrel physiology or survival. In this study we validated an enzyme immunoassay (EIA) to measure fecal cortisol metabolites (FCMs) in gray squirrels; assessed their stress response relative to the positioning of the live trap (sun, shade, or control); and assessed the impact of translocation on their long-term stress, movement patterns, and survival using FCM levels, body mass changes, and radiotelemetry. We found that a 5α-pregnane-3β,11β,21-triol-20-one EIA reliably detected acute stress in gray squirrel feces 12–24 h after the stressor; live traps positioned in the sun resulted in higher peak FCM levels compared with traps positioned in the shade; translocated squirrels experienced a 10% mortality rate, compared with no mortality in the controls, although overall fates were the same; translocated squirrels initially explored more and dispersed farther than controls, but after 2 weeks made similar movements; and after controlling for the effect of season, translocation did not affect long-term FCM levels or body mass; this conclusion must be tempered by the low number of recaptures. Our study demonstrates the utility of the FCM assay; that gray squirrels are extremely sensitive to capture, handling, and confinement; and that live-capture must be done in a way that minimizes exposure to additive environmental stressors.
Navarro-Castilla Á., Barja I., Olea P. P., Piñeiro A., Mateo-Tomás P., Silván G., Illera J. C. (2014): Are degraded habitats from agricultural crops associated with elevated faecal glucocorticoids in a wild population of common vole (Microtus arvalis)?. Mammalian Biology 79: 36-43.
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The severe impact of agriculture on species’ abundance and diversity is widely recognized. However, its effects on the physiology of wild animal populations are poorly known. We analyzed faecal glucocorticoids levels in wild common voles (Microtus arvalis) living in a farmland landscape to test whether living in degraded habitats, such as crops, is correlated with increased glucocorticoids. Other factors such as sex, reproductive status, and population density were also considered. We captured voles with Sherman traps in crops and in their field margins which were comprised of semi-natural vegetation. We collected fresh faecal samples from captured individuals and quantified their levels of faecal corticosterone metabolites (FCM) in the laboratory. The quantification of FCM concentrations was performed by competitive enzyme immunoassay. Individuals captured within the crops had higher levels of FCM than those in field margins; females and breeding individuals exhibited higher FCM levels. In addition, FCM concentrations positively correlated with abundance of voles. Our results suggest that degraded habitats in agricultural landscapes are associated with increased glucocorticoid levels on common voles likely caused by a higher disturbance from agricultural practices and a lesser vegetation cover in crops compared with field margins.
Wey T., Lin L., Patton M., Blumstein D. (2015): Stress hormone metabolites predict overwinter survival in yellow-bellied marmots. Acta Ethologica 18: 181-185.
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Biological assays of stress, in particular noninvasive measures, are valuable tools for wildlife management. Chronic stress can have negative impacts on fitness outcomes, and an area of particular interest is if elevated baseline stress levels are predictive of survival outcomes. We examined the relationship between fecal glucocorticoid metabolites (FGMs) measured from samples collected during routine trapping and overwinter survival in a wild population of yellow-bellied marmots (Marmota flaviventris). In particular, we asked if elevated FGMs were associated with reduced survival probability. Both higher yearly FGM levels averaged over several months and higher late season FGM levels averaged over the several weeks before hibernation were associated with lower probability of survival. Additionally, there was an interaction between late-season FGM levels and body mass, such that the association between late-season FGMs and survival was much stronger in animals with lower body mass (i.e., in poorer condition). This study highlights the promise of using stress hormone metabolites, a noninvasive measure, for studying factors affecting survival in this and potentially other natural wildlife systems.
Ramahlo M., Chimimba C., Pirk C., Ganswindt A. (2019): Non‐invasive monitoring of adrenocortical activity in free‐ranging Namaqua rock mice Micaelamys namaquensis from South Africa in response to anthropogenic land use and season. Wildlife Biology 2019: wlb.00544.
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Stress in animals has been linked to behavioural and physiological changes in response to environmental, social and anthropogenic stimuli. Hence, stress-related responses in animals, especially in rodents, have been used as biological indicators of ecosystem health. This study aimed to establish an enzyme immunoassay (EIA) for monitoring adrenocortical activity in free-ranging Namaqua rock mice Micaelamys namaquensis (Rodentia: Muridae) using faeces as a prerequisite for assessing the effects of anthropogenic land use and season on faecal glucocorticoid metabolite (fGCM) concentration. Rodents were live-trapped seasonally across four land use types: an agricultural crop farm, an agricultural livestock farm, a human-populated site and a nature reserve; all situated in Limpopo Province, South Africa. Determined fGCM concentrations from capture and recapture events were used for biologically validating an EIA detecting steroids with a 5α-3β-11β-diol structure. Recapturing resulted in a significant overall 40% elevation of individual fGCM concentrations demonstrating the effectiveness of the chosen EIA to reliably detect glucocorticoid output in the study species. Neither land use type nor season affected fGCM concentrations in the species, suggesting that land use and season-related environmental changes do not necessarily act as stressors for M. namaquensis, presumably due to their adaptive and resilient nature. Such species can be used to identify ecosystems affected by human-mediated disturbances and allow insights into the management and restoration of these threatened ecosystems and their associated species.
Santangeli A., Wistbacka R., Morosinotto C., Raulo A. (2019): Hair cortisol concentration in Siberian flying squirrels is unrelated to landscape and social factors. The Science of Nature 106: 29.
