fGCM Concentrations Research Articles

Previous assessments of faecal glucocorticoid metabolites in Cape mountain zebra (Equus zebra zebra) were flawed

Abstract Steroid hormones, especially glucocorticoids (GCs), are widely used to assess physiological responses to stressors. As steroid hormones are heavily metabolised prior to excretion, it is essential to validate enzyme immunoassays (EIAs) for measuring faecal glucocorticoid metabolites (fGCMs). Although problems with unvalidated assays have been raised repeatedly, their use persists widely. Lea et al. (2017) used an unvalidated corticosterone assay (CJM006) to relate fGCM concentrations to habitat quality, demography and population performance in the Cape mountain zebra (Equus zebra zebra). Here, we revisit their findings and evaluate the validity of their conclusions using a validated EIA. First, we evaluate the biological sensitivity of six EIAs (three group‐specific metabolite assays and three corticosterone assays, including CJM006) through a biological validation experiment (translocation) for two sub‐species of mountain zebra, Cape mountain and Hartmann's mountain zebra (E. z. hartmannae). Second, we reanalyse the faecal extracts from Lea et al. (2017) using a validated EIA. fGCM concentrations consistently increased following translocation, when using two 11‐oxoaetiocholanolone (lab codes: 72T and 72a) and an 11ß‐hydroxyaetiocholanolone (69a) EIA, but did not with three different corticosterone EIAs. All corticosterone EIAs (including CJM006) failed to detect an increase in fGCMs within the critical 48–72‐h period post translocation. Therefore, the CJM006 EIA utilised in Lea et al. (2017) does not sensitively measure hypothalamic–pituitary–adrenal (HPA) axis activity in CMZ faeces. Using a validated assay (72T), fGCM concentrations were no longer associated with adult sex ratio or habitat quality (measured by grassiness) and these variables were dropped from predictive models. fGCM concentrations now varied between seasons and were negatively associated with female fecundity (foal:mare ratio). Consequently, we can conclude that the results of the previous study are unreliable. We introduce the terms “insensitive” and “sub‐optimal” to categorise assays that are tested but fail validation, and assays that are comparatively poor at detecting relevant hormone changes, respectively. We discuss how both “insensitive” and “sub‐optimal” assays could lead to incorrect inferences about population stressors and counterproductive conservation recommendations. Read the free Plain Language Summary for this article on the Journal blog.

Feasibility of a Sprague???Dawley Rat Model for Investigating the Effects of Seated Whole-body Vibration.

Vehicular whole-body vibration (WBV) can have long-term adverse effects on human quality of life. Animal models can be used to study pathophysiologic effects of vibration. The goal of this study was to assess animal cooperation and well-being to determine the feasibility of a novel seated rat model for investigating the effects of WBV on biologic systems. Twenty-four male Sprague???Dawley rats were used. The experiment consisted of an acclimation phase, 2 training phases (TrP1 and TrP2), and a testing phase (TeP), including weekly radiographic imaging. During acclimation, rats were housed in pairs in standard cages without vibration. First, experimental (EG; n = 18) and control group 1 (C1; n = 3) rats were placed in a vibration apparatus without vibration, with increasing duration over 5 d during TrP1. EG rats were exposed to vertical random WBV that was increased in magnitude over 5 d during TrP2 until reaching the vibration signal used during TeP (15min, 0.7m??s-2 root mean square, unweighted). C1 rats were placed in the vibration apparatus but received no vibration during any phase. Control group 2 (C2; n = 3) rats remained in the home cages. Cooperation was evaluated with regard to rat-apparatus interactions and position compliance. Behavior, weight, and fecal glucocorticoid metabolite concentrations (fGCM) were used to evaluate animal well-being. We observed good cooperation and no behavioral patterns or weight loss between phases, indicating little or no animal stress. The differences in fGCM concentration between groups indicated that the EG rats had lower stress levels than the control rats in all phases except TrP1. Thus, this model elicited little or no stress in the conscious, unrestrained, seated rats.

The dissection of a despotic society: exploration, dominance and hormonal traits.

Naked mole-rats (Heterocephalus glaber) live in large colonies with one breeding female (queen), one to three breeding males (BMs) and the remainder are non-reproductive subordinates. The animals have a linear dominance rank with the breeders at the top of the hierarchy. We investigated how dominance rank in naked mole-rats differs with exploration (the propensity to explore a novel environment) and related endocrine markers. Exploration behaviour, faecal progestagen metabolite (fPM), faecal glucocorticoid metabolite (fGCM), faecal androgen metabolite (fAM) and plasma prolactin concentrations were quantified in breeding, high-, middle- and low-ranked females and males from five naked mole-rat colonies. There were no significant differences between the dominance rank and exploration behaviour. Interestingly, the queens and high-ranking females had higher fGCM and fAM concentrations compared with middle- and low-ranked females. The queens had significantly higher fPM concentrations than all other ranked females, since they are responsible for procreation. In the males, the BMs had higher fGCM concentrations compared with high- and low-ranked males. In addition, BMs and middle-ranking males had overall higher prolactin levels than all other ranked males, which could be linked to cooperative care. Overall, the results suggest that physiological reproductive suppression is linked to high dominance rank.

Validation of Fecal Glucocorticoid Metabolites as Non-Invasive Markers for Monitoring Stress in Common Buzzards (Buteo buteo).

For wild animals, being in captivity in wildlife centers can cause considerable stress. Therefore, it is necessary to establish and validate non-invasive tools to measure chronic stress during rehabilitation. Eight Common Buzzards which lived in permanent husbandry were placed individually into prepared aviaries and their feces were collected before, during and after a stress event for biological validation over a period of seven days. The extracted fecal glucocorticoid metabolites (fGCMs) were analyzed with three different enzyme immune assays (EIA) to find the most suitable one. Additionally, we aimed to investigate the stability of fGCM levels after defecation because further metabolization by bacterial enzymes can lead to changed results. The Cortisone-EIA performed best in males and females and showed that the stress event led to an fGCM increase of 629% (557% in females and 702% in males) in relation to basal values. We found no significant differences between the sexes, but observed significant differences between different times of day. FGCM concentration significantly changed after eight hours at room temperature. Our study successfully validated the non-invasive measurement of fGCM as a stress indicator in Common Buzzards and could therefore lay the foundation for future studies providing new insights for animal welfare research in Buzzards.

The paradox of spring: Thyroid and glucocorticoid responses to cold temperatures and food availability in free living Carneddau ponies

In seasonal environments, maintaining a constant body temperature poses challenges for endotherms. Cold winters at high latitudes, with limited food availability, create opposing demands on metabolism: upregulation preserves body temperature but depletes energy reserves. Examining endocrine profiles, such as thyroid hormone triiodothyronine (T3) and glucocorticoids (GCs), proxies for changes in metabolic rate and acute stressors, offer insights into physiological trade-offs. We evaluated how environmental conditions and gestation impact on faecal hormone metabolites (fT3Ms and fGCMs) from late winter to spring in a free-living population of Carneddau ponies. Faecal T3Ms were highest in late February and March, when temperatures were lowest. Then, fT3Ms concentrations decreased throughout April and were at the lowest in May before increasing towards the end of the study. The decline in fT3M levels in April and May was associated with warmer weather but poor food availability, diet diversity and diet composition. On the other hand, fGCM levels did not display a clear temporal pattern but were associated with reproductive status, where pregnant and lactating females had higher fGCM levels as compared to adult males and non-reproductive females. The temporal profile of fT3Ms levels highlights metabolic trade-offs in a changing environment. In contrast, the ephemeral but synchronous increase in fGCM concentrations across the population suggest a shared experience of acute stressors (i.e., weather, disturbance or social). This multi-biomarker approach can evaluate the role of acute stressors versus energy budgets in the context of interventions, reproduction, seasonality and environmental change, or across multiple scales from individuals to populations.

Physiological changes in captive elephants in northern Thailand as a result of the COVID-19 tourism ban-stress biomarkers.

The international travel ban instituted by the Thai government in March 2020 in response to the COVID-19 pandemic greatly affected how tourist camp elephants were managed, with reductions in exercise opportunities, longer chaining hours, and diminished food provisioning. This study was conducted to determine how those changes affected health and welfare biomarkers in individual elephants over the 2 years of the countrywide lockdown (April 2020-April 2022). Blood and fecal samples were collected from 58 elephants at six camps (monthly in Year 1, quarterly in Year 2) and analyzed for stress biomarkers - fecal glucocorticoid metabolites (fGCM), serum oxidative stress [malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)], and stress leukograms. Overall, fGCM concentrations increased within the first few months and remained higher than pre-COVID levels, as did the H/L ratio, a measure affected by cortisol. Serum 8-OHdG, an indicator of DNA oxidative damage, also increased over time, while monocytosis and lymphopenia further suggested alterations in immune function as a result of stress. By contrast, another marker of oxidative stress, serum MDA, declined, possibly in response to reduced roughage and supplement intake. A notable finding was a seasonal pattern of fGCM that was significantly different from previous studies. Whereas higher fGCM during the rainy season were observed in this study, previously, concentrations were highest during the winter, high tourist season. Thus, ironically, both the presence and absence of tourists have been associated with increased fGCM concentrations, albeit for different reasons. Camp management factors negatively affecting stress outcomes included shorter chain lengths, longer chain hours, lack of exercise, and reduced roughage and supplements. Overall, it was clear that camps struggled to maintain adequate care for elephants during the COVID-19 pandemic, highlighting the importance of tourist income and need for contingency plans to cope with potential future disruptions to tourism.

Analytical and biological validation of a noninvasive measurement of glucocorticoid metabolites in feces of Geoffroy's spider monkeys (Ateles geoffroyi).

We report on an analytical and biological validation of a commercial cortisol enzyme immunoassay (EIA)to measure glucocorticoids (GC)in feces of Geoffroy's spider monkeys (Ateles geoffroyi). Validation of endocrinological methods for each sample matrix and study species is crucial to establish that the methods produce reliable results. For the analytical validation of the EIA, we assessed parallelism, accuracy, and precision. We carried out a biological validation based on three well-studied GC patterns with the following predictions: (1) increased fecal GC metabolite (fGCM)concentrations after veterinary intervention; (2) increased fGCM concentrations during early morning hours; and (3) higher fGCM concentrations during gestation than in other female reproductive states. For the first prediction, we sampled feces of two zoo-housed females 2 days before, the day of, and 2 days after a veterinary intervention. For the second prediction, we analyzed 284 fecal samples collected from 12 wild males using a linear mixed model (LMM).For the third prediction, we analyzed 269 fecal samples of eight wild females using an LMM. Analytical validation revealed that the EIA showed parallelism, was accurate, and precise within each assay. However, there was elevated variation in between-assay precision. The biological validation supported all predictions: (1) the two zoo-housed females showed a substantial increase in fGCM concentrations 2.5and 11 h after veterinary intervention; (2) there was a negative effect of sample collection time on fGCM concentrations (i.e., higher concentrations during early morning); (3) gestating females had significantly higher fGCM concentrations than lactating females. Thus, we analytically validated the commercial EIA and, despite between-assay variation, we were able to find three biologically relevant GC signals in captive and wild settings, and in males and females. We are therefore confident that the method can be used to noninvasively address behavioral endocrinology questions in Geoffroy's spider monkeys.

Adrenal and metabolic hormones demonstrate risk-reward trade-offs for African elephants foraging in human-dominated landscapes.

A key driver of the African savannah elephant population decline is the loss of habitat and associated human-elephant conflict. Elephant physiological responses to these pressures, however, are largely unknown. To address this knowledge gap, we evaluated faecal glucocorticoid metabolite (fGCM) concentrations as an indicator of adrenal activity and faecal thyroid metabolite (fT3) concentrations as an indicator of metabolic activity in relation to land use, livestock density, and human landscape modification, while controlling for the effects of seasonality and primary productivity (measured using the normalized difference vegetation index). Our best-fit model found that fGCM concentrations to be elevated during the dry season, in areas with higher human modification index values, and those with more agropastoral activities and livestock. There was also a negative relationship between primary productivity and fGCM concentrations. We found fT3 concentrations to be higher during the wet season, in agropastoral landscapes, in locations with higher human activity, and in areas with no livestock. This study highlights how elephants balance nutritional rewards and risks in foraging decisions when using human-dominated landscapes, results that can serve to better interpret elephant behaviour at the human-wildlife interface and contribute to more insightful conservation strategies.

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.

Seasonal patterns of oxidative stress markers in captive Asian elephants in Thailand and relationships to elephant endotheliotropic herpesvirus shedding.

Oxidative stress refers to an imbalance between oxidant and antioxidant activity and accumulation of reactive oxygen species, which can have detrimental effects on animal health. Annual fluctuations in oxidative stress status can occur, increasing disease susceptibility during certain time periods. However, a full understanding of factors related to oxidative stress in Asian elephants and how to mitigate the negative consequences is lacking. This study measured six serum oxidative stress markers [reactive oxygen species (ROS), malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), albumin, glutathione peroxidase (GPx), and catalase] and two stress markers [serum cortisol and fecal glucocorticoid metabolites (fGCM)] in 23 captive Asian elephants in Thailand over a 12 months period to examine relationships with age and season. Seasonal variations were observed, with several markers exhibiting significantly higher concentrations in the summer (ROS, MDA, 8-OHdG, albumin) and lower values during the rainy/winter seasons (MDA, 8-OHdG, albumin, catalase). By contrast, GPx was the only marker to be highest during the rainy season. For the stress markers, higher fGCM concentrations were noted during the rainy season, which contrasts with earlier studies showing more activity in the winter (tourist season). Positive correlations were found between the temperature-humidity index and ROS, GPx, and fGCM, while a negative correlation was observed with serum albumin. Elephant endotheliotropic herpesvirus (EEHV) shedding events were associated with higher concentrations of ROS and MDA. A moderate negative correlation was observed between 8-OHdG and the PCR threshold cycle of EEHV shedding (Ct), indicating DNA damage may be involved in EEHV shedding in elephants. Results revealed significant age and seasonal effects on several oxidative stress markers, indicating those factors should be considered in study design and data interpretation. There also may be physiological adaptations in oxidative stress conditions in relation to environmental changes that could impact health outcomes.

Dog barks influence the physiological stress and behavior of a wild primate

Non-lethal impacts of dogs on primates have seldom been assessed. We used an experimental approach to determine if mantled howler monkeys (Alouatta palliata) perceive dog barks as an aversive stimulus and thus display physiological and behavioral responses toward simulated barks. For one year (1754 h of observations) we studied 16 adult males belonging to five groups in Los Tuxtlas (Mexico), and recorded the occurrence of naturally occurring dog barks, their sound pressure level (SPL), and the behavioral responses of howler monkeys to barks. We then exposed males to bark playbacks at two SPL treatments, 40 and 80 dB in a total of 50 experiments. We assayed glucocorticoid metabolite concentrations in fecal samples (fGCM) as a marker of the physiological stress response of males. We also recorded the duration of vigilance, vocalizations, and flight in relation to playbacks. Naturally occurring barks were frequent and usually elicited behavioral responses by males. fGCM concentrations increased after bark playbacks and with stimuli intensity. Time spent vigilant increased following playbacks independently of stimuli intensity but both vocalizations and flight were linked to stimuli intensity: vocalizations were the longest after barks played-back at 80 dB, but males spent more time fleeing in response to 40 dB bark playbacks. These results provide evidence that dog barks are pervasive in the habitat of mantled howler monkeys living at Los Tuxtlas and disturb males, both physiologically and behaviorally. Although the potential costs of physiological and behavioral responses could not be determined, there is sufficient evidence to assume that they do have negative impacts on individuals. Therefore, our study provides avenues for future research on dog-wildlife interactions and valuable information for the design of conservation actions aimed at mitigating the impact of dogs on mantled howler monkeys.

Measures of Adrenal and Gonadal Hormones in Relation to Biological and Management Factors among Captive Red Pandas in Indian Zoos.

Animals in human care are affected by stressors that can ultimately reduce fitness. When reproduction is affected, endangered species conservation programs can be severely compromised. Thus, understanding factors related to stress and reproduction, and related hormones, is important to ensure captive breeding success. Red pandas (Ailurus fulgens) are endangered, and populations in the wild are threatened with extinction. A global captive breeding program has been launched to conserve the species with the goal of reintroduction. However, there is little information on how stressors impact physiological aspects of the species. This study measured fecal glucocorticoid (fGCM), progestagen (fPM), and androgen (fAM) metabolite concentrations in 12 female and 8 male red pandas at 3 zoos in India to determine predictors of adrenal and gonadal steroid activity, and the influence of fGCM on reproduction. Based on the generalized linear mixed model (GLMM), fGCM concentrations were positively correlated with the number of visitors, number of nests and enclosure areas, and negatively related to frequency of feedings, log density, and social time, while fPM concentrations were negatively associated with enclosure areas. A confounder for enclosure areas and number of nests was the fact that these spaces were relatively barren, with limited hiding spaces, compared to the smaller enclosures. By contrast, no significant relationships were found for fAM, perhaps due to the smaller sample size. A negative relationship between fGCM and fPM was observed, indicating increasing adrenal hormones may decrease reproductive function among female red pandas. Results suggest that zoo management should consider increasing feeding frequency, providing larger enclosures with more enrichment and more nests in larger spaces, and regulating visitor numbers to support good welfare and potentially improve reproductive fitness of red pandas in captivity.

Fecal cortisol metabolites reflect transport stress in 3-month-old dairy calves pre- and postweaning: A pilot study

Measurement of fecal cortisol metabolites (FGCM) is a well-established, noninvasive method to assess stress in adult dairy cattle. However, this procedure has not yet been validated for unweaned dairy calves, and it can be expected that the milk proportion of the diet may influence the resulting FGCM concentrations. The aim of this study was therefore to assess whether a peak in FGCM concentrations in response to a stressor can be measured in unweaned dairy calves on a largely milk-based diet. If so, further objectives were to examine whether maximum FGCM concentrations, as well as the time lag until they are reached, are comparable to the values in the same calves on a solid-based diet after weaning. For this study, 5 German Holstein calves of about 3 mo of age (93 to 102 d preweaning) were exposed to a 45 min transport stressor once before and once after weaning, which was 3 wk apart. All voided fecal samples were collected for 24 h after termination of the transport. Fecal cortisol metabolites were analyzed with an 11-oxoetiocholanolone enzyme immunoassay and changes in FGCM concentrations relative to the individual baseline (FGCMrel) were calculated. Results showed a clear peak in FGCM concentrations on both diet types. The peak FGCMrel concentrations tended to be higher when the calves were on the preweaning diet (at peak: +233 ± 25% increase relative to baseline) in comparison to the postweaning diet (+124 ± 23%). Considering the whole 24 h sampling period, the FGCMrel concentrations for all calves were significantly higher on the preweaning diet than on the postweaning diet. There was also a numerical difference in the delay between occurrence of the stressor and appearance of the peak FGCMrel concentrations in feces, as the time lag was 1.5 ± 1.2 h longer when the calves were on the preweaning diet compared with the postweaning diet. In conclusion, our results suggest that FGCM concentrations are a useful stress marker for unweaned dairy calves in the same way they are for older cattle, but that FGCMrel concentrations tend to be higher in unweaned than in weaned calves and are thus not directly comparable.

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.

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.

Assessing the drivers of gut microbiome composition in wild redfronted lemurs via longitudinal metacommunity analysis

The gut microbiome influences host’s immunity, development, and metabolism and participates in the gut–brain axis, thus impacting the health of the host. It is a dynamic community varying between individuals and within individuals at different time points. Hence, determining the factors causing this variability may elucidate their impact on host’s health. However, understanding the drivers of variation has proven difficult particularly as multiple interactions occur simultaneously in the gut microbiome. We investigated the factors shaping the gut microbiome by applying the metacommunity concept where the gut microbiome is considered as a microbial community shaped by the interactions within the community, with the host and microbial communities outside the host, this through a longitudinal study in a wild primate. Focal behavioral data were collected for 1 year in four groups of redfronted lemurs to determine individual social and feeding behaviors. In addition, regular fecal samples were collected to assess bacteria, protozoa, and helminths through marker gene analysis and to measure fecal glucocorticoid metabolite (fGCM) concentrations to investigate the impact of physiological stress on the gut microbiome. Higher consumption of leaves and elevated fGCM concentrations correlated with higher alpha diversity, which also differed among groups. The major drivers of variation in beta diversity were group membership, precipitation and fGCM concentrations. We found positive and negative associations between bacterial genera and almost all studied factors. Correlations between bacterial indicator networks and social networks indicate transmission of bacteria between interacting individuals. We detected that processes occurring inside the gut environment are shaping the gut microbiome. Host associated factors such as, HPA axis, dietary changes, and fluctuations in water availability had a greater impact than interactions within the microbial community. The interplay with microbial communities outside the host also shape the gut microbiome through the exchange of bacteria through social relationships between individuals and the acquisition of microorganisms from environmental water sources.

Can stress and anxiety be assessed in African elephants (Loxodonta africana) using self-directed behaviour?

