Do multiple experimenters improve the reproducibility of animal studies?

The credibility of scientific research has been seriously questioned by the widely claimed "reproducibility crisis". In light of this crisis, there is a growing awareness that the rigorous standardisation of experimental conditions may contribute to poor reproducibility of animal studies. Instead, systematic heterogenisation has been proposed as a tool to enhance reproducibility, but a real-life test across multiple independent laboratories is still pending. The aim of this study was therefore to test whether heterogenisation of experimental conditions by using multiple experimenters improves the reproducibility of research findings compared to standardised conditions with only one experimenter. To this end, we replicated the same animal experiment in 3 independent laboratories, each employing both a heterogenised and a standardised design. Whereas in the standardised design, all animals were tested by a single experimenter; in the heterogenised design, 3 different experimenters were involved in testing the animals. In contrast to our expectation, the inclusion of multiple experimenters in the heterogenised design did not improve the reproducibility of the results across the 3 laboratories. Interestingly, however, a variance component analysis indicated that the variation introduced by the different experimenters was not as high as the variation introduced by the laboratories, probably explaining why this heterogenisation strategy did not bring the anticipated success. Even more interestingly, for the majority of outcome measures, the remaining residual variation was identified as an important source of variance accounting for 41% (CI95 [34%, 49%]) to 72% (CI95 [58%, 88%]) of the observed total variance. Despite some uncertainty surrounding the estimated numbers, these findings argue for systematically including biological variation rather than eliminating it in animal studies and call for future research on effective improvement strategies.

Correction: Do multiple experimenters improve the reproducibility of animal studies?

[This corrects the article DOI: 10.1371/journal.pbio.3001564.].

Repeated social defeat stress differently affects arthritis-associated hypersensitivity in male and female mice.

Chronic stress enhances the risk of neuropsychiatric disorders and contributes to the aggravation and chronicity of pain. The development of stress-associated diseases, including pain, is affected by individual vulnerability or resilience to stress, although the mechanisms remain elusive. We used the repeated social defeat stress model promoting susceptible and resilient phenotypes in male and female mice and induced knee mono-arthritis to investigate the impact of stress vulnerability on pain and immune system regulation. We analyzed different pain-related behaviors, measured blood cytokine and immune cell levels, and performed histological analyses at the knee joints and pain/stress-related brain areas. Stress susceptible male and female mice showed prolonged arthritis-associated hypersensitivity. Interestingly, hypersensitivity was exacerbated in male but not female mice. In males, stress promoted transiently increased neutrophils and Ly6Chigh monocytes, lasting longer in susceptible than resilient mice. While resilient male mice displayed persistently increased levels of the anti-inflammatory interleukin (IL)-10, susceptible mice showed increased levels of the pro-inflammatory IL-6 at the early- and IL-12 at the late arthritis stage. Although joint inflammation levels were comparable among groups, macrophage and neutrophil infiltration was higher in the synovium of susceptible mice. Notably, only susceptible male mice, but not females, presented microgliosis and monocyte infiltration in the prefrontal cortex at the late arthritis stage. Blood Ly6Chigh monocyte depletion during the early inflammatory phase abrogated late-stage hypersensitivity and the associated histological alterations in susceptible male mice. Thus, recruitment of blood Ly6Chigh monocytes during the early arthritis phase might be a key factor mediating the persistence of arthritis pain in susceptible male mice. Alternative neuro-immune pathways that remain to be explored might be involved in females.

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.

Coping with extreme free cortisol levels: Seasonal stress axis changes in sympatric North American flying squirrels.