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Intact ecosystems are being lost or modified worldwide, and many animal species are now forced to live in altered landscapes. A large amount of scientific studies have focused on understanding direct effects of habitat alterations on species occurrence, abundance, breeding success, and other life history aspects. Much less attention has been placed on understanding how habitat alterations impact on the physiology of species, e.g., via elevated chronic stress when living in an altered landscape. Here, we quantify the effects of individual age and sex, as well as effects of landscape and social factors on chronic stress of an endangered forest specialist species, the Siberian flying squirrel Pteromys volans. We collected hair samples over 2 years from across 192 flying squirrels and quantified their chronic stress response via cortisol concentrations. We then ran statistical models to relate cortisol concentrations with landscape and social factors. We show that cortisol concentrations in flying squirrels are neither affected by habitat amount and connectivity, nor by the density of conspecifics in the area. We however found that cortisol concentration was higher in adults than in pups, and in males compared with females. Lack of an effect of environmental factors on cortisol concentrations may indicate low physiological sensitivity to alterations in the surrounding environment, possibly due to low densities of predators that could induce stress in the study area. Further research should focus on possible effects of varying predator densities, alone and in interaction with landscape features, in shaping chronic stress of this and other species.
Stewart N. D., Mastromonaco G. F., Burness G. (2020): No island-effect on glucocorticoid levels for a rodent from a near-shore archipelago. PeerJ 8: e8590.
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Island rodents are often larger and live at higher population densities than their mainland counterparts, characteristics that have been referred to as “island syndrome”. Island syndrome has been well studied, but few studies have tested for island-mainland differences in stress physiology. We evaluated island syndrome within the context of stress physiology of white-footed mice (Peromyscus leucopus) captured from 11 islands and five mainland sites in Thousand Islands National Park, Ontario, Canada. Stress physiology was evaluated by quantifying corticosterone (a stress biomarker), the primary glucocorticoid in mice, from hair and its related metabolites from fecal samples. White-footed mice captured in this near-shore archipelago did not display characteristics of island syndrome, nor differences in levels of hair corticosterone or fecal corticosterone metabolites compared with mainland mice. We suggest that island white-footed mice experience similar degrees of stress in the Thousand Islands compared with the mainland. Although we did not find evidence of island syndrome or differences in glucocorticoid levels, we identified relationships between internal (sex, body mass) and external (season) factors and our hormonal indices of stress in white-footed mice.
Veitch J. S., Bowman J., Mastromonaco G., Schulte-Hostedde A. I. (2021): Corticosterone response by Peromyscus mice to parasites, reproductive season, and age. General and Comparative Endocrinology 300: 113640.
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A common response to parasite infestations is increased production of glucocorticoid hormones that regulate immune function. We examined relationships between ectoparasite infestations and fecal corticosterone metabolites (FCM) in deer mice (Peromyscus maniculatus). Furthermore, we experimentally removed fleas to determine if reductions in ectoparasites affected FCM production. Individuals were assigned to control (no flea removal) or treatment (anti-flea application, physical combing) groups and individuals were recaptured to assess changes in FCM concentrations. There was a significant and negative effect of number of anti-flea treatment applications on FCM concentrations of deer mice. However, models including host biology traits and environmental predictors had a better model fit compared to models containing ectoparasite predictors. In particular, there was a significant relationship of deer mouse FCM with date and host age, where glucocorticoid production decreased towards the end of the breeding season and increased with age. Overall, adverse events associated with reproduction and age class, rather than ectoparasites, may be more important to variation in glucocorticoids of deer mice.
Tranquillo C., Villa F., Wauters L. A., Dantzer B., Palme R., Preatoni D., Martinoli A., Santicchia F. (2022): Physiological stress and spatio-temporal fluctuations of food abundance and population density in Eurasian red squirrels. Hystrix 33: 58-64.
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In continuously changing environments, variation of different ecological factors could affect the functioning of the hypothalamic-pituitary-adrenal (HPA) axis in wild mammals, increasing the secretion of glucocorticoids (GCs). In different animal species, GC concentrations are often used as a measure of the physiological stress response to environmental pressures, such as fluctuations in food abundance, population density, intra- and interspecific competition, and predation risk. However, previous studies reported contrasting results or did not find clear associations between physiological stress and environmental variables. Here, we used concentrations of faecal glucocorticoid metabolites (FGMs) as an integrated measure of physiological stress in wild Eurasian red squirrels (Sciurus vulgaris) from three study areas in the Italian Alps, to investigate whether variations in conifer-seed crop size and/or population density affected HPA axis activity. Squirrel density was estimated in each trapping session using the minimum number of animals alive, and annual counts of fresh cones from different conifer species were used to estimate annual food abundance (MJ/ha). We expected higher FGMs in response to increasing population density and/or decreasing food abundance, since these two variables could act as environmental stressors. Our results showed a lack of association between population density and FGMs and a significant effect of food abundance on FGMs. When conifer seed-crops were poor to moderate, FGMs increased with food abundance, while in the range of high seed-crops, FGMs remained first constant and then slightly decreased with a further increase in seed abundance. We also found differences in FGMs among seasons, as previously observed in this species. Our study adds further evidence that physiological stress can be influenced in different ways by environmental pressures and that long-term studies using individually marked animals are needed to disentangle the potential adaptive outcome of the physiological stress response in pulsed resource systems.
Zenth F., Corlatti L., Giacomelli S., Saleri R., Cavalli V., Andrani M., Donini V. (2022): Hair cortisol concentration as a marker of long-term stress: sex and body temperature are major determinants in wild-living Alpine marmots. Mammalian Biology 102: 2083-2089.
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Hair cortisol concentration (HCC) has recently gained popularity as an easy-to-measure biomarker of long-term stress in wild and domestic animals. Hair integrates cortisol over long time periods within a single sample and it can be collected non-invasively, which makes its use particularly interesting for wildlife studies. Interpreting HCC values, however, is challenging, because they are determined by the interplay of multiple factors. Here, were explore potential determinants of HCC in the Alpine marmot Marmota marmota. We tested the relationship of sex, age class, physical condition and body temperature with the hair cortisol concentration of free-ranging marmots. We found marked sex difference in HCC, with higher levels in females. This might be related to sex-specific variation in social stress or resulting from physiological difference, e.g., in baseline and stress-induced levels of cortisol secretion. Interestingly, body temperature was also positively related to HCC, possibly hinting at individual short- and long-term stress reactivity as part of coping styles. Although further work is needed to entangle possible mechanisms underlying the neuro-endocrinological modulation on HCC, our results emphasize that determinants such as sex and body temperature in Alpine marmots should be accounted for, when using HCC as marker of chronic stress.
Zhang V. Y., Buck C. L. (2022): Seasonal patterns in behavior and glucocorticoid secretion of a specialist Holarctic tree squirrel (Sciurus aberti). Journal of Comparative Physiology B 192: 541-559.
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Seasonally breeding mammals must make constant adjustments in behavior and physiology to manage energetic trade-offs between survival and reproduction. Despite encountering high levels of climate and resource variability across the year, specialist Abert’s squirrels (Sciurus aberti), lack the capacity to express hibernation or pronounced morphological adaptations to seasonality. Using accelerometer and GPS devices, we assessed how abiotic environmental factors, reproduction, and resource abundance influenced levels of activity and daily range size in a rural and food-supplemented suburban population of squirrels. We also quantified fecal cortisol metabolites (FCM) in squirrels to assess patterns of glucocorticoid secretion. While changes in weather predicted activity levels in both populations, seasonal variation in activity levels were reduced in food-supplemented compared to rural squirrels. In contrast to activity, daily range size was not affected by weather but was a better predictor of sex-specific reproductive investment. Comparisons between populations suggest that food-supplemented squirrels forage more efficiently within smaller areas. Across both sexes and populations, squirrels showed no sexual dimorphism in body size, no major patterns of seasonal weight change, and no associations between body mass and FCM concentrations; however, FCMs were lower in the food-supplemented compared to rural population during late-spring. Taken together, activity levels and FCM concentrations appear primarily influenced by weather and seasonal fluctuations in food availability, whereas daily range size reflects sexual asymmetries in seasonal reproductive investment. Overall, squirrels appear to rely largely on behavioral adjustments to cope with novel environmental heterogeneity, rather than changes in morphology or GC secretion.
UNGULATES
Millspaugh J. J., Woods R. J., Hunt K. E., Raedeke K. J., Brundige G. C., Washburn B. E., Wasser S. K. (2001): Fecal glucocorticoid assays and the physiological stress response in elk. Wildlife Society Bulletin 29: 899-907.
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Fecal glucocorticoid assays provide a potentially useful, noninvasive means to study physiological responses of wildlife to various stressors. Consequently we quantified fecal glucocorticoid concentrations among free-ranging elk (Cervus elaphus) by subherd, sex, and season and determined their relationship to various human activities and environmental conditions. Using a validated technique, we assayed 558 fecal samples collected from 30 radiocollared elk in the Black Hills, South Dakota, from June 1995 to September 1997. Fecal glucocorticoid measures were least in winter (x̄=17.41 ng/g, SE=2.97 for bull subherds and 18.9 ng/g, SE=2.85 for cow subherds) and increased to peak concentrations in summer (x̄=33.6 ng/g, SE=3.42 for bull subherds and 34.21 ng/g, SE=3.71 for cow subherds). Vehicle use along primary roads, primary road density, and mean temperature each independently explained a significant portion of the variation in fecal glucocorticoid concentrations (r2=0.61, F2, 557=286.13, P<0.001). Annual glucocorticoid secretion also may be related to normal seasonal metabolic rhythms. Though more research is needed on fecal glucocorticoid concentrations of undisturbed elk, we hypothesize that human activities, high temperatures, or normal seasonal metabolic rhythms may have elevated summer glucocorticoid concentrations. Our findings suggested that fecal glucocorticoid assays, if coupled with population performance measures, could assess physiological effects of natural and human-induced disturbances on free-ranging elk. Also, our study illustrated the need for caution when interpreting fecal glucocorticoid measurements, because several confounding factors may influence interpretation.
Millspaugh J. J., Washburn B. E., Milanick M. A., Beringer J., Hansen L. P., Meyer T. M. (2002): Non-invasive techniques for stress assessment in white-tailed deer. Wildlife Society Bulletin 30: 899-907.
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Measuring stress hormones (i.e., glucocorticoids) in large free-ranging vertebrates such as white-tailed deer (Odocoileus virginianus) is difficult, and invasive procedures (e.g., animal capture and blood collection) have been the only tools available. Non-invasive techniques, including fecal and saliva glucocorticoid measurements, are currently being developed that offer advantages over traditional techniques. Using adrenocorticotropic hormone (ACTH) challenges, we determined that fecal glucocorticoid metabolite and salivary cortisol assays measured adrenal activity (i.e., stress hormones) in white-tailed deer. For each of four ACTH trials, we housed captive deer singly for 72 hr in an outdoor pen to which they had been exposed previously. After 24 hours, we injected a total of 50 I.U. of ACTH (Cortrosyn™) intramuscularly (2 25-I.U. injections in experiments 1 and 2; 1 50-I.U. injection in experiments 3 and 4). All available fecal samples were collected every hour, homogenized, and frozen. During the last 2 experiments, we collected saliva samples at 10-min intervals for 2 hr prior to and 6 hr after the ACTH injection. Fecal glucocorticoid metabolites were extracted from fecal samples and assayed using a commercially available I125 corticosterone radioimmunoassay. We used an enzyme immunoassay to measure salivary cortisol. A peak in fecal glucocorticoids occurred at 20-24 hr (experiments 1 and 2) and 10-13 hr (experiments 3 and 4) post-ACTH injection, physiologically validating the assay’s ability to detect biologically important changes in adrenocortical activity. Salivary cortisol levels increased 30-60 min after ACTH injection, confirming the assay’s ability to detect changes in cortisol concentrations. These findings demonstrated that fecal glucocorticoid and salivary cortisol assays provide an index of physiological stress in white-tailed deer. These techniques may prove useful in addressing conservation issues surrounding white-tailed deer, provided additional sampling questions are addressed.
Pelletier F., Bauman J., Festa-Bianchet M. (2003): Fecal testosterone in bighorn sheep (Ovis canadensis): behavioural and endocrine correlates. Canadian Journal of Zoology 81: 1678-1684.
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Noninvasive endocrine techniques allow repeated sampling of the same individual to study causes and consequences of variation in individual behaviour and physiology. In this study, radioimmunoassay was used to measure fecal testosterone and to assess the repeatability of the testosterone assay for bighorn rams (Ovis canadensis). Fecal samples were collected from marked males during the pre-rut and the rut over 2 years. Results were highly repeatable for samples of the same ram within a day (r = 0.93). Fecal testosterone peaked during the pre-rut (when social relationships are established) and then declined from the pre-rut to the rut. For both years of study, fecal testosterone was correlated with social rank (2001: r = 0.73, P < 0.0001; 2002: r = 0.54, P = 0.007) and age (2001: r = 0.65, P = 0.002; 2002: r = 0.53, P = 0.008) of individual rams. When age was accounted for, however, the relationship between social rank and testosterone was no longer significant. Aggressiveness (measured as hourly interaction rate) was weakly correlated with fecal testosterone (r = 0.44, P = 0.039). There was no association between aggressiveness and social rank (r = 0.13, P = 0.591). To our knowledge, this is the first report of an association between testosterone levels and individual social rank in wild ungulates.
Dalmau A., Ferret A., Chacon G., Manteca X. (2007): Seasonal changes in fecal cortisol metabolites in Pyrenean chamois. The Journal of Wildlife Management 71: 190-194.
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We studied seasonal changes in fecal cortisol metabolites (FCM), which have been widely used as indicators of stress, in a population of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in the Cadí Range of northeastern Spain. We collected fecal samples from 2001 to 2003 in 3 particular locations with different altitudes and male or female presence, and we analyzed them for FCM and fecal nitrogen as an indicator of diet quality. We observed a clear seasonal pattern, with the highest FCM in winter, and we obtained correlations between FCM and monthly mean minimum temperatures and fecal nitrogen. We observed no effects of tourism presence, trophy hunting, or rut season on FCM. Analysis of cortisol metabolites in feces can be a good measure of winter stress in Pyrenean chamois.
Franceschini M. D., Rubenstein D. I., Low B., Romero L. M. (2008): Fecal glucocorticoid metabolite analysis as an indicator of stress during translocation and acclimation in an endangered large mammal, the Grevy’s zebra. Animal Conservation 11: 263-269.
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Fecal glucocorticoid metabolite (FGM) analysis provides a non‐invasive method for studying the physiological response of wildlife to a variety of stressors and is a ground‐breaking monitoring technique in wildlife management and conservation. The conservation benefits of successful wildlife translocation restocking efforts are significant but understandably stressful for the animals being captured, removed from familiar habitat, held in captivity in many cases and subsequently released into an unfamiliar environment. It is imperative that we identify non‐invasive methods for evaluating stress in translocated animals, especially in endangered species. Twenty Grevy’s zebra Equus grevyi were translocated to Meru National Park as part of a Kenya Wildlife Service re‐population initiative. FGMs were monitored from the time of capture, during captivity and post‐release as an indicator of the stress of translocation and acclimation to the new environment. FGMs from representative non‐translocated zebra were used as a further control. When held in pens at Meru Park 3–4 and 5–6 weeks after capture, the zebra had higher FGMs (25.1±1.2 and 23.4±1.3 ng.g−1) than either at the time of capture (14.6±2.1 ng.g−1) or non‐translocated controls (16.2±1.2 ng.g−1). This suggests that the stress of captivity elevated FGMs. FGM concentrations returned to pre‐capture concentrations c 11–18 weeks after the zebra were released into Meru Park. The return of FGM concentrations to baseline suggests successful acclimation to the new environment. This study supports the use of FGM analysis as an assessment technique in wildlife management projects involving the movement of endangered large mammals with application for monitoring stress in a wide array of conservation projects involving translocation, reintroduction and rehabilitation.
Chinnadurai S. K., Millspaugh J. J., Matthews W. S., Canter K., Slotow R., Washburn B. E., Woods R. J. (2009): Validation of fecal glucocorticoid metabolite assays for South African herbivores. The Journal of Wildlife Management 73: 1014-1020.
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Fecal glucocorticoid metabolite (FGM) assays are a popular means of monitoring adrenocortical activity (i.e., physiological stress response) in wildlife. Species‐specific differences in glucocorticoid metabolism and excretion require assay validation, including both laboratory and biological components, before assay use in new species. We validated a commercially available radioimmunoassay (MP 125I corticosterone RIA kit [MP Biomedicals, Solon, OH]) for measuring FGMs of several South African herbivores, including giraffe (Giraffa camelopardalis ), impala (Aepyceros melampus ), nyala (Tragelaphus buxtoni ), kudu (Tragelaphus strepsiceros ), wildebeest (Connochaetes taurinus ), and zebra (Equus burchelli ). These herbivores are important in South African parks and reserves for ecotourism and as a prey base for predators and serve an integral role in ecosystem processes. Standard biochemical validations (e.g., recovery of exogenous corticosterone, intra‐ and interassay variation, and parallelism) demonstrated that the assay accurately and precisely measured FGMs of all 6 herbivore species. Our biological validations demonstrated that the assay was sensitive enough to detect changes in FGM production associated with season. Samples collected during the dry season (Jun‐Aug) contained higher FGM concentrations than those from the wet season (Dec‐Feb) in all species. We established optimal sample dilutions and reference FGM levels for these 6 herbivores, which can now be used to monitor the effects of management and ecotourism activities on the stress responses of these herbivores.
Flauger B., Krueger K., Gerhards H., Möstl E. (2010): Simplified method to measure glucocorticoid metabolites in faeces of horses. Veterinary Research Communications 34: 185-195.
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Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3alpha,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two-step clean up procedure of the samples (Merl et al. 2000). In addition, the 3alpha,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species.
Konjević D., Janicki Z., Slavica A., Severin K., Krapinec K., Božić F., Palme R. (2011): Non-invasive monitoring of adrenocortical activity in free-ranging fallow deer (Dama dama L.). European Journal of Wildlife Research 57: 77-81.
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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.
Morden C. J. C., Weladji R. B., Ropstad E., Dahl E., Holand Ø., Mastromonaco G., Nieminen M. (2011): Fecal hormones as a non‐invasive population monitoring method for reindeer. The Journal of Wildlife Management 75: 1426-1435.
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Proper management of threatened species requires knowledge of population sizes and structures, however current techniques to gather this information are generally impractical and costly and can be stressful on the animals. Non‐invasive methods that can produce high quality and accurate results are better alternatives. In winter 2010, we collected blood and fecal samples from 2 reindeer (Rangifer tarandus) populations (Kaamanen, Finland and Svalbard, Norway) to investigate the feasibility of using fecal progesterone metabolites to help estimate the reproductive status, the sex, and the age structures of the populations. We first examined the relationship between plasma progesterone and fecal progesterone metabolite concentrations. We further assessed whether fecal progesterone metabolite levels would clearly differ among calf, yearling, and adult and between pregnant and non‐pregnant females. We quantified fecal progesterone metabolites (using enzyme immunoassay) and plasma progesterone (using radio immunoassay) of females and males of different ages from the 2 herds. We found in both populations that fecal progesterone metabolite levels reflected plasma progesterone concentrations. However, the range of fecal progesterone metabolite concentration was much wider in Finland than in Svalbard, possibly due to differences in diet or body condition. We determined a threshold value of 1.31 ng/ml plasma progesterone and 2025.93 ng/g dried fecal progesterone metabolites to identify pregnant reindeer from non‐pregnant animals with 100% accuracy. We found a significant difference in fecal progesterone metabolite concentrations only between calves and yearlings/adults in Finland. We could not differentiate among males, non‐pregnant adults, or calves of either sex; therefore identification of sex may have to rely on the use of DNA techniques. Our results suggest that hormone concentration, in combination with fecal DNA and pellet morphometry techniques, may provide important population parameters and is a valuable tool for the monitoring of reindeer and may have an application for threatened populations of woodland caribou throughout the winter and early spring.
Hadinger U., Haymerle A., Knauer F., Schwarzenberger F., Walzer C. (2015): Faecal cortisol metabolites to assess stress in wildlife: evaluation of a field method in free‐ranging chamois. Methods in Ecology and Evolution 6: 1349-1357.
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Non‐invasive faecal cortisol metabolite (FCM) analysis is a well‐established tool to quantify stress in captive and free‐ranging species. While the method has great potential, its suitability in field studies might be limited when faecal samples from unknown individuals are used. Possible factors affecting final results and thus jeopardizing correct data interpretation are individual and sex‐specific variation, storage conditions and uneven distribution of metabolites in the faeces. We tested these factors on a population of free‐ranging Alpine chamois Rupicapra rupicapra rupicapra in the Austrian Alps. Faecal samples (n = 183) were analysed with an established enzyme immunoassay (EIA). To further validate the assay for FCM in chamois, a high‐performance liquid chromatography (HPLC) was performed. Sex‐specific differences in metabolite excretion were evaluated. Effects of storage length and temperature on FCM were tested with two experiments. The distribution of metabolites in the faeces was determined by the analysis of subsamples of single faecal samples. Potential individual effects on FCM levels and individually variable reactions to stressful events were evaluated with a simulation experiment. Patterns of immunoreactive peaks after HPLC separation were similar for different faecal samples, except in one sample of a male. In the stability tests, storage time at ambient temperature prior to freezing and the individual were the most important variables in modulating FCM. Concentrations within single samples varied significantly between pellets. Analysis of faecal samples collected from June to October showed a highly significant seasonal trend (P < 0·001) and a considerable variance of FCM levels within the population. Simulations confirmed that individual reactions to stressors in terms of varying gradients and FCM levels can explain the observed FCM patterns. Using FCM to assess adrenocortical function requires measuring extensively metabolized products of glucocorticoids, whose excretion and detection in faeces depend on several environmental, endogenous and methodological factors. In free‐ranging wildlife, these factors and the intrinsic individual differences in FCM excretion generate systemic noise and substantially distort final results. Therefore, sampling of unknown individuals inevitably jeopardizes meaningful interpretation of data, if the above named factors are not taken into consideration.