Captive African elephants used in the tourism industry face numerous welfare issues which are often stress related and linked to high numbers of tourists or human-elephant interactions. Elephant welfare is commonly assessed by quantifying faecal glucocorticoid metabolite (fGCM) concentrations, and monitoring stereotypic behaviour, which are either costly or problematic in identifying underlying causes. Self-directed behaviours (SDBs), a form of displacement activity, have been substantially linked with stress and anxiety in primates, and could be a potential but not yet utilised behavioural marker in elephants. We thus explored the usage of several trunk, tail, and body related SDBs in a group of 7 semi-captive African elephants maintained at the Knysna Elephant Park, a tourist destination offering a variety of close contact experiences. Using continuous behavioural sampling (focal following), SDBs were recorded as they occurred, along with the numbers of nearby tourists (<5 m to focal; with 3 levels: None, Low 1–5, and High >5), and the nature of the tourist interaction (5 levels: None, Feed, Touch, Walk, and Ride). We then compared SDB rates with fGCM concentrations (n = 115) determined following observations (1 day and 2 days after behavioural sampling). Data were analysed using Generalized Linear Mixed effects Models. SDB rates (p/min) significantly increased during elephant Walks (p < 0.001) and Rides (p < 0.001), indicating a correlation with their usage during potentially stressful scenarios. The Touch interaction significantly decreased SDBs (p < 0.05), whilst the Feed interaction had a non-significant effect (p > 0.05), which could indicate the element of control in terms of perceived stress is an important component in welfare, as elephants were free to move away during these interactions. Interestingly, SDBs significantly decreased when tourist numbers were High (p = < 0.05), potentially due to greater quantities of high value food. Additionally, no correlation was found between SDBs and fGCM concentrations determined on day one (p > 0.05), and day two (p > 0.05). Hence, SDBs may be correlated more with anxiety or lower-level stress, which is not significant enough to activate GC production, and may therefore act as a coping strategy utilised to maintain physiological homeostasis during anxiety-inducing situations. Additional research would benefit from coupling SDB observations with different forms of physiological assessments to better understand the internal motivations, and formally establish SDBs as a reliable, cost-effective, and non-invasive welfare index to identify stressed individuals in real time and ensure optimum husbandry.

Exploring the effect of auditory stimuli on activity levels, milk yield and faecal glucocorticoid metabolite concentrations in Holstein cows

Health and welfare are inextricably linked within efficient and sustainable dairy production, and several potential risk factors may affect the well-being of dairy cows, including chronic stress. Although auditory stimuli could be used as a tool to decrease the potential stress that cows might experience, it is seldom applied to livestock production systems due to the perception that enrichment is an unnecessary expense. This study aimed to explore the effect of auditory stimuli as a form of enrichment in a Holstein herd by monitoring fecal glucocorticoid metabolite (fGCM) concentrations (a non-invasive, stress-associated biomarker). Cow activity level and milk yield were also measured. Nine cows in their second and third lactation were divided into 3 groups, using a Latin Square experimental design, exposing each cow group to each of the 3 treatments, namely constant exposure (CE), limited exposure (LE), and no exposure (NE) to classical music. FGCMs were quantified using a group-specific enzyme immunoassay detecting 11,17-dioxoandrostanes. Compared to LE and NE animals, cows exposed to constant music had significantly lower fGCM concentrations (P = 0.012), as well as higher milk yields (P < 0.0001) and lowered activity levels during the morning (P = 0.005) and the evening activity period (P = 0.048). These findings indicate that auditory stimuli in the form of classical music may have a positive effect on the welfare of cows as well as milk yield, which hold economic benefits for the producer and potentially reduces the number of cows needed for profitable production.

Social support correlates with glucocorticoid concentrations in wild African elephant orphans

Social relationships have physiological impacts. Here, we investigate whether loss of the mother/offspring relationship has lasting effects on fecal glucocorticoid metabolite (fGCM) concentrations in wild African elephant orphans several years following their mothers’ deaths. We find no difference in fGCM concentrations between orphans and nonorphans, but find lower fGCM concentrations in elephants with more age mates in their family. We also unexpectedly identify lower concentrations in orphans without their natal family versus nonorphans and natal orphans, which we speculate may be due to the development of hypocortisolism following a prolonged period without familial support. An index of plant productivity (i.e. food) shows the largest correlation with fGCM concentrations. Our findings indicate no lasting differences in glucocorticoid concentrations of surviving orphan elephants who are with their family, suggest the presence of age mates may reduce glucocorticoid concentrations in elephants, and emphasize that basic survival needs are the primary regulators of the stress response.

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.

Preliminary Findings on How Different Management Systems and Social Interactions Influence Fecal Glucocorticoid Metabolites in White Rhinoceros (Ceratotherium simum).

Simple SummaryIn recent years, interest in improving the welfare of wild species in captivity has grown. The aim of this investigation was to evaluate the effects of different social environments and management systems on the cortisol secretion of fourteen white rhinoceroses (Ceratotherium simum) living under different conditions by using use noninvasive methods. The fecal glucocorticoid metabolite secretion was found to be affected by both management systems and social interactions. Additionally, sex is another factor that seems to influence. This research provides a deeper understanding of glucocorticoid production in white rhinoceroses, but more studies are needed to fully understand its influence on reproductive biology.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.