Fecal Glucocorticoid Metabolite Levels Research Articles

The Comparative Effect of Occupational and Musical Enrichment on Fecal Glucocorticoid Metabolite Levels in a Captive Colony of Stumptail Macaques (Macaca arctoides).

Environmental enrichment improves captive animal welfare by reducing stress-related behaviors. Previous studies in a captive colony of stumptail macaques (Macaca arctoides) reported a reduction of aggression, coprophilia, and stereotypic behaviors after an occupational enrichment program; however, the effect on stress hormones such as glucocorticoids has not been investigated yet. The goal of this study was to compare the effect of sex, age, and social rank on changes in fecal glucocorticoid metabolites (fGCM) after applying two kinds of enrichments (occupational vs. musical) in a captive colony of stumptail macaques. We collected 234 fecal samples from 25 stumptail macaques under the following conditions: (1) basal (no enrichment), (2) three weeks of occupational enrichment, and (3) three weeks of relaxing/classical music. The Generalized Estimated Equation Model showed an increase in fGCM levels after the occupational enrichment only in adult subjects (p = 0.003 compared to basal). The fGCM levels reached by the adults after the occupational enrichment was higher than that of juveniles (p = 0.002) and subadults (p = 0.02). Occupational and musical enrichment decreased fGCM levels only in middle-ranking individuals (p < 0.001 and p = 0.013, respectively). No sex differences were found. In conclusion, there were age and rank differences in individuals' physiological reactivity to the effects of environmental enrichment which need to be considered when planning enrichment programs.

Changes in Fecal Glucocorticoid Metabolites in Captive Coyotes (Canis latrans): Influence of Gender, Time, and Reproductive Status.

Reproduction is considered an energetically and physiologically demanding time in the life of an animal. Changes in physiological stress are partly reflected in changes in glucocorticoid metabolites and can be measured from fecal samples. We examined levels of fecal glucocorticoid metabolites (fGCMs) in 24 captive coyotes (Canis latrans) to investigate responses to the demands of reproduction. Using 12 pairs of coyotes (five pairs produced pups, seven pairs did not), we analyzed 633 fecal samples covering 11 biological periods (e.g., breeding, gestation, and lactation). Levels of fGCMs showed high individual variability, with females having higher fGCM levels than males. The production of pups showed no statistical effect on fGCM levels among females or males. Among females, fGCM levels were highest during 4-6 weeks of gestation compared to other periods but were not significantly different between pregnant and nonpregnant females. Among males, the highest fGCM levels were during 1-3 weeks of gestation compared to other periods, but were not significantly different between males with a pregnant mate versus nonpregnant mate. Of females producing pups, litter size did not influence fGCM levels. Given that they were fed ample food throughout the year, we found that the demands of producing pups did not appear to statistically influence measures of fGCM concentrations in captive coyotes.

Reaction to Novel Objects and Fecal Glucocorticoid Metabolite Levels in Two Species of Nocturnal Geckos.

Many reptiles are maintained in captivity and heavily traded, although welfare measures for many species are not well established and are under-researched compared to other animals. In this study, we focused on two of these species: crested geckos (Correlophus ciliatus) and leopard geckos (Eublepharis macularius). To better interpret their behavior in captivity, the individual reaction to novel objects and the fecal glucocorticoid metabolite levels were measured in an attempt to identify the potential correlation between them. Also, we explored if some characteristic of the objects (e.g., color, shape, or smell) resulted in being more attractive to some species and/or individuals. Equivalent responses to different objects were not obtained for all the geckos, the behavioral response being highly individual and context-dependent, although modulated by the species. Individuals which manipulated earlier and interacted longer with novel objects showed lower basal fecal corticosterone metabolite (FCM) levels. Differences according to the species suggested that crested geckos have significantly greater and more variable FCM levels than leopard geckos. Our results can help to understand the reaction of geckos to novelty and have the potential to serve in their welfare assessment, although more studies are needed to proper establish welfare protocols.

Identifying potential measures of stress and disturbance during a captive to wild African elephant reintegration.

There is increasing evidence of compromised welfare for elephants managed in captivity. Should such facilities eventually close, more elephants will need to be rehabilitated and reintegrated into the wild. The goal of such reintegration would be to restore any physical or psychological aspects of the elephant that may have been compromised in captivity, followed by introduction into a free-roaming system where they can interact with other elephants. However, to achieve this goal, the reintegration methods implemented need to be assessed to ensure that welfare remains the priority. The objective of this study was to test whether parameters generally associated with stress and disturbance in African elephants, respond to changes in potentially stressful environmental conditions, assessed at multiple temporal scales ranging from minutes to months. The main changes in environmental conditions that were investigated included the different phases of reintegration of a group of elephants from captivity into the wild. Stress and disturbance related parameters used for comparisons included physiological responses, namely the extent of temporal gland secretions (eTGS) and faecal glucocorticoid metabolite (fGCM) levels as well as behavioural responses, namely the display of stereotype and stress-related behaviours. Results showed that eTGS significantly increased during the initial release of the elephants compared to when in captivity. Stereotypic behaviours were only recorded during the captive phase and immediately ceased after release. Faecal GCM levels spiked in the first year after release before decreasing back to pre-reintegration levels during the third year. These findings indicate that fGCM levels, the eTGS and disturbance related behaviours all proved effective in explaining the changes in stress and disturbance experienced by elephants during the initial years after being reintegrated from captivity into the wild.

Effects of Tourist and Researcher Presence on Fecal Glucocorticoid Metabolite Levels in Wild, Habituated Sulawesi Crested Macaques (Macaca nigra).

Ecotourism managers and researchers often assume that apparently habituated primate groups no longer experience adverse consequences of prolonged exposure to tourists or researchers. We examined the effects of tourists and researchers on fecal glucocorticoid metabolite output (FGCM) in three critically endangered, wild crested macaque (Macaca nigra) groups in Tangkoko Nature Reserve, Sulawesi, Indonesia. We assayed FGCM from 456 fecal samples collected from thirty-three adults. Tourists can walk through and among macaque groups freely. Hence, we examined the possible effects of tourists both (1) in the reserve when away and not interacting with the study groups and (2) when they were present within the macaque groups. Generalized Linear Mixed Model (GLMM) analysis indicated that when tourists were present in the forest, but not directly among the macaques, FGCM levels in the macaque tourism groups were higher in months with more tourists. When tourists were among the macaque groups, some female macaques experienced rises and subsequent postexposure decreases in FGCM levels, consistent with predictions for acute stress. Male FGCM levels increased with tourist numbers within the group. Nevertheless, they were not significantly different from levels during undisturbed or postexposure conditions. FGCM responses related to researchers in groups varied by group, sex, and tourist presence. However, the temporal patterning of FGCM responses showed little evidence of chronic stress from tourism at this site.

Analytical Validation and Assessment of Baseline Fecal Glucocorticoid Metabolites in Northern Sea Otters (Enhydra lutris kenyoni) in Human Care

Fecal glucocorticoid metabolites (FGMs) have been used as a non-invasive and indirect measurement of the complex stress response in a variety of species. Animals in facilities under managed care allow for the longitudinal study of FGMs in a controlled environment. Animal histories often include environmental, husbandry, and medical notes that can be matched to FGM concentrations to aid in the physiological validation of adrenal stimulation and response. The goal of this study was to demonstrate analytical validations using two enzyme-linked immunosorbent assays (EIA) to measure FGMs from northern sea otters (Enhydra lutris kenyoni) under human care (Seattle Aquarium, Seattle, WA, USA) and to determine baseline and stress response spike levels for individual sea otters. Individual variation was found among the four subjects in the study with fecal baseline levels ranging from 20.2 to 83.7 ng/g for cortisol-immunoreactive metabolites and 52.3 to 102 ng/g for corticosterone-immunoreactive metabolites. As a retrospective study, 39 percent of hormone peaks were associated with notes and most FGM spikes were associated with veterinary procedures or days in which enrichment items were provided and produced an excitatory response. Monitoring baseline FGMs levels and events associated with hormone peak values may provide insight into effective husbandry management to improve the overall welfare of sea otters and other marine mammals.

Faecal glucocorticoid metabolite levels of intensive captive, free‐range captive and wild Tasmanian devils

AbstractCaptivity can alter the stress physiology and behaviour of an animal in both the short‐ and long‐term through repetitive exposure to novel stressors and, subsequently, may reduce the success of conservation efforts such as translocation and reintroduction. The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction from a fatal facial tumour disease which has led to the establishment of an insurance meta‐population designed for future reintroductions of disease‐free devils. The meta‐population is comprised of intensive captive and free‐range captive environments; however, no study has yet examined the long‐term physiological implications of captivity on devils. We used non‐invasive faecal glucocorticoid metabolite (FGM) monitoring to determine if there were any differences in adrenal activity among intensive captive, free‐range captive and wild devils. FGM levels were not age or sex dependent, and we found that all population types had similar intra‐population variability and mean FGMs. In conclusion, both types of captive environment appear to maintain stress profiles similar to wild devils.

Diurnal fecal glucocorticoid metabolite rhythms in a cathemeral primate, the red-bellied lemur (Eulemur rubriventer), and across mammalian species.

Measuring glucocorticoids is one of the most reliable and widely used techniques to monitor stress responses, however invasive techniques to collect plasma samples may not be applicable for wild populations. Monitoring excreted glucocorticoids is an effective noninvasive technique that researchers have used increasingly over the past two decades, and it has allowed the investigation of glucocorticoids in a variety of species with a range of activity patterns. Many species exhibit predictable circadian patterns of glucocorticoid secretion in accordance with their daily activity pattern. There remains a gap in our understanding of how excreted glucocorticoid metabolites vary throughout the day and across species, despite the utility of this information when developing sampling protocols and analyzing data. We investigated circadian patterns of glucocorticoid excretion in a cathemeral primate species, Eulemur rubriventer (red-bellied lemur), in Ranomafana National Park, Madagascar. We collected fecal samples from 10 individuals throughout the day and analyzed fecal glucocorticoid levels across three time points (Early, Midday, and Late), and again across two time points (Morning and Afternoon). We also investigated whether activity pattern, sex (as a control variable), and other traits associated with gut passage rate (diet, body mass) could help predict the presence and timing of circadian patterns of fecal glucocorticoid metabolites across mammal species. We found that fecal glucocorticoid metabolite levels in E. rubriventer fluctuate throughout the day, with lowest levels in the morning and peak levels in the afternoon. None of the variables that we tested predicted whether daily fecal glucocorticoid metabolites changed significantly throughout the day, nor when levels were likely to peak, across species. We stress the importance of controlling for sampling time and reporting these results as standard practice in studies of fecal glucocorticoid metabolites.

Factors affecting captive female giraffe stress response: Male presence, small enclosure, and low temperature.

To improve animal welfare based on suitable social housing conditions, it is important to understand the factors that trigger high-stress responses. Wild giraffes live in a fission-fusion society and males and females are rarely in the same herd for a long period. The captive condition of belonging to a herd with the same individuals for months or years is uncommon in nature. To understand the effect of male presence on female stress levels, fecal glucocorticoid metabolite (fGCM) levels and social interactions in two captive female giraffes were investigated. Additionally, the effect of enclosure size and temperature on fGCM level and social interactions were examined. The results showed no significant difference in the fGCM levels of females based on male presence. The frequency of agonistic behavior by the dominant female toward the subordinate female was significantly increased when a male was present. The subordinate female was significantly less likely to approach the dominant female and showed decreased affiliative and agonistic interactions toward the dominant female when a male was present. The frequencies of agonistic interactions between females were higher in the small enclosure regardless of male presence. Low temperature triggered higher fGCM levels and increased agonistic interaction in an aged female. The findings of this study suggest that these multiple factors should be considered individually to promote the welfare of captive giraffes.

Linking glucocorticoid variations to monthly and daily behavior in a wild endangered neotropical primate.

Identifying the factors swaying physiological stress levels in wild animals can help depict how they cope with environmental and social stressors, shedding light on their feeding ecology, behavioral plasticity, and adaptability. Here, we used noninvasive methods to explore the link between glucocorticoid levels and behavior in an endangered neotropical primate facing habitat fragmentation pressure, the black lion tamarin (Leontopithecus chrysopygus). We investigated monthly and day-to-day glucocorticoid variations independently to attempt to disentangle the complex nature of the adrenocortical activity. Between May 2019 to March 2020, we followed two groups of black lion tamarins in two different areas, a continuous forest and a small fragment, and gathered behavioral data (over 95 days in total; 8.6 ± 3.9 days/month) and fecal samples (Nsamples = 468; 4.93 ± 3.5 samples/day) simultaneously. Preliminary analyses enabled us to identify circadian variations linked to the biological rhythm, which were taken into account in subsequent models. Monthly analyses revealed that black lion tamarin fecal glucocorticoid metabolite levels vary according to changes in activity budget associated with the fruit consumption, movement, and resting time of the groups. At a day-to-day level, while intergroup encounters led to increases in fecal glucocorticoid metabolite concentrations, we found that changes in food intake or activity level did not trigger physiological stress responses. These findings suggest that diet and ranging patterns, driven by food availability and distribution, influence physiological stress at a seasonal scale, while acute stressors such as interspecific competition trigger short-term stress responses. Exploring fecal glucocorticoid metabolite variations over different timescales can help uncover the predictive and reactive facets of physiological stress in wild species. Moreover, having a comprehensive understanding of the physiological state of species is a valuable conservation tool for evaluating how they cope in changing environments.

Assessing stress in wild black-and-white colobus monkeys non-invasively

Analysis of glucocorticoid profiles serves as a valuable, multi-faceted tool for insight into the behavior and physiology of wild populations. Recently, the measurement of fecal glucocorticoid metabolites (FCMs) has exploded in popularity due to its compatibility with noninvasive techniques and remote environments A critical first step is to perform a biological validation to ensure that the assay accurately reflect changes in FCM levels. We use an enzyme immunoassay (EIA) to perform a biological validation on samples collected from two males and six females in a wild population of Colobus vellerosus in response to three naturally occurring potential stressors. We also describe the FCM response pattern in the week following parturition in three females and examine the influence of sex, reproductive state, and time of day on the concentrations of baseline samples collected daily from 13 adult individuals over a period of four months. We validated the assay: FCM levels increase in response to natural stressors with a two-day lag. In the two days surrounding parturition, FMC levels increased. Baseline concentrations were affected by collection time and female reproductive state, with lactating females having lower concentrations than pregnant or cycling females. Thus, we successfully carried out the first validation and characterization of FCMs in a wild African colobine. This will serve as an essential foundation for future studies of C. vellerosus and similar wild primates whose objective is to investigate the role glucocorticoids play in responses to social and ecological challenges.

Between hunter and climate: the effects of hunting and environmental change on fecal glucocorticoid metabolite levels in two sympatric ungulate species in the Ruaha-Rungwa ecosystem, Tanzania.

Understanding the drivers of animal population decline is a key focus of conservation biologists. Anthropogenic activities such as hunting have long been established as potentially detrimental to a population's persistence. However, environmental perturbations such as increased temperature variability, exacerbated by climate change, can also have important effects on animal populations. Animals can respond to these challenges by adjusting both their behavior and physiology. We measured fecal glucocorticoid metabolites (FGMs) of common impala (Aepyceros melampus) and greater kudu (Tragelaphus strepsiceros), both currently in stable populations, to examine effects of hunting, forage availability, daily variability in temperature and group size on their physiological stress response. The study was conducted across two adjacent protected areas, (i) one non-hunted area (Ruaha National Park; RNP) and (ii) one area used for trophy hunting (Rungwa Game Reserve; RGR). Both impala and kudu had significantly higher FGM levels in the area that allows hunting, while FGM levels decreased with increasing forage availability and increasing daily temperature. Moreover, impala (but not kudu) had lower FGM levels with larger group sizes. Our results indicate that the management regime can significantly alter the physiological state of wild ungulate populations. We also highlight the importance of considering the combined effects of anthropogenic, environmental and social contexts when studying the stress response of wild populations. Our results emphasize the value of protected areas and continued monitoring of hunting quota in order to maintain ungulate populations that are less vulnerable to population declines.