Caslini C., Comin A., Peric T., Prandi A., Pedrotti L., Mattiello S. (2016): Use of hair cortisol analysis for comparing population status in wild red deer (Cervus elaphus) living in areas with different characteristics. European Journal of Wildlife Research 62: 713-723.
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We tested a method to measure Hair Cortisol Concentration (HCC) in 174 red deer (Cervus elaphus) culled in the hunting season 2011/12 in four areas of Central Italian Alps, with different population densities (SPN, 11.8 deer/km2; HD-AV, 3.6 deer/km2; HD-SO, 2.1 deer/km2; HD-MO, 2.0 deer/km2) and environmental conditions. Our hypothesis was that higher population densities, associated with more difficult environmental conditions, may result in higher allostatic load for these wild ungulates. No significant differences in HCC were detected between sexes (males, 4.77 ± 0.69 pg/mg; females, 5.75 ± 0.63 pg/mg) nor among age classes (calves, 6.17 ± 0.66 pg/mg; yearlings, 4.47 ± 0.83 pg/mg; adults, 5.15 ± 0.74 pg/mg; least square mean ± SE), but HCC difference between calves and yearlings was close to statistical significance (P = 0.059). HCC showed high individual variation, but on average it was higher in areas with higher deer density (SNP, 7.45 ± 1.01 pg/mg; HD-AV, 6.07 ± 0.89 pg/mg; HD-SO, 4.67 ± 1.14 pg/mg; HD-MO, 2.87 ± 1.56 pg/mg), with significant differences between HD-AV and HD-MO (P = 0.01). Carcass weight was significantly lower in SNP (46.74 ± 1.49 kg) than in HD-MO (62.71 ± 4.01 kg), HD-SO (61.73 ± 2.9 kg) and HD-AV (62.07 ± 2.04 kg) (P < 0.001). These results seem to confirm our hypothesis that allostatic load is higher in areas with higher density and harder environmental conditions. We suggest that the methodology used in this study to measure HCC provides good information on long-term HPA axis activity and allostatic load and constitutes a highly promising, reliable and non-invasive method in wildlife management for assessing HPA axis activity over extended time periods.
Ping X., Liu N., Jiang Z., Xu H., Bai J., Zhang S., Zhong Z., Li C. (2018): Assaying progesterone, estradiol and cortisol concentrations in hair of Père David deer hinds: an alternative way to reflect seasonality of steroid secretion. Biological Rhythm Research 49: 422-430.
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The use of a hair hormone concentration assay is increasingly recognized as a useful and noninvasive technique for monitoring the endocrinological status of animals. However, few studies have focused on reproductive and stress hormones together. We used a chemiluminescent immunoassay to determine whether the progesterone, estradiol, and cortisol concentrations could be measured from hair and whether these hormone concentrations varied in different hair segments of captive Père David deer hinds. We found that progesterone, estradiol, and cortisol could be measured in the hair samples and that the progesterone concentration varied but the estradiol and cortisol concentrations did not among different hair segments. Contrary to the segmental decline in hair cortisol found in many studies, we found that progesterone concentration was higher near the tip than at the base of hair in Père David deer. This suggests that the variation in segmental hair steroid hormone concentration in seasonal molting animals may be mainly due to internal reproductive cycles and that hair steroid hormones may reflect long-term physiological changes and can thus be used for the conservation and management of wildlife.
Seeber P. A., Franz M., Dehnhard M., Ganswindt A., Greenwood A. D., East, M. L. (2018): Plains zebra (Equus quagga) adrenocortical activity increases during times of large aggregations in the Serengeti ecosystem. Hormones and Behavior 102: 1-9.
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Adverse environmental stimuli (stressors) activate the hypothalamic-pituitary-adrenal axis and contribute to allostatic load. This study investigates the contribution of environmental stressors and life history stage to allostatic load in a migratory population of plains zebras (Equus quagga) in the Serengeti ecosystem, in Tanzania, which experiences large local variations in aggregation. We expected higher fGCM response to the environmental stressors of feeding competition, predation pressure and unpredictable social relationships in larger than in smaller aggregations, and in animals at energetically costly life history stages. As the study was conducted during the 2016 El Niño, we did not expect food quality of forage or a lack of water to strongly affect fGCM responses in the dry season. We measured fecal glucocorticoid metabolite (fGCM) concentrations using an enzyme immunoassay (EIA) targeting 11β-hydroxyetiocholanolone and validated its reliability in captive plains zebras. Our results revealed significantly higher fGCM concentrations 1) in large aggregations than in smaller groupings, and 2) in band stallions than in bachelor males. Concentrations of fGCM were not significantly higher in females at the energetically costly life stage of late pregnancy/lactation. The higher allostatic load of stallions associated with females, than bachelor males is likely caused by social stressors. In conclusion, migratory zebras have elevated allostatic loads in large aggregations that probably result from their combined responses to increased feeding competition, predation pressure and various social stressors. Further research is required to disentangle the contribution of these stressors to allostatic load in migratory populations.
Hunninck L., May R., Jackson C. R., Palme R., Røskaft E., Sheriff M. J. (2020): Consequences of climate-induced vegetation changes exceed those of human disturbance for wild impala in the Serengeti ecosystem. Conservation Physiology 8: coz117.