Most environments exhibit predictable yearly changes, permitting animals to anticipate them. The hypothalamic-pituitary-adrenal (HPA) axis is a key physiological pathway that enables animals to cope with such changes. Monitoring glucocorticoid (the end products of the HPA axis) levels in wild animals throughout the year can improve our understanding of how this pathway responds to different conditions. For this study, we collected 18 months of data on two species of North American flying squirrels (Glaucomys sabrinus and G. volans) living in a southern Ontario forest where temperature and food availability fluctuate dramatically throughout the year. These squirrels are active year-round, nest communally, and rely on scatter hoarded foods in the winter months. Flying squirrels have extremely high levels of free plasma cortisol relative to other mammals, but it is unknown how these levels are affected by environmental and reproductive factors. For both species, our goals were to (1) validate an enzyme immunoassay (EIA) to measure their fecal glucocorticoid metabolite (FGM) concentrations and (2) assess yearly differences, seasonal changes, and the influence of sex, reproduction, and ambient temperature on FGM concentrations in each species. In the lab, we successfully validated the use of antibody 5α-pregnane-3ß, 11ß, 21-triol-20-one EIA for FGM analysis in both species. In the field, neither sex nor reproductive status (breeding condition or not) were linked to FGM concentrations in either species. FGM concentrations were higher in autumn compared to the spring and summer. There were no other seasonal differences. We discuss possible explanations for the autumn peak in FGM concentrations (increased energy expenditure and social nesting changes), as well as outline possible avenues for future research. Understanding how individuals and populations respond to environmental change is a critical goal in evolutionary ecology, particularly in the context of a rapidly changing Anthropocene.

Weathering the storm: Decreased activity and glucocorticoid levels in response to inclement weather in breeding Columbian ground squirrels.

Inclement weather can rapidly modify the thermal conditions experienced by animals, inducing changes in their behavior, body condition, and stress physiology, and affecting their survival and breeding success. For animals living in variable environments, the extent to which they have adapted to cope with inclement weather is not established, especially for hibernating species with a short active season that are constrained temporally to breed and store energy for subsequent hibernation. We examined behavioral (foraging activity) and physiological (body mass and fecal cortisol metabolites) responses of Columbian ground squirrels (Urocitellus columbianus), small hibernating rodents inhabiting open meadows in Rocky Mountains, to 3 events of inclement weather (two snow storms in May 2021 and May 2022, one heavy rainfall in June 2022). We found that individuals adapted to inclement weather conditions by (1) reducing above-ground activity, including foraging, (2) decreasing the mobilization of stored resources as indicated by a decrease in the activity of the hypothalamo-pituitary-adrenal (HPA) axis and lower fecal cortisol metabolites in the hours/days following periods of inclement weather; and (3) compensating through increased foraging and more local activity when favorable conditions resumed. As a result, body mass and growth did not decrease following short periods of inclement weather. Columbian ground squirrels were well-adapted to short periods of inclement weather, coping via modifications of their behavior and the activity of the HPA axis.

Is chronic stress a causal mechanism for small mammal population cycles? Reconciling the evidence.

Chronic stress has long been hypothesized to play a role in driving population cycles. Christian (1950) hypothesized that high population density results in chronic stress and mass "die-offs" in small mammal populations. Updated variations of this hypothesis propose that chronic stress at high population density may reduce fitness, reproduction, or program aspects of phenotype, driving population declines. We tested the effect of density on the stress axis in meadow voles (Microtus pennsylvanicus) by manipulating population density in field enclosures over three years. Using fecal corticosterone metabolites as a non-invasive measure of glucocorticoid (GC) concentrations, we found that density alone was not associated with GC differences. However, we found that the seasonal relationship of GC levels differed by density treatment, with high-density populations having elevated GC levels early in the breeding season and decreasing towards late summer. We additionally tested hippocampal glucocorticoid receptor and mineralocorticoid receptor gene expression in juvenile voles born at different densities, with the hypothesis that high density may reduce receptor expression, altering negative feedback of the stress axis. We found that females had marginally higher glucocorticoid receptor expression at high density, no effect in males, and no detectable effect of density on mineralocorticoid receptor expression in either sex. Hence, we found no evidence that high density directly impairs negative feedback in the hippocampus, but rather female offspring may be better equipped for negative feedback. We compare our findings with prior studies to attempt to disentangle the complicated relationship between density, seasonality, sex, reproduction and the stress axis.

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.

Age and spatio-temporal variations in food resources modulate stress-immunity relationships in three populations of wild roe deer.

Living in variable and unpredictable environments, organisms face recurrent stressful situations. The endocrine stress response, which includes the secretion of glucocorticoids, helps organisms to cope with these perturbations. Although short-term elevations of glucocorticoid levels are often associated with immediate beneficial consequences for individuals, long-term glucocorticoid elevation can compromise key physiological functions such as immunity. While laboratory works highlighted the immunosuppressive effect of long-term elevated glucocorticoids, it remains largely unknown, especially in wild animals, whether this relationship is modulated by individual and environmental characteristics. In this study, we explored the co-variation between integrated cortisol levels, assessed non-invasively using faecal cortisol metabolites (FCMs), and 12 constitutive indices of innate, inflammatory, and adaptive immune functions, in wild roe deer living in three populations with previously known contrasting environmental conditions. Using longitudinal data on 564 individuals, we further investigated whether age and spatio-temporal variations in the quantity and quality of food resources modulate the relationship between FCMs and immunity. Negative covariation with glucocorticoids was evident only for innate and inflammatory markers of immunity, while adaptive immunity appeared to be positively or not linked to glucocorticoids. In addition, the negative covariations were generally stronger in individuals facing harsh environmental constraints and in old individuals. Therefore, our results highlight the importance of measuring multiple immune markers of immunity in individuals from contrasted environments to unravel the complex relationships between glucocorticoids and immunity in wild animals. Our results also help explain conflicting results found in the literature and could improve our understanding of the link between elevated glucocorticoid levels and disease spread, and its consequences on population dynamics.

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.

Welfare Assessment on Healthy and Tumor-Bearing Mice after Repeated Ultrasound Imaging.

INTRODUCTION: Ultrasound (US) imaging enables tissue visualization in high spatial resolution with short examination times. Thus, it is often applied in preclinical research. Diagnostic US, including contrast-enhanced US (CEUS), is considered to be well-tolerated by laboratory animals although no systematic study has been performed to confirm this claim. Therefore, the aim of this study was to screen for possible effects of US and CEUS examinations on welfare of healthy mice. Additionally, the potential influence of CEUS and molecular CEUS on well-being and therapy response to regorafenib was investigated in breast cancer-bearing mice. MATERIAL AND METHODS: Forty healthy Balb/c mice were randomly assigned for examination with US or CEUS (3×/week) for 4 weeks. Untreated healthy mice and mice receiving only isoflurane anesthesia served as controls (n = 10/group). Ninety-four 4T1 tumor-bearing Balb/c mice were allocated randomly to the following groups: no imaging, isoflurane anesthesia, CEUS, and molecular CEUS. They either received 10 mg/kg regorafenib or vehicle solution daily by oral gavage. Animals were examined three times within 2 weeks. CEUS measurements were performed using phospholipid microbubbles, and phospholipid microbubbles targeting the vascular endothelial growth factor receptor-2 were applied for molecular CEUS. Welfare evaluation was performed by daily observational score sheets, measuring the heart rate, Rotarod performance, and fecal corticosterone metabolites twice a week. On the last day, pathological changes in serum corticosterone concentrations, hemograms, and organ weights were obtained. Moreover, a potential influence of isoflurane anesthesia, CEUS, and molecular CEUS on regorafenib response in tumor-bearing mice was examined. Analysis of variance and Dunnett's post hoc test were performed as statistical analyses. RESULTS: Severity parameters were not altered after repeated US and CEUS examinations of healthy mice, but spleen sizes were significantly lower after isoflurane anesthesia. In tumor-bearing mice, no effect on animal welfare after repeated CEUS and molecular CEUS could be observed. However, leukocyte counts and spleen weights of tumor-bearing mice were significantly lower in animals examined with CEUS and molecular CEUS compared to the control groups. This effect was not visible in regorafenib-treated animals. CONCLUSIONS: Repeated US and (molecular) CEUS have no detectable impact on animal welfare in healthy and tumor-bearing mice. However, CEUS and molecular CEUS in combination with isoflurane anesthesia might attenuate immunological processes in tumor-bearing animals and may consequently affect responses to antitumor therapy.

Does Sex Matter in Liver Surgery? Comparison of Severity Assessments between Female and Male Rats after Partial Hepatectomy: A Pilot Study.

INTRODUCTION: Current animal-based biomedical research, including studies on liver function and disease, is conducted almost exclusively on male animals to mitigate confounding effects of the estrous cycle. However, liver diseases afflict both men and women, so translational research findings should also be applicable to female patients. This pilot study investigated sex differences in objective and subjective severity assessment parameters in rats following 50% partial hepatectomy. MATERIALS AND METHODS: This study was performed using Wistar Han rats, in which measurements of body weight, spontaneous motor activity in the open field (OF) (movement distance, movement velocity, rearing frequency), and fecal corticosterone metabolites were conducted at baseline and at multiple times after partial hepatectomy. Subjective postsurgical severity assessments were conducted using modified score sheets. Blood parameters such as leukocyte count and serum aspartate aminotransferase, as well as estrogens and testosterone were measured from samples obtained during partial hepatectomy and at sacrifice. In addition, the amount of resected liver tissue was measured at partial hepatectomy, and the proliferated liver was weighed at sacrifice. RESULTS: Fecal corticosterone metabolite concentrations differed significantly between males and females at baseline and following hepatectomy. Also, leukocyte counts and estrogen concentrations were significantly different between sexes before partial hepatectomy. Alternatively, there were no sex differences in severity assessments, body weight changes, and behavior in the OF at any measurement time point. Liver weight was significantly different in males and females at the time point of partial hepatectomy and sacrifice. CONCLUSION: The results of this pilot study suggest that males and females respond similarly following partial hepatectomy. Examination of both sexes is very important for translation to humans, where both men and women suffer from liver disease. Furthermore, the use of both sexes in animal-based research would improve the utilization of the animal breeding in terms of the 3 Rs. However, due to some limitations, larger scale investigations including a broader spectrum of pathophysiolological, behavioral, and pharmacokinetic measures are planned.

Toward Evidence-Based Severity Assessment in Mouse Models with Repeated Seizures: (II.) Impact of Surgery and Intrahippocampal Kainate.

INTRODUCTION: Chronic epilepsy models require neurosurgical procedures including depth electrode implants. The intrahippocampal kainate model is a frequently used chronic paradigm, which is based on chemoconvulsant administration and status epilepticus induction during the surgical procedure. This experimental approach raises the question of the extent to which this approach affects postsurgical recovery. In addition to the short- and long-term impact of the surgical intervention, a potential impact of highly frequent electrographic seizure events needs to be considered in the context of severity assessment. METHODS: Various behavioral, biochemical, and telemetric parameters were analyzed in four experimental groups of mice: 1st naive, 2nd with transmitter implants, 3rd with transmitter and electrode implants, and 4th with transmitter implants, electrode implants, and kainate-induced status epilepticus. RESULTS: During the early postsurgical phase, transmitter implants caused a transient impact on Mouse Grimace scores and intragroup increase of fecal corticosterone metabolites. Additional craniotomy was associated with an influence on total heart rate variability and fecal corticosterone metabolites. Heart rate and Irwin score increases as well as a prolonged increase in Mouse Grimace scores pointed to an added burden related to the induction of a nonconvulsive status epilepticus. Data from the chronic phase argued against a relevant influence of frequent electrographic seizures on behavioral patterns, fecal corticosterone metabolites, heart rate, and its variability. However, Irwin scores indicated long-term changes in some animals with increased reactivity, body tone, and Straub tail. Interestingly, selected behavioral and telemetric data from the early post-status epilepticus phase correlated with the frequency of electrographic seizure events in the chronic phase. CONCLUSION: In conclusion, our findings argue against the pronounced impact of highly frequent electrographic seizures on the well-being of mice. However, an increased level of nervousness in a subgroup of animals should be considered for handling procedures and refinement measures. In the early postsurgical phase, several parameters indicate an influence of the interventions with evidence that the nonconvulsive status epilepticus can negatively affect the recovery. Thus, the development and validation of refinement efforts should focus on this experimental phase. Finally, the datasets suggest that simple readout parameters may predict the long-term consequences of the epileptogenic insult. Respective biomarker candidates require further validation in the follow-up studies in models with subgroups of animals with or without epilepsy development.

Severity Assessment in Rats Undergoing Subarachnoid Hemorrhage Induction by Endovascular Perforation or Corresponding Sham Surgery.

INTRODUCTION: Animal models for preclinical research of subarachnoid hemorrhage (SAH) are widely used as much of the pathophysiology remains unknown. However, the burden of these models inflicted on the animals is not well characterized. The European directive requires severity assessment-based allocation to categories. Up to now, the classification into predefined categories is rather subjective and often without underlying scientific knowledge. We therefore aimed at assessing the burden of rats after SAH or the corresponding sham surgery to provide a scientific assessment. METHODS: We performed a multimodal approach, using different behavior tests, clinical and neurological scoring, and biochemical markers using the common model for SAH of intracranial endovascular filament perforation in male Wistar rats. Up to 7 days after surgery, animals with SAH were compared to sham surgery and to a group receiving only anesthesia and analgesia. RESULTS: Sham surgery (n = 15) and SAH (n = 16) animals showed an increase in the clinical score the first days after surgery, indicating clinical deterioration, while animals receiving only anesthesia without surgery (n = 5) remained unaffected. Body weight loss occurred in all groups but was more pronounced and statistically significant only after surgery. The analysis of burrowing, open field (total distance, erections), balance beam, and neuroscore showed primarily an effect of the surgery itself in sham surgery and SAH animals. Only concerning balance beam and neuroscore, a difference was visible between sham surgery and SAH. The outcome of the analysis of systemic and local inflammatory parameters and of corticosterone in blood and its metabolites in feces was only robust in animals suffering from larger bleedings. Application of principal component analysis resulted in a clear separation of sham surgery and SAH animals from their respective baseline as well as from the anesthesia-only group at days 1 and 3, with the difference between sham surgery and SAH being not significant. DISCUSSION/CONCLUSION: To our knowledge, we are the first to publish detailed clinical score sheet data combined with advanced behavioral assessment in the endovascular perforation model for SAH in rats. The tests chosen here clearly depict an impairment of the animals within the first days after surgery and are consequently well suited for assessment of the animals' suffering in the model. A definitive classification into one of the severity categories named by the EU directive is yet pending and has to be performed in the future by including the assessment data from different neurological and nonneurological disease models.

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.

Native species exhibit physiological habituation to invaders: a reason for hope.

Animals cope with environmental perturbations through the stress response, a set of behavioural and physiological responses aimed to maintain and/or return to homeostasis and enhance fitness. Vertebrate neuroendocrine axis activation in response to environmental stressors can result in the secretion of glucocorticoids (GCs), whose acute increases may be adaptive, while chronic elevation may be detrimental. Invasive grey squirrels (Sciurus carolinensis) act as a stressor eliciting elevation of GCs in native red squirrels (Sciurus vulgaris). Here we used 6-year data of variation in faecal glucocorticoid metabolite (FGM) concentrations following invasion by grey squirrels in three red squirrel populations, to identify if red squirrels showed physiological habituation to this stressor. The decrease in FGMs over time was more pronounced shortly after invasion and at high densities of grey squirrels while it decreased less strongly and was no longer influenced by the invader density as time since invasion elapsed. At the individual level, FGMs also decreased more markedly as each red squirrel experienced prolonged contact with the invader. Our study provides compelling new data suggesting that native species in the wild can habituate to prolonged contact with invasive species, showing that they may avoid the potentially harmful effects of chronic elevations in GCs.

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.

Invasive alien species as an environmental stressor and its effects on coping style in a native competitor, the Eurasian red squirrel.

Free-living animals cope with environmental stressors through physiological and behavioural responses. According to the unidimensional model, these responses are integrated within a coping style: proactive individuals (bold, active-explorative and social) have a lower hypothalamic-pituitary-adrenal (HPA) axis reactivity than reactive ones (shy, less active-explorative, less social). These associations may change when individuals are exposed to human-induced rapid environmental change (HIREC), such as the introduction of invasive alien species (IAS). Here, we studied Eurasian red squirrels to investigate the relationship between personality traits and one integrated measure of HPA axis activity, both in areas uncolonized (natural populations) and colonized by an IAS, the Eastern grey squirrel (invaded populations). We expected an association between physiological and behavioural responses, and that activity, exploration and social tendency would covary, forming a behavioural syndrome in natural populations, while competition with the IAS was predicted to disrupt these associations. We used faecal glucocorticoid metabolites (FGMs) as an integrated measure of adrenocortical activity, and measured the levels of four personality traits (exploration, activity, activity-exploration and social tendency) with an open field test and a mirror image stimulation test. We found no correlation between FGMs and personality traits, neither in natural nor invaded populations. However, we found correlations among personality traits in areas without interspecific competition, indicating a behavioural syndrome, which was disrupted in invaded populations. This is one of the few studies showing that an IAS, acting as an environmental stressor, alters a native species' behavioural syndrome, but does not influence its coping style.

The glucocorticoid response to environmental change is not specific to agents of natural selection in wild red squirrels.

Evolutionary endocrinology aims to understand how natural selection shapes endocrine systems and the degree to which endocrine systems themselves can induce phenotypic responses to environmental changes. Such responses may be specialized in that they reflect past selection for responsiveness only to those ecological factors that ultimately influence natural selection. Alternatively, endocrine responses may be broad and generalized, allowing organisms to cope with a variety of environmental changes simultaneously. Here, we empirically tested whether the endocrine response of female North American red squirrels (Tamiasciurus hudsonicus) was specialized or generalized. We first quantified the direction and magnitude of natural selection acting on three female life history traits (parturition date, litter size, offspring postnatal growth rate) during 32 years of fluctuations in four potential ecological agents of selection (food availability, conspecific density, predator abundance, and temperature). Only three of the four variables (food, density, and predators) affected patterns of natural selection on female life history traits. We then quantified fecal glucocorticoid metabolites (FGMs) across 7 years and found that all four environmental variables, regardless of their effects on patterns of selection, were associated with glucocorticoid production. Our results provide support for a generalized, rather than specific, glucocorticoid response to environmental change that can integrate across multiple co-occurring environmental stressors.

Territorial scent-marking effects on vigilance behavior, space use, and stress in female Columbian ground squirrels.

Social environments can profoundly affect the behavior and stress physiology of group-living animals. In many territorial species, territory owners advertise territorial boundaries to conspecifics by scent marking. Several studies have investigated the information that scent marks convey about donors' characteristics (e.g., dominance, age, sex, reproductive status), but less is known about whether scents affect the behavior and stress of recipients. We experimentally tested the hypothesis that scent marking may be a potent source of social stress in territorial species. We tested this hypothesis for Columbian ground squirrels (Urocitellus columbianus) during lactation, when territorial females defend individual nest-burrows against conspecifics. We exposed lactating females, on their territory, to the scent of other lactating females. Scents were either from unfamiliar females, kin relatives (a mother, daughter, or sister), or their own scent (control condition). We expected females to react strongly to novel scents from other females on their territory, displaying increased vigilance, and higher cortisol levels, indicative of behavioral and physiological stress. We further expected females to be more sensitive to unfamiliar female scents than to kin scents, given the matrilineal social structure of this species and known fitness benefits of co-breeding in female kin groups. Females were highly sensitive to intruder (both unfamiliar and kin) scents, but not to their own scent. Surprisingly, females reacted more strongly to the scent of close kin than to the scent of unfamiliar females. Vigilance behavior increased sharply in the presence of scents; this increase was more marked for kin than unfamiliar female scents, and was mirrored by a marked 131% increase in free plasma cortisol levels in the presence of kin (but not unfamiliar female) scents. Among kin scents, lactating females were more vigilant to the scent of sisters of equal age, but showed a marked 318% increase in plasma free cortisol levels in response to the scent of older and more dominant mothers. These results suggest that scent marks convey detailed information on the identity of intruders, directly affecting the stress axis of territory holders.