Stress-associated metabolites vary with both season and habitat across populations of a climate sentinel species

ABSTRACT Relating physiological stress to habitat quality could refine conservation efforts. Habitat quality, which is often inferred from patch occupancy or demographic rates, might be measured in a more timely and nuanced way using metrics of physiological stress. To understand whether stress-associated hormones vary with metrics of habitat quality, we measured fecal glucocorticoid metabolite (FGM) levels in the American pika (Ochotona princeps), a small mammal with well-defined habitat (talus), which can vary in quality depending on the presence of subsurface ice features. In spring and fall 2018, we collected feces noninvasively from pika territories in taluses “with” or “without” subsurface ice to capture seasonal variation in FGM between habitat types. We used linear mixed effects models to explore the interactions among season, habitat metrics (including subsurface ice status), and subsurface temperature as predictors of FGM. We found support for interacting effects on FGM levels, which covaried with season, elevation, putative ice presence, graminoid to forb ratio, graminoid cover, and measures of acute subsurface heat exposure. However, only one subsurface temperature metric differed according to putative presence of subsurface ice. Our results contribute to the growing evidence that FGMs might be developed as a tool to assess habitat quality.

Fecal glucocorticoid analysis as a health monitoring tool for endangered African penguins (Spheniscus demersus)

African penguins (Spheniscus demersus) are an endangered species, with approximately 70,000 mature adults remaining in the wild. Population loss is linked to a combination of environmental and anthropogenic stressors. The aim of the study was to validate a commercially available enzyme immunoassay (EIA) to assess adrenal activity and measure the response to stressors in the feces of African penguins. Fecal samples (n = 609) were collected from 12 African penguins housed at Mystic Aquarium throughout their natural lifecycle, including breeding and molt, where measurable changes in fecal glucocorticoid metabolite (FGM) levels are predicted to occur. Fecal samples collected post-veterinary exam were used for biological validation. Longitudinal analysis shows a significant difference (p = <0.0001) between the average FGM levels during baseline and breeding season, 33.97 ± 1.30 ng/g and 50.21 ± 3.18 ng/g, respectively. Females displayed significantly higher FGM levels than males during both baseline (p = 0.0386; females = 38.80 ± 2.19 ng/g; males = 29.34 ± 1.37 ng/g) and breeding periods (p = 0.0175; females = 57.53 ± 4.84 ng/g; males = 42.69 ± 3.95 ng/g). Average FGM levels decreased significantly over the three-week molting period, from 85.40 ± 20.35 ng/g at week one to 20.23 ± 5.30 ng/g at week three. A seasonal difference in FGM levels was observed in both male and female fecal samples, with Fall having the highest average FGM levels, 54.38 ± 3.64 ng/g, and Summer the lowest, 30.87 ± 2.21 ng/g. General linear mixed model analysis determined that lifecycle (females) and visitor presence (males) were the two factors which best explained the variation in FGM levels observed, however neither factor was found to be significant. These results show FGM analysis can detect physiologically meaningful changes in endocrine activity in African penguins and can be used to monitor health for penguins in aquaria and in the wild, thus contributing to conservation efforts for the survival of the species.

Fecal glucocorticoid metabolite levels in captive Indian leopards (Panthera pardus fusca) housed under three different enrichment regimes.

Environmental enrichment improves the health and wellbeing of zoo animals. To test this hypothesis, we used Indian leopards (Panthera pardus fusca), one of the popular zoo animals, as a model organism to understand effects of active and passive enrichment elements on stress hormone levels of captive individuals. We included three enrichment categories, category 'A' (having both active: cage size of 1204 m3 with raised platforms and earthen flooring, and passive: controlled temperature, playback of forest sounds and sound proof glass to filter visitors' noise, enrichment elements), category 'B' (active enrichment type I, cage size of 264 m3 with air coolers), and category C (active enrichment type II, cage size of 517 m3 without air coolers) for leopards (n = 14) housed in two Indian zoos. We used a group-specific enzyme immunoassay to measure fecal glucocorticoid metabolites (fGCM) in captive leopards. For comparison, we analysed samples from free-ranging leopards, as well. fGCM levels (Mean±SEM) were 10.45±2.01 and 0.95±0.003 μg/g dry feces in captive and free-ranging leopards, respectively. Our results revealed that fGCM levels of leopards in categories B and C were significantly (P<0.05) different from each other, thus, indicating cage size (an active enrichment element) as an important factor in influencing the physiology of the sampled animals. Overall, the findings of the study will contribute towards informed policies for management of captive Indian leopards.

How individual, social, and ecological conditions influence dispersal decisions in malevervet monkeys.

Dispersal between social groups reduces the risk of inbreeding and can improve individuals'reproductive opportunities. However, this movement has costs, such as increased risk of predation and starvation, loss of allies and kin support, and increased aggression associated with entering the new group. Dispersal strategies, such as the timing of movement and decisions on whether to transfer alone or in parallel with a peer, involve different costs and benefits. We used demographic, behavioral, hormonal, and ecological data to examine the causes and consequences of 36 dispersal events from 29 male vervet monkeys (Chlorocebus pygerythrus) at Lake Nabugabo, Uganda. Adult males'secondary dispersal coincided with the conception season in females, and males improved their potential access to females by moving to groups with higher female-to-male sex ratios and/or by increasing their dominance rank. Males that dispersed with a peer had lower fecal glucocorticoid and androgen metabolite levels than lone dispersers.Subadult males were not more likely to engage in parallel dispersals compared to adult males. Dispersal was also used as a mechanism to avoid inbreeding, but changes in hormone levels did not seem to be a trigger of dispersal in our population. Our findings illustrate the complex individual strategies used during dispersal, how many factors can influence movement decisions, as well as the value of dominance and hormone analyses for understanding these strategies.

Validation of an Enzyme Immunoassay to Measure Faecal Glucocorticoid Metabolites in Common Brushtail Possums (Trichosurus vulpecula) to Evaluate Responses to Rehabilitation.

Simple SummaryLittle is known about how exposure to novel stimuli during rescue and rehabilitation could affect the physiology of native wildlife. We investigated this question in a species commonly rescued for rehabilitation, the common brushtail possum (Trichosurus vulpecula). Glucocorticoids (major hormones involved in stress responses) are metabolised in the body and excreted in the form of faecal glucocorticoid metabolites, which can be measured as a way of evaluating the response of animals to potential stressors. Comparing five enzyme immunoassay options, we found that the 11-oxoaetiocholanolone (abbreviation: 72a) EIA was the most suitable for measuring these metabolites in brushtail possums. This assay was then used to measure faecal glucocorticoid metabolite concentrations in 20 possums during rehabilitation. The probability of a physiological “stress” response occurring within five days of a potentially stressful event was about 50%, regardless of the type of event. There was a high level of variation in hormone profiles between possums. Our study has demonstrated that injured and orphaned possums show detectable changes in faecal glucocorticoid metabolites during captivity and rehabilitation, and has identified events that can induce a physiological response in some individuals. This is the first step toward understanding the relationship between these responses during rehabilitation and survival.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.

Impact of Indigestible Materials on the Efficiency of Fecal Corticosterone Immunoassay Testing in Pituophis Species.

Simple SummaryEvaluating stress in animals is important for improving animal welfare and husbandry. However, it has been challenging to establish reliable and noninvasive methods for quantifying stress. Steroid hormones released during prolonged periods of stress are metabolized by the liver and excreted in feces. In snakes, corticosterone is the primary hormone of stress and is often measured in fecal samples collected from these animals. Assays can be used to measure the metabolites of corticosterone from feces, but are limited by compounding factors (season, reproductive status, diet, etc.) that influence the ability to confidently conclude the results. Our overall objective is to standardize these methods by first improving the extraction methodology. We found that 75% of the fecal sample contained indigestible materials (hair, teeth, bone, etc.) and, therefore, interferes with the extraction process. After removing the indigestible material, we found that we had a 95% improvement in overall yield. These findings alleviate one limitation to using fecal samples to measure stress in animals. Measuring fecal glucocorticoid metabolites (FGM) has recently become a sought-after method for assessing stress in animals. While there are many benefits to this methodology, there are also recognized limitations, including the apprehensive interpretation of results. While many factors can influence FGM levels, we aimed to standardize and improve these methods in snakes. Fecal samples were collected from Pituophis species and FGMs were extracted by two different sample collection methods: (1) fecal sample containing undigested materials and (2) fecal samples with undigested materials removed. These extracts were then used to quantify FGM concentrations using a corticosterone EIA kit. The results indicated that the samples with the undigestible materials removed had a 95% increase in overall yield (p < 0.01). Since the collected fecal samples contain 75% undigestible materials by weight, these results support our hypothesis that removing these materials will improve extraction methods for a more reliable measurement of corticosterone. This is the first step towards standardizing the methods for assessing stress by measuring fecal glucocorticoid metabolites in snakes.

Patterns of faecal glucocorticoid metabolite levels in captive roan antelope (Hippotragus equinus) in relation to reproductive status and season

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.

Seasonal Variation in Fecal Glucocorticoid Levels and Their Relationship to Reproductive Success in Captive Populations of an Endangered Parrot

Many species are threatened with extinction, and captive breeding programs are becoming more common to avoid this outcome. These programs serve to prevent extinction and produce individuals for eventual reintroduction to natural populations in historical habitat. Captive animals experience different energetic demands than those in the wild, however, and as a result may have different levels of glucocorticoid hormones. Glucocorticoids help with responses to energetically expensive and potentially stressful situations. Elevated glucocorticoid levels can also potentially alter reproduction and other key behaviors, thus complicating successful captive breeding. The Puerto Rican parrot (Amazona vittata) is a critically endangered parrot that currently exists in only two wild and two captive populations. Its recovery program provides a good platform to better understand how glucocorticoid levels may relate to reproductive success under captive conditions. We validated a corticosterone assay in this species and used non-invasive techniques of measuring fecal glucocorticoid metabolites of males and females from two captive populations (Rio Abajo and El Yunque) of Puerto Rican parrots over two consecutive breeding seasons, 2017 and 2018, and the pre-breeding season of 2018, which occurred just after Hurricane Maria struck Puerto Rico. Our results show that levels of fecal glucocorticoid metabolites of males measured during the breeding season of 2018 negatively correlated to the number of total eggs and fertile eggs laid by pairs. In contrast, there was a positive relationship of female fecal glucocorticoid metabolite levels during the pre-breeding season of 2018 with total eggs laid. In males from the Rio Abajo population, we found seasonal differences in fecal glucocorticoid metabolite levels, with higher levels during the pre-breeding season of 2018 compared to both 2017 and 2018 breeding seasons. There was no difference in the mean value of male fecal glucocorticoid metabolites between the 2017 breeding season and 2018 breeding season which started four months after Hurricane Maria struck Puerto Rico. We did find sex differences during the pre-breeding season of 2018 in birds from the Rio Abajo population. Adjustments in the care routine of both populations that could reduce circulating baseline glucocorticoids and avoid frequent, sudden elevations of glucocorticoids should be considered. These results provide a baseline for future comparison with reintroduced populations of this endangered species and other species with captive breeding programs.