Fecal Cortisol Metabolite Concentrations Research Articles

Predicting Sex in White Rhinoceroses: A Statistical Model for Conservation Management.

Ensuring the effective management of every rhinoceros population is crucial for securing a future for the species, especially considering the escalating global threat of poaching and the challenges faced in captive breeding programs for this endangered species. Steroid hormones play pivotal roles in regulating diverse biological processes, making fecal hormonal determinations a valuable non-invasive tool for monitoring adrenal and gonadal endocrinologies and assessing reproductive status, particularly in endangered species. The purpose of this study was to develop a statistical model for predicting the sex of white rhinoceroses using hormonal determinations obtained from a single fecal sample. To achieve this, 562 fecal samples from 15 individuals of the Ceratotherium simum species were collected, and enzyme immunoassays were conducted to determine the concentrations of fecal cortisol, progesterone, estrone, and testosterone metabolites. The biological validation of the method provided an impressive accuracy rate of nearly 80% in predicting the sex of hypothetically unknown white rhinoceroses. Implementing this statistical model for sex identification in white rhinoceroses would yield significant benefits, including a better understanding of the structure and dynamics of wild populations. Additionally, it would enhance conservation management efforts aimed at protecting this endangered species. By utilizing this innovative approach, we can contribute to the preservation and long-term survival of white rhinoceros populations.

Welfare impacts associated with using aversive geofencing devices on captive Asian elephants

Animal-borne aversive geofencing devices (AGDs, or satellite-linked shock collars) are commercially available and used on livestock to restrict their movement within a virtual boundary. This technology has potential application as a human-wildlife conflict mitigation tool, where problem animals might be conditioned to avoid human-dominated habitats by associating an audio warning with a subsequent electric shock, which is delivered if the audio warning is ignored. Ensuring that high standards of animal welfare are maintained when implementing such tools is important for acquiring manager and community acceptance of such approaches. We conducted two pilot experiments with eight captive Asian elephants using mild electric shocks from a modified dog-training collar fitted around the neck, as part of an ongoing effort to develop AGDs suitable for mitigating human-elephant conflict. As part of these experiments, we assessed elephants' behavioural and physiological stress before, during and after the experiments. During the experiments elephants wore collars for up to nine consecutive days and received a small number of electric shocks on 1–3 consecutive days. Bootstrapped principal component analysis showed that daily activity budgets of individual elephants on experiment days were not different from the pre-experiment days. Generalised linear mixed-effects model (GLMM) showed that anxiety/stress behaviours increased on the first day of acclimatising to the collar and on testing days (i.e. days they received shocks) of the first experiment, but not during the second experiment relative to pre-experiment days. Analysis of faecal cortisol metabolite (FCM) concentrations using GLMM showed that FCM concentrations were higher in samples collected ∼24 hrs and ∼48 hrs after testing days compared to baseline levels as expected given the lag time for excretion of cortisol metabolites. These elevated anxiety/stress behaviours and FCM concentrations returned to baseline levels shortly after the experiment. Therefore, we conclude that AGDs did not produce lasting behavioural or physiological stress effects in elephants during this short-term study but recommend further studies with a larger sample of elephants to confirm the transferability of these findings.

Season-dependent impact of forage quality on stress in alpine chamois.

Chronically heightened stress levels in wildlife species may have detrimental effects on individual life history traits, for example, through the increased likelihood of disease, parasitic infections, and overall reduced fitness. Understanding the drivers of stress may thus have great potential for informing wildlife conservation. Although the role of climate and individual status is well studied in stress ecology, the impact of related stressors such as dietary quality is of increasing interest to wildlife research and conservation. In this study, fecal cortisol metabolites (FCMs) in Alpine chamois Rupicapra r. rupicapra used as bioindicators of stress, and their relationship with forage quality-measured as the percentage of fecal crude protein (CP)-were investigated. Data collection took place in 2011 and 2012 in the Gran Paradiso National Park (Western Italian Alps), on 22 individually marked adult males. The relationship between FCMs and CPs was analyzed through linear models and separated between winter and summer months, accounting for the effect of potentially confounding exogenous and endogenous variables. After AICc-based model selection, we found that forage quality was negatively related to FCM levels in Alpine chamois during the summer months, meaning that higher quality forage was associated with the decreased expression of stress hormones. However, during the winter months, we did not find a significant relationship, potentially as a result of forage quality being ubiquitously poor. Although the mechanisms through which dietary variations impact FCM concentrations in wildlife populations are largely unknown, the occurrence of significant relationships between forage quality and stress levels supports potentially important implications for the long-term effect of climatic changes on the fitness of wildlife populations.

Effects of OmniGen AF feed supplementation on glucocorticoids, blood leukocyte messenger RNA abundance, and energy metabolism in overstocked Holstein dairy cows

The physiological consequences of overstocking require more investigation, and no research has explored whether dietary supplements could mitigate the anticipated negative physiological effects. OmniGen AF (OG, Phibro Animal Health Corporation, Teaneck, NJ, USA) is a nutritional supplement that has been shown to support the immune system of cattle following internal and environmental stressors. This study aimed to determine if a 45-day period of OG feed supplementation would influence whole blood leukocyte messenger RNA abundance, energy metabolism and glucocorticoid concentration, during a two-week period of overstocking. Two stocking density treatments (control: one headlock and lying stall per cow; overstocked: 0.5 headlocks and 0.5 lying stalls per cow) and two diet treatments (control: no added supplement; and OG: 56 g/cow per day) were investigated. Four pens of 15 cows were fed their assigned diet (two pens per diet; control stocking density) for 45 days after which each stocking density treatment was applied for a 14-day period using a cross-over design; this study design was replicated twice. During each 14-day period, blood was collected on day four to measure whole blood leukocyte messenger RNA abundance (cluster of differentiation 80, interleukin 8 receptor-beta, interleukin 10 receptor-beta and L-selectin) and fecal samples were collected every two days to measure fecal cortisol metabolite concentration (11,17-dioxoandrostanes). At the end of each 14-day period, eight cows from each pen were selected for an intravenous glucose tolerance test; glucose, insulin and non-esterified fatty acids were measured. There were no effects of diet or stocking density on leukocyte messenger RNA abundance. Fecal cortisol metabolite concentrations were highest for overstocked cows on the control diet on day four of the stocking density treatment; however, by day 10, overstocked cows fed OG had the highest fecal cortisol metabolite concentrations. Overstocked cows, regardless of diet, had an attenuated insulin response during the glucose tolerance test, represented by a lower area under the curve estimate. Cows fed OG but not overstocked, had a lower non-esterified fatty acid nadir during the glucose challenge, compared to all the other treatments. In conclusion, overstocking prompts a physiological stress response and alters energy metabolism by decreasing the insulin response to an intravenous glucose challenge. Feeding OG during overstocking delayed the increase in fecal cortisol metabolites by several days; however, it is unclear if this altered glucocorticoid response benefited the cow, as OG had no effect on insulin responses or immune parameters.

Laissez-Faire Stallions? Males' Fecal Cortisol Metabolite Concentrations Do Not Vary with Increased Female Turnover in Feral Horses (Equus caballus).

Stress responses can be triggered by several physical and social factors, prompting physiological reactions including increases in glucocorticoid concentrations. In a population of feral horses (Equus caballus) on Shackleford Banks, North Carolina, females previously immunized with the immunocontraceptive agent porcine zona pellucida (PZP) change social groups (bands) more often than unimmunized females, disrupting the social stability within the population. We assessed the effects of increased female group changing behavior (or female turnover) on individual male stress by comparing fecal cortisol metabolite (FCM) concentrations among stallions experiencing varying amounts of female group changing behavior. FCM concentrations did not significantly correlate with female turnover. Similarly, FCM concentrations were not dependent upon the timing of female group changing behavior. These findings suggest that female turnover rate has little influence on physiological measures of stress in associated stallions. That said, Shackleford stallions experiencing increased female turnover do engage in behaviors typically associated with stress (increased vigilance, highly escalated male-male conflicts). Future work should compare FCM concentrations across time within populations and among populations managed under different strategies to better isolate factors influencing stallion stress physiology. Such studies are especially important if we are to determine how changes in female behavior related to immunocontraception impact physiological and behavioral indicators of stress for non-target animals. Finally, our study highlights the importance of considering both physiological and behavioral measures when investigating animal responses to potentially challenging situations.

Resident wild koalas show resilience to large-scale translocation of bushfire-rescued koalas.

Wildlife translocation is increasingly utilized as a conservation management action, to mitigate the immediate negative effects of habitat loss and fragmentation (e.g. from land clearing or bushfires). Previous research has shown that stress responses can help or hinder survival in translocated wildlife and determine the efficacy of translocation as a conservation action. Yet these translocated animals are only one side of the equation, with translocation also potentially impacting the animals in the recipient population. We measured physiological markers of stress (faecal cortisol metabolite concentrations and neutrophil-lymphocyte ratios) and assessed health condition in a wild koala population one year after a major translocation of bushfire-rescued koalas on Kangaroo Island. We expected to find a high population density at the site (>0.75 koalas per hectare) and that resident koalas would show signs of chronic stress and ill health as a result of territorial conflict over food trees and reproductive opportunities. In contrast, we found that only one-fifth of the population remaining at the site were translocated koalas. The overall population density was also much lower (0.21 koalas per hectare) than anticipated. With no evidence of mass mortality at the site, we suggest that the majority of translocated koalas dispersed away from the site. Our stress marker measurements did not differ between the wild koalas and a sample of captive (non-display) koalas at the nearby Kangaroo Island Wildlife Park and were generally low compared to other studies. Veterinary examinations found that most koalas were in good body condition with very few diagnostic indicators of systemic ill health. Overall, our results suggest that, if there is adequate landscape-scale habitat connectivity and opportunity for dispersal, translocated koalas are likely to disperse from the site of release, with limited impacts on recipient koala populations at translocation release sites.

The use of fecal cortisol for enrolment of free-roaming dogs in Singapore to a national rehabilitation-rehoming program: A possible indicator of allostasis

Singapore's trap-neuter-release-management program strives to rehome as many free-roaming dogs as possible. However, before rehoming, most free-roaming dogs require rehabilitation and this requires sheltering them for some time. Therefore, an individual's suitability for rehoming hinges on his or her adaptability to shelter conditions. Currently, this is being assessed qualitatively across behavioral contexts. Augmenting the current assessment with a known bioindicator of stress, such as cortisol, would increase its robustness. However, the serum measurement of cortisol can be invasive. Canine fecal samples have presented an attractive alternative for measuring cortisol indirectly in various contexts. Nevertheless, it is unclear whether the resolution of fecal cortisol metabolites (FCM) is sufficient for evaluating Singapore free-roaming dogs’ adaptability to shelter conditions. To elucidate this, we performed linear mixed-effects model on 27 candidate models to compare FCM between pet and recently sheltered dogs, with sex, state of the feces, and time as covariates. Subsequently, we ran generalized mixed-effected model on 12 candidate models to examine the influence of between- and within-subject variations in FCM on the prognosis of sheltered dogs—based on our current assessment. Our analyses showed that FCM levels of recently sheltered dogs were higher than pet individuals. However, unlike several studies, FCM concentrations of recently sheltered dogs continued to rise, indicating that individuals have yet to adapt. Furthermore, the difference in FCM levels between newly sheltered individuals and pet dogs was moderated by sex. Unexpectedly, we found the average FCM levels of dogs with a good prognosis to be higher than those that received a poor prognosis, with sex and state of the fecal samples as important covariates. This may be due to the difference in allostasis between the 2 groups of dogs. Thus, our results showed FCM not only has sufficient resolution but is useful in evaluating dogs’ adaptability to shelter conditions.

Covariation between glucocorticoids, behaviour and immunity supports the pace-of-life syndrome hypothesis: an experimental approach.

The biomedical literature has consistently highlighted that long-term elevation of glucocorticoids might impair immune functions. However, patterns are less clear in wild animals. Here, we re-explored the stress-immunity relationship considering the potential effects of behavioural profiles. Thirteen captive roe deer (Capreolus capreolus) were monitored over an eight-week period encompassing two capture events. We assessed how changes in baseline faecal cortisol metabolite (FCM) concentrations following a standardized capture protocol and an immune challenge using anti-rabies vaccination affected changes in 13 immune parameters of innate and adaptive immunity, and whether these changes in baseline FCM levels and immune parameters related to behavioural profiles. We found that individuals with increased baseline FCM levels also exhibited increased immunity and were characterized by more reactive behavioural profiles (low activity levels, docility to manipulation and neophilia). Our results suggest that the immunity of large mammals may be influenced by glucocorticoids, but also behavioural profiles, as it is predicted by the pace-of-life syndrome hypothesis. Our results highlight the need to consider covariations between behaviour, immunity and glucocorticoids in order to improve our understanding of the among-individual variability in the stress-immunity relationships observed in wildlife, as they may be underpinned by different life-history strategies.

The Adrenal Cortisol Response to Increasing Ambient Temperature in Polar Bears (Ursus maritimus).

Simple SummaryHuman-caused climate change is proceeding rapidly and providing challenges to wildlife species, especially those adapted to colder temperatures. We investigated the cortisol response of polar bears to increasing ambient temperatures to improve our knowledge of the physiology of this Arctic species with the goal of informing management in zoos and in the wild. In adult polar bears temperatures above 20 °C were associated with an increase in the hormone cortisol to accommodate increased thermoregulatory demands. This temperature threshold was surprisingly high for an Arctic-adapted species. Zoos can provide sufficient housing options to prevent overheating in polar bears exposed to warmer temperatures but our results are concerning for wild polar bears. The number of days reaching 20 °C in the Arctic has increased significantly over the past 30 years and the associated increase in thermoregulatory costs add to the physiological burden many wild polar bears are already facing with the loss of sea ice hunting opportunities. We recommend that the management of polar bears in the wild and under human care be adapted to reflect the increased cortisol concentrations associated with thermoregulatory challenges in warmer temperatures.Our objective was to identify the upper ambient temperature threshold that triggers an increase in cortisol in response to increased thermoregulatory demands in polar bears. The results reported here include endocrine data collected over two years from 25 polar bears housed in 11 accredited zoological institutions across North America. The effects of ambient temperature, sex, age group (juvenile, adult, elderly), breeding season and humidity on fecal cortisol metabolite (FCM) concentrations (N = 8439 samples) were evaluated using linear mixed models. Ambient temperatures were placed into five different categories: <5 °C, 6–10 °C, 11–15 °C, 16–20 °C, and >20 °C. Ambient temperature and humidity had a significant (p < 0.05) effect on FCM concentrations with significant (p < 0.05) interactions of sex, age and breeding season. Once biotic factors were accounted for, there was a significant (p < 0.05) increase in FCM concentrations associated with ambient temperatures above 20 °C in adult polar bears. The implications of these findings for the management of both zoo and wild polar bears are discussed.

Seasonal patterns in behavior and glucocorticoid secretion of a specialist Holarctic tree squirrel (Sciurus aberti).

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.

Faecal Cortisol Metabolites as an Indicator of Adrenocortical Activity in Farmed Blue Foxes.

Simple SummaryThe measurement of faecal cortisol metabolites (FCMs) is increasingly used to monitor physiological stress responses in different animal species. Before this method is applied in coming stress- and welfare-related studies of farmed blue foxes (Vulpes lagopus), a species-specific validation is first required. In the current study, a 5α-pregnane-3ß,11ß,21-triol-20-one enzyme immunoassay was found suited to measure FCMs and thus hypothalamus–pituitary–adrenal (HPA) axis activity in farmed blue foxes. FCMs can therefore serve as a valid indicator of stress in future welfare studies of blue foxes.Welfare studies of blue foxes would benefit from a measurement of faecal cortisol metabolites (FCMs) as a non-invasive, physiological stress parameter reflecting hypothalamus–pituitary–adrenal (HPA) axis activity. Before implementation, a species-specific validation of such a method is required. Therefore, we conducted a physiological validation of an enzyme immunoassay (EIA) to measure FCMs in blue foxes. Twenty individuals (nine males and eleven females) were injected with synthetic adrenocorticotrophic hormone (ACTH) and faecal samples were collected every third h for two days. The FCM baseline levels were assessed based on the first sampling day (control period, 144 samples), followed by the ACTH injection and the second day of sampling (treatment period, 122 samples). FCMs were analysed with a 5α-pregnane-3ß,11ß,21-triol-20-one EIA. We compared the estimated mean FCM concentrations of the treatment samples to the baseline average. All samples for the two periods were collected at the same time of the day, which enabled to test the data also with an hourly pairwise comparison. With the two statistical approaches, we tested whether a possible diurnal fluctuation in the FCM concentrations affected the interpretation of the results. Compared to the baseline levels, both approaches showed 2.4–3.2 times higher concentrations on time points sampled 8–14 h after the ACTH injection (p < 0.05). The estimated FCM concentrations also fluctuated slightly within the control period (p < 0.01). Inter-individual variations in FCM levels were marked, which highlights the importance of having a sufficient number of animals in experiments utilising FCMs. The sampling intervals of 3 h enabled forming of informative FCM curves. Taken together, this study proves that FCM analysis with a 5α-pregnane-3ß,11ß,21-triol-20-one EIA is a valid measurement of adrenocortical activity in the farmed blue foxes. Therefore, it can be utilised as a non-invasive stress indicator in future animal welfare studies of the species.

Evaluation of Fecal Glucocorticoid Metabolite Levels in Response to a Change in Social and Handling Conditions in African Lions (Panthera leo bleyenberghi).

Simple SummaryNon-invasive determination of cortisol metabolite concentrations in feces is widely used to evaluate the influence of housing and handling conditions on the stress physiology of wildlife in captivity. The present study aimed to assess the physiological response of a lion pride to a change in management and social conditions after the death of the dominant male of the pride. Before the dominant male died, weekly management routines between the indoor and outdoor enclosures were conducted to avoid cohabitation problems between the two males of the pride. After the death of the dominant male, these weekly management dynamics ceased, leading to a decrease in the daily management routine of the lion pride. An individualized sampling of the animals through the utilization of indigestive markers was conducted, and fecal samples were collected before and after the death of the dominant male. Significant lower cortisol metabolite concentrations in feces were detected after the death of the dominant male, suggesting a positive impact of a decrease in daily management routines, together with a more stable social environment. In addition, assessment of individualized hormone concentrations throughout the study revealed variable physiological responses among lions, providing evidence of the importance of monitoring hormonal profiles individually.Monitoring the hypothalamic–pituitary–adrenal (HPA) axis through determination of fecal cortisol metabolite (FCM) levels is a non-invasive method useful for understanding how handling and social conditions may affect the physiological status of zoo animals. The present study used FCM analysis to evaluate whether the HPA axis activity of a lion pride was modified by a change in social and handling conditions after the death of the dominant male. Five African lions (Panthera leo bleyenberghi), two males and three females, were included in the study. Fecal samples were collected before and after the death of the dominant male. To avoid cohabitation conflicts between males before the dominant male died, subgroups were established and subjected to weekly changes between indoor and outdoor facilities. After the death of the dominant male, these management dynamics ceased, and the remaining four lions were kept together outdoors. Significant lower group FCM concentrations (p < 0.001) were detected after the decease of the dominant male, probably associated with a decrease in daily handling, together with a more stable social environment. Overall, the present study indicates the effect of different management scenarios on the HPA axis activity and differentiated physiological responses to the same situation between individuals.

Resident European ground squirrels exhibit higher stress levels than translocated individuals after conservation reinforcement

The European ground squirrel (Spermophilus citellus) is a rodent inhabiting the open grasslands of central and southeastern Europe. The species is endangered, and various conservation activities are being conducted to preserve and improve the populations’ status, especially during the recent years including translocations of individuals. The success of these measures has varied and is potentially strongly influenced by the chronic and acute stress associated with the translocation process itself. This stress load, however, has rarely been investigated in the framework of such conservation projects, neither in translocated nor in resident individuals. To fill this gap, we investigated glucocorticoid metabolite concentrations in translocated ground squirrels and compared these with concentrations in resident animals of a reinforced colony in southeastern Bulgaria. Between 2017 and 2019, we sampled a total of 252 individuals for faecal cortisol metabolite concentrations (FCM). The results showed that FCM decreased after the release in translocated individuals and that the time span after the translocation event was the key variable explaining the decrease. Surprisingly, the FCM levels were higher in the resident ground squirrels than in the translocated individuals throughout the active season and in the following spring. Furthermore, we found no effect of FCM concentrations on dispersal distances or body mass gain in translocated individuals.

Fecal Cortisol Metabolites in Dairy Cows: A Cross-Sectional Exploration of Associations with Animal, Stockperson, and Farm Characteristics.

Simple SummaryDairy cows are exposed to various potentially stressful situations in the daily farm routine, which might considerably impair their welfare and performance. On 25 German organic dairy farms, we explored associations of cows’ physiological stress levels by means of cortisol metabolite concentrations in feces (1) with different potentially influencing farm factors including human–animal contact, (2) cows’ fear behaviors towards humans, and (3) milk production and udder health. Cortisol metabolite levels were decreased on farms that did not separate diseased cows, possibly reflecting less regrouping stress. Levels were also lower on farms with straw yards compared to raised cubicles, and on farms with generous compared to suboptimal lying space, underlining the importance of resting comfort for cattle. Increased human–animal contact was associated with decreased cortisol metabolite levels. However, against expectations, levels were higher, when the farm provided concentrate feed by hand and habituated young cows to milking, requiring specific experimental investigations to draw conclusions on causal associations.To date, little is known about influences on cows’ physiological stress levels on farms. The present study explored associations of fecal cortisol metabolite concentrations (FCM) with (1) farm factors including human–animal contact, (2) cows’ fear behaviors towards humans, and (3) milk production and udder health, involving 25 dairy farms and repeated fecal samples (n = 2625) from 674 focal cows. Farm factors via interviews and observations, avoidance distance (AD) and qualitative behavior assessment (QBA) during a human–animal interaction were recorded. Milk yield and somatic cell scores (SCS) were calculated from milk recordings. Levels of FCMs were in general relatively low. No associations with AD and milk yield could be detected. Correlations between FCMs and QBA and SCS were significant, but on a low level. Against expectations, FCMs were higher, when the farm provided concentrates by hand and habituated heifers to milking, in part possibly due to reversed cause–effect relations. Decreased FCM levels were found on farms that did not separate diseased cows, possibly due to the avoidance of social stress following changes in group structure. Additionally, straw yards compared to raised cubicles and generous compared to suboptimal lying space were associated with decreased levels, underlining the importance of comfort around resting. Moreover, FCMs were decreased with increased human contact time per cow. The different associations detected in this study provide a basis for further experimental investigations that moreover might provide insights into causal relationships.

Glucocorticoids and activity in free-living arctic ground squirrels: Interrelationships between weather, body condition, and reproduction

The dynamic relationship between glucocorticoids and behavior are not well understood in wild mammals. We investigated how weather, body condition, and reproduction interact to affect cortisol levels and activity patterns in a free-living population of arctic ground squirrels (Urocitellus parryii). As a proxy for foraging and escape behaviors, collar-mounted accelerometers and light loggers were used to measure above-ground activity levels and the amount of time squirrels spent below the surface, respectively. Fecal cortisol metabolites (FCMs) were quantified to assess glucocorticoid secretion in squirrels. Male and female squirrels differed in above-ground activity levels and time spent below-ground across the active season, with males being most active during mating and females most active during lactation. We also found that female, but not male, squirrels exhibited seasonal variation in FCM levels, with concentrations highest during mid-lactation and lowest after the lactation period. In female squirrels, the seasonal relationships between breeding stage, activity, and FCM levels were also consistent with changes in maternal investment and the preparative role that glucocorticoids are hypothesized to play in energy mobilization. Body condition was not associated with FCM levels in squirrels. As predicted, deteriorating weather also influenced FCM levels and activity patterns in squirrels. FCM concentrations were affected by an interaction between temperature and wind speed when seasonal temperatures were lowest. In addition, above-ground activity, but not time spent below-ground, positively correlated with FCM levels. These results suggest that, although ground squirrels avoid inclement weather by remaining below-ground, activation of the stress axis may stimulate foraging activity.

Evaluation of long-term 11-oxoaetiocholanolone stability in red deer faecal samples under different storage conditions

Faecal cortisol metabolite (FCM) analysis is a reliable non-invasive method used in field endocrinology studies to assess levels of stress in animals. It is known that weather and, above all, humidity, can affect FCM concentrations in faecal samples. In addition, the prolonged storage of samples and delay in their analysis may alter metabolite concentrations. Intrinsic factors such as the heterogeneous distribution of FCMs within scats may likewise cause intra-sample variation. All of these sources of variation in FCM concentrations need to be addressed if we are to interpret results correctly. The aim of this study was to assess the effects of lyophilisation and storage temperature on the long-term stability of 11-oxoaetiocholanolone (11-o) in red deer (Cervus elaphus) faecal samples. After pre-cleaning with hexane and extraction with methanol, 11-o levels were analysed using high-performance liquid chromatography coupled with tandem mass spectrometry HPLC-MS/MS at 1, 2, 4, 6, 8, 16 and 32 weeks post-collection. We used linear mixed models to explore the effects of temperature and storage time on concentrations of faecal 11-o in wet and dry samples. Our results showed significant variations in 11-o concentrations in wet faecal samples over time and at different storage temperatures. By contrast, the 11-o values of dry samples were more stable in terms of storage temperatures. Lyophilising red deer faecal samples and storage at − 80 °C guarantees the stability of 11-o for several months.

Changes in behavior and fecal cortisol metabolites when dairy cattle are regrouped in pairs versus individually after calving

Regrouping occurs when dairy cows are moved between pens, and this can negatively affect cow behavior and production. Pen changes occur frequently around calving, a time when cows are vulnerable to health problems. Regrouping cows along with a familiar conspecific after calving may make social integration easier, but little work has tested this hypothesis. The objective of this study was to compare lying behavior, feeding behavior, social behavior, and fecal cortisol metabolite concentrations (a physiological indicator of stress) between cows regrouped individually and cows regrouped with a familiar partner after calving. Holstein (n = 8) and Jersey (n = 26) cows were monitored from approximately 2 wk before until 7 d after calving. Cows were held in 1 of 2 identical maternity pens until they calved. After calving, cows were held in a transition pen for 3 d before being regrouped into the main lactation pen (regrouping of interest). On d 3 postpartum, cows were assigned to a treatment, balancing for parity and breed: regrouped into the lactation pen alone (individual; n = 17) or with a familiar partner (paired; n = 17). A familiar partner was defined as a cow that spent 3 d in the transition pen with the focal cow. Using live observation, we quantified the time cows spent feeding, standing or lying in the lying stalls, grooming, and standing in different locations in the pen during the 1 h immediately after regrouping. The frequency of competitive interactions at the feed bunk was also measured during this observation period. Lying behavior was monitored from 2 d before until 5 d after regrouping using a data logger. Fecal samples were collected for 5 d beginning on the day of regrouping to assess fecal cortisol metabolite concentrations (11,17-dioxoandrostane, 11,17-DOA). We found no effects of regrouping treatment on behaviors observed during the 1-h period immediately following regrouping. Cows that were regrouped with a partner had more lying bouts than cows moved individually (9.2 ± 0.4 bouts/d vs. 7.5 ± 0.4 bouts/d) and shorter lying bout durations (66.6 ± 3.9 vs. 78.1 ± 3.5 min/d); however, we also observed these differences during the day before regrouping, suggesting that the differences in lying behavior could not be explained by the regrouping treatment alone. Individually regrouped primiparous cows had higher 11,17-DOA concentrations than individually regrouped multiparous cows but we found no differences in 11,17-DOA between parities when cows were moved with a familiar partner. In addition, 11,17-DOA was higher in primiparous cows moved alone compared with primiparous cows moved with a partner. More research is needed to understand the mechanisms by which social familiarity affects behavior and the physiological stress response following regrouping; this may provide new insight into how to better transition cows into new environments.

Timed environmental exposure indicates sample stability for reliable noninvasive measurement of fecal cortisol metabolite concentrations in sheep

The use of noninvasive techniques to evaluate stress responses in animals has become an increasingly popular method of animal welfare assessment in both production animals and wildlife. In particular, using fecal samples to measure fecal cortisol metabolites (FCMs) as a quantitative measure has proven ideal as samples can be collected remote to the animal after defecation without the need for invasive procedures. Colorimetric enzyme immunoassays (EIAs) have been shown to have a high level of selectivity and sensitivity for FCM concentration analysis, equivalent to the traditionally used radioimmunoassay. Regardless of the assay system used, species- and sample-specific validation must be undertaken to ensure the reliability of results, particularly where sampling is undertaken in a novel species or where environmental conditions might impact FCM stability in the fecal sample. To determine the limit of environmental exposure acceptable for analysis of FCM concentrations in ovine scat samples collected from a paddock under conditions of stable heat and humidity, this study quantified FCMs in ovine feces shortly after defecation (2–7 h) and after timed environmental exposure (1–9 d). Samples were determined to show stable FCM concentrations for up to 5 d by this analysis. Understanding the impacts of environmental exposure, and therefore the viability of remote fecal collection methods for quantitative analysis of FCM by EIA, is important to assess the utility of noninvasive measures of endocrine status in animals where the exact timing of defecation may not be known.

Virtual Fencing Is Comparable to Electric Tape Fencing for Cattle Behavior and Welfare.

Virtual fencing technology restricts animal movement via communicated signals without physical boundaries. Specifically, the eShepherd™ automated virtual fencing system operates via GPS technology and provides stimuli via a neckband device. An audio warning tone is emitted at the virtual boundary which is followed by an electrical pulse if the animal continues moving forward. Animal welfare is a priority consideration for the commercial implementation of virtual fencing systems. The current study assessed the effects of a virtual fence, in comparison to an electric tape fence, to contain eight groups of eight 12–14 month old steers within a 6-ha area across eight separate paddocks for 4 weeks following 1 week acclimation to the paddocks. Cattle were assessed across two cohorts (four groups/cohort) from January until March 2019 in Australia. Body weight and fecal samples from each animal were taken weekly. Fecal samples were processed for fecal cortisol metabolite (FCM) concentrations. IceQube R®'s fitted to the leg measured individual lying and standing time and the virtual fencing neckbands recorded GPS location and all administered audio and electrical stimuli. Cattle were maintained within their allocated area by both fence types across the 4-week period and those with the virtual fences were responding correctly to the audio cue with an average of 71.51 ± 2.26% of all cues across all animals being audio only. There was individual variation in rate of learning. The electric tape groups in cohort 1 showed a greater increase in body weight over 4 weeks than the virtual fence groups (P < 0.001) but this difference was not confirmed in cohort 2. The fence type statistically influenced the total daily lying time (P = 0.02) with less lying in cattle from the virtual fence groups but this difference equated to an average of <20 min per day. There were no differences between fence types in FCM concentrations (P = 0.39) and the concentrations decreased across time for all cattle (P < 0.001). These results indicate that virtual fencing technology effectively contains animals in a prescribed area across 4 weeks without substantial behavioral and welfare impacts on the cattle.

Physiology and behaviour of juvenile snowshoe hares at the start of the 10-year cycle

Snowshoe hares, Lepus americanus, are the dominant prey species in the boreal forest and they undergo regular, predator-driven population cycles every 8–11 years. Previous research has found that hare stress physiology is highly sensitive to both within-year (seasonal/litter group) and across-year (cycle phase) variation in predation risk and that maternal–offspring stress profiles are tightly correlated. Thus, both pre- and postnatal environments may interact to shape offspring physiological phenotype. If changes in the function and reactivity of the maternal stress axis are associated with variation in offspring behaviours that increase survival during periods of high predation risk, then the maternal effects on offspring physiology seen previously could represent a mechanistic route of adaptive maternal programming. To examine the relationship between physiology and behaviour in snowshoe hares, we monitored free-ranging adult and juvenile stress hormone levels in the first and second litters of the breeding season and assessed open field exploratory behaviour in weaning-age and independent juveniles in the southwestern Yukon (Canada) during 2013–2015, when predator density was low but increasing. Thus, our study spanned the late low phase (2013) and the early increase phase (2014–2015) of the hare population cycle. We found that increased concentrations of faecal cortisol metabolites (FCMs) were associated with aspects of risk avoidance in weaning-age hares. Juveniles with higher stress hormone levels spent more time under cover and were less active during open field trials, highlighting a potential mechanistic route to allow individuals to sensitively cope with a changing environment. Although average FCM levels of breeding females and juveniles were not correlated with one another and litter-based differences in physiology and behaviour were not present during these low-risk phases of the cycle, the association between stress hormone levels and behaviour sets the stage for adaptive maternal effects on offspring behaviour and survival as the cycle progresses and predation risk intensifies.