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In East Africa, climate change is predicted to reduce vegetation quality, and pervasive human disturbance has already resulted in significant declines in biodiversity. We studied the combined effects of reduced forage quality and human disturbance on faecal glucocorticoid metabolite (FGM) concentrations. We predicted that decreasing nutritional quality and increasing human disturbance would have an additive positive effect on FGM levels in wild impala (Aepyceros melampus). Employing a space-for-time approach, we used normalized difference vegetation index (NDVI) as a measure of forage quality, combined with spatially explicit proxies of human disturbance across areas of different protection management strategies in the Serengeti ecosystem. We collected 639 faecal samples, spread over 4 years, including both wet and dry seasons. Impala FGM levels increased significantly with declining NDVI and, to a lesser extent, with increasing proxies for human disturbance. However, we found no interaction between the two, such that impala had elevated FGM levels with low NDVI and low FGM levels with high NDVI regardless of human disturbance levels. This implies that impala will have high FGM levels if forage quality is poor, even with significant protection and reduced human disturbance. Understanding how animals respond to and cope with changes in forage quality and human land use across different protected areas is important for conservationists and managers to better protect species at risk and predict population viability.
Liu R., Shi J., Liu D., Dong S., Zhang Y., Wu Y., Guo D. (2020): Effect of group size and reproductive status on faecal glucocorticoid concentration and vigilance in a free-ranging population of Przewalski’s gazelle. Conservation Physiology 8: coaa027.
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Elevated glucocorticoid (GC) concentration and increased vigilance are two common responses to predation risk in mammals. Chronic high-level GC concentration and vigilance occur at the expense of other life maintenance and reproduction activities, reflecting a trade-off between individual survival and future fecundity. Przewalski’s gazelle (Procapra przewalskii) is a group-living ungulate endemic to the high-altitude Qinghai Lake region of China. Group-size effect on gazelle vigilance has been examined, yet little is known about how their GC concentration is affected by group size or reproductive status. In this study, we examined the effect of group size and reproductive status on faecal glucocorticoid metabolite (FGM) concentrations and individual vigilance during different stages of the reproduction cycle (i.e. non-breeding, lambing and rutting) in free-ranging adult female Przewalski’s gazelles. Group size did not influence FGMs significantly, but mean vigilance duration increased with group size. The gazelles’ FGMs and vigilance peaked in lambing season. FGMs showed no difference between rutting season and non-reproductive season, but vigilance was lowest in the rutting season. FGMs correlated with vigilance frequency and vigilance duration. Antipredator responses of female Przewalski’s gazelles appear to change with reproductive status but not with group size in free-ranging females. Management measures should be taken in the lambing season to minimize stress on mother gazelles.
Rosenblatt E., DeBow J., Blouin J., Donovan T., Murdoch J., Creel S., Rogers W., Gieder K., Fortin N., Alexander C. (2021): Juvenile moose stress and nutrition dynamics related to winter ticks, landscape characteristics, climate-mediated factors and survival. Conservation Physiology 9: coab048.
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Moose populations in the northeastern United States have declined over the past 15 years, primarily due to the impacts of winter ticks. Research efforts have focused on the effects of winter tick infestation on moose survival and reproduction, but stress and nutritional responses to ticks and other stressors remain understudied. We examined the influence of several environmental factors on moose calf stress hormone metabolite concentrations and nutritional restriction in Vermont, USA. We collected 407 fecal and 461 snow urine samples from 84 radio-collared moose calves in the winters of 2017–2019 (January–April) to measure fecal glucocorticoid metabolites (fGCM) concentrations and urea nitrogen:creatinine (UN:C) ratios. We used generalized mixed-effects models to evaluate the influence of individual condition, winter ticks, habitat, climate and human development on stress and nutrition in calf moose. We then used these physiological data to build generalized linear models to predict calf winter survival. Calf fGCM concentrations increased with nutritional restriction and snow depth during adult winter tick engorgement. Calf UN:C ratios increased in calves with lighter weights and higher tick loads in early winter. Calf UN:C ratios also increased in individuals with home ranges composed of little deciduous forests during adult winter tick engorgement. Our predictive models estimated that winter survival was negatively related to UN:C ratios and positively related to fGCM concentrations, particularly in early winter. By late March, as winter ticks are having their greatest toll and endogenous resources become depleted, we estimated a curvilinear relationship between fGCM concentrations and survival. Our results provide novel evidence linking moose calf stress and nutrition, a problematic parasite and challenging environment and winter survival. Our findings provide a baseline to support the development of non-invasive physiological monitoring for assessing environmental impacts on moose populations.
Martínez L., Silván G., Cáceres S., Caperos J. M., Fernández-Morán J., Casares M., Crespo B., de Andrés P. J., Illera J. C. (2022): Preliminary findings on how different management systems and social interactions influence fecal glucocorticoid metabolites in white rhinoceros (Ceratotherium simum). Animals 12: 897.
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White rhinoceroses (Ceratotherium simum) are the most social and gregarious species of all rhinoceroses known worldwide. One of the most critical effects of elevated glucocorticoid concentrations, especially in threatened species, is its relation to chronic stress, which could potentially lead to immunosuppression and reduced reproductive activity. Our aim is to determine how different social environments and management systems might be influencing the secretion of fecal glucocorticoids in white rhinoceroses. We have analyzed the concentration of fecal glucocorticoid metabolites in 658 fecal samples from 14 white rhinoceroses, seven free-ranging rhinos, and seven rhinos from two different managed captive populations. HPLC techniques were used to determine the main glucocorticoid metabolite found in this species, and a competitive EIA was used to establish fecal 5α-pregnan-3 3β, 11β, 21-triol-20-oneglucocorticoid metabolite (fGCM) levels. Our results reveal that management systems and social interactions had an influence on fGCM levels, suggesting that the more restrictive the management and social conditions are, the higher the glucocorticoid concentrations that are found. Additionally, sex was also found to influence fGCM levels, as in females, fGCM concentrations were higher than in males. We conclude that the analysis of glucocorticoids in relation to other factors is a powerful tool to assess adrenocortical response in white rhinoceros in order to broaden the knowledge of their reproductive biology and improve the management of the species.
Pero E. M., Chitwood M. C., Hildreth A. M., Keller B. J., Millspaugh R. J., Sumners J. A., Hansen L. P., Isabelle J. L., Breuner C. W., Millspaugh J. J. (2022): Physiological acclimation of elk during population restoration in the Missouri Ozarks, USA. Conservation Physiology 10: coac009.
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Conservation translocations—the intentional movement of animals to restore populations—have increased over the past 30 years to halt and reverse species declines and losses. However, there are many challenges translocated animals face that should be considered for restoration programs to be successful. Understanding how long it takes for translocated animals to acclimate to these challenges and their new landscape is a critical component of post-release population management. Physiological measures such as hormone responses are increasingly used to assess animal responses and acclimation to disturbances including translocation. We determined the physiological acclimation period of elk (Cervus canadensis) translocated to the Missouri Ozarks, USA, as part of a restoration effort. From 2011 to 2013, we translocated 108 GPS-radio-collared elk from Kentucky, USA, to Missouri, USA, and collected faecal samples for glucocorticoid metabolite extraction to use as an indicator of physiological acclimation. We modelled the response of population-wide faecal glucocorticoid metabolites (fGCMs) across the initial 9 years of the restoration in response to days following release and additional site-specific covariates. Presence of white-tailed deer (Odocoileus virginianus) hunts and monthly precipitation levels were positively and negatively associated with fGCM levels, respectively. Concurrent with influences from site-specific conditions on the release landscape, fGCM levels declined following release. We identified a breakpoint in fGCM decline at ~42 days following translocation releases suggesting elk acclimated physiologically relatively quickly compared to other species. The fast physiological acclimation by Missouri elk suggests effective use of temporary post-release management efforts. Determining how quickly animals acclimate following translocations allows researchers to tailor post-release management plans to each species’ needs, thus maximizing the success of future translocation efforts while minimizing costs.
Kamgang V. W., Bennett N. C., van der Goot A. C., Majelantle T. L., Ganswindt A. (2022): Patterns of faecal glucocorticoid metabolite levels in captive roan antelope (Hippotragus equinus) in relation to reproductive status and season. General and Comparative Endocrinology 325: 114052.
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Populations of roan antelope (Hippotragus equinus) in southern Africa have experienced a drastic decline over the past few decades and this situation has led to the development of intensive breeding programmes to support conservation efforts. However, little is known about related welfare aspects, including stress-related physiological biomarkers. The present study set out to establish a non-invasive method to monitor faecal glucocorticoid metabolite (fGCM) concentrations as a measure of stress and determine fGCM concentrations in relation to male reproductive activity and female reproductive status in the roan antelope. An adrenocorticotrophic hormone challenge was performed using two adult roan antelope (one male and one female) at Lapalala Wilderness Nature Reserve, South Africa, to determine the suitability of five enzyme immunoassays (EIA) for monitoring adrenocortical function in roan antelope. An 11-oxoaetiocholanolone I EIA detecting 11,17 dioxoandrostanes performed best showing 17–20 folds increases in fGCM concentrations after 12 h-17 h post-injection. The identified EIA was then used to monitor fGCM concentrations during active and non-active reproductive periods in males (n = 3), and during periods of cyclicity, gestation, and postpartum in females (n = 10). Males showed an overall 80% increase in fGCM concentrations when reproductively active and females showed a progressively significant increase in fGCM levels throughout pregnancy, with overall fGCM concentrations being 1.5 to 2.6-fold higher than the respective fGCM concentrations during periods of postpartum and cyclicity, respectively. Furthermore, fGCM concentrations remained above baseline for up to 21 days post-partum. A correlation between ecological parameters (rainfall and temperature) and fGCM concentrations revealed elevated fGCM concentrations during the dry season for males, but not females. The non-invasive method validated in this study provides a valuable tool to quantify stress-related biomarkers in roan antelope, and findings can be used to support management decisions in conservation breeding facilities.
Ramos A., Robin J. P., Manizan L., Audroin C., Rodriguez E., Kemp Y. J., Sueur C. (2022): Glucocorticoids of European bison in relation to their status: age, dominance, social centrality and leadership. Animals 12: 849.
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Stress is the body’s response to cope with the environment and generally better survive unless too much chronic stress persists. While some studies suggest that it would be more stressful to be the dominant individual of the group, others support the opposite hypothesis. Several variables can actually affect this relationship, or even cancel it. This study therefore aims to make the link between social status and the basal level of stress of 14 wild European bison (Bison bonasus, L. 1758) living together. We collected faeces and measured the faecal glucocorticoid metabolites (FGM). We showed that FGM is linked to different variables of social status of European bison, specifically age, dominance rank, eigenvector centrality but also to interactions between the variables. Preferential leaders in bison, i.e., the older and more dominant individuals which are more central ones, are less stressed compared to other group members. Measurement of such variables could thus be a valuable tool to follow and improve the conservation of species by collecting data on FGM and other social variables and adapt group composition or environmental conditions (e.g., supplement in food) according to the FGM concentration of herd individuals.