Faecal Corticosterone Metabolites Levels Research Articles

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

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

Acclimation and Blood Sampling: Effects on Stress Markers in C57Bl/6J Mice.

Blood sampling in rodents is common practice in scientific studies. Some of the refined methods widely used are the puncture of the saphenous vein or tail vein, or even tail docking. The handling needs of these different blood sampling methods are different and can directly affect stress, increasing the variability of the study. Moreover, there is less aversion and stress if the animal is accustomed to the environment, handling and technique. Therefore, our study aimed to assess the influence of these three blood sampling techniques (saphenous puncture, tail vein puncture and tail vein docking) and the use of previous acclimation on different indicators of animal stress, assessing blood glucose concentrations and faecal corticosterone metabolites (FCMs). Twenty-four young adult male and female C57Bl6/J mice were divided in three groups by sampling method: tail docking (TD), saphenous vein puncture (SV) and caudal vein puncture (CV) groups. All mice were studied with and without acclimation, which was performed during 9 consecutive days. The results showed that both males and females present very similar responses to the different handling and sampling methods without significant differences. Nevertheless, acclimation in all sampling methods decreased glucose and FCM levels significantly. The method that obtained the lowest glucose and FCM levels with significance was saphenous vein puncture. Therefore, we can say that it causes less stress when performing prior acclimation, even when this involves greater handling of the animal. Our results contribute to refinement within the 3R concept and could serve researchers to programme and select a good handling technique and a welfare-friendly blood sampling method for their experiments.

The power of a touch: Regular touchscreen training but not its termination affects hormones and behavior in mice.

Touchscreen-based procedures are increasingly used in experimental animal research. They not only represent a promising approach for translational research, but have also been highlighted as a powerful tool to reduce potential experimenter effects in animal studies. However, to prepare the animals for a touchscreen-based test, an often time-consuming training phase is required that has itself been shown to cause increased adrenocortical activity and anxiety-like behavior in mice. While these findings point at a potentially negative effect of touchscreen training at first glance, results have also been discussed in light of an enriching effect of touchscreen training. The aim of the present study was therefore to shed more light on recently reported touchscreen training effects, with a particular focus on the termination of the training routine. Specifically, we investigated whether the termination of regular touchscreen training could constitute a loss of enrichment for mice. Thus, we assessed fecal corticosterone metabolites (FCMs), exploratory-, anxiety-like and home cage behavior in touchscreen-trained mice in comparison to food restricted and ad libitum fed mice, as a restricted diet is an integral part of the training process. Furthermore, we compared these parameters between mice that were continuously trained and mice whose training was terminated 2 weeks earlier. Our results confirm previous findings showing that a mild food restriction increases the animals' exploratory behavior and shifts their activity rhythm. Moreover, touchscreen training was found to increase FCM levels and anxiety-like behavior of the mice. However, no effect of the termination of touchscreen training could be detected, a finding which contradicts the enrichment loss hypothesis. Therefore, we discuss two alternative explanations for the findings. Yet, the current state of knowledge is not sufficient to draw final conclusions at this stage. In compliance with the refinement endeavors for laboratory animals, further research should assess the severity of touchscreen procedures to ensure a responsible and well-founded use of animals for experimental purposes.

Individualized Housing Modifies the Immune-Endocrine System in CD1 Adult Male Mice.

In the last years, different research groups have made considerable efforts to improve the care and use of animals in research. Mice (Mus musculus) are the most widely used animal species in research in the European Union and are sociable and hierarchical creatures. During experiments, researchers tend to individualize males, but no consideration is given to whether this social isolation causes them stress. The aim of this study was, therefore, to explore whether 4 weeks of social isolation could induce changes in different physiological parameters in adult Crl:CD1(ICR) (CD1) males, which may interfere with experimental results. Body weight, blood cells, and fecal corticosterone metabolites levels were the analyzed parameters. Blood and fecal samples were collected at weeks 1 and 4 of the experimental procedure. Four weeks of single housing produced a significant time-dependent decrease in monocytes and granulocytes. Fecal corticosterone metabolite levels were higher in single-housed mice after 1 week and then normalized after 4 weeks of isolation. Body weight, red blood cells, and platelets remained unchanged in both groups during this period. We can, therefore, conclude that social isolation affects some immune and endocrine parameters, and that this should be taken into account in the interpretation of research data.

Ablation of Gabra5 Influences Corticosterone Levels and Anxiety-like Behavior in Mice.

Stress responses are activated by the hypothalamic-pituitary-adrenal axis (HPA axis), culminating in the release of glucocorticoids. During prolonged periods of secretion of glucocorticoids or inappropriate behavioral responses to a stressor, pathologic conditions may occur. Increased glucocorticoid concentration is linked to generalized anxiety, and there are knowledge gaps regarding its regulation. It is known that the HPA axis is under GABAergic control, but the contribution of the individual subunits of the GABA receptor is largely unknown. In this study, we investigated the relationship between the α5 subunit and corticosterone levels in a new mouse model deficient for Gabra5, which is known to be linked to anxiety disorders in humans and phenologs observed in mice. We observed decreased rearing behavior, suggesting lower anxiety in the Gabra5-/- animals; however, such a phenotype was absent in the open field and elevated plus maze tests. In addition to decreased rearing behavior, we also found decreased levels of fecal corticosterone metabolites in Gabra5-/- mice indicating a lowered stress response. Moreover, based on the electrophysiological recordings where we observed a hyperpolarized state of hippocampal neurons, we hypothesize that the constitutive ablation of the Gabra5 gene leads to functional compensation with other channels or GABA receptor subunits in this model.

High-quality habitats and refuges from tourism reduce individual stress responses in a forest specialist

Context The assessment of suitable habitat for a species is often evaluated based merely on where the species is most abundant. However, a variety of environmental factors such as temperature, resources and human disturbances might force animals to avoid otherwise high-quality habitats. Additionally, individuals’ capacities to cope with such factors – as reflected in physiological stress responses – may also contribute to avoidance. Combining measures of stress response of individual animals with habitat suitability models may contribute essential insights to the development of species conservation plans. Aims We used a landscape-level approach to assess whether habitat quality, abiotic factors and human disturbance affect the physiological stress response of a forest specialist, the capercaillie (Tetrao urogallus), and to what extent these relationships are determined by variation between individuals. Methods We applied a non-invasive approach by collecting 1096 droppings across the entire species range in two adjacent national parks spanning the border between the Czech Republic and Germany. We measured faecal corticosterone metabolites (FCMs) to evaluate the physiological stress response. To test the influences of temperature, habitat quality, season and sex, as well as forest management and tourism intensity on FCM levels, we used generalised additive mixed effect models (GAMMs). The same models were applied for a reduced subset of randomly selected samples that were attributed to individuals via DNA profiling using 10 microsatellite loci. This approach allowed us to control for possible individual differences (through repeated measures within individual genotypes). Key results Both models revealed decreasing FCM levels with increasing habitat quality and showed no relationship between increasing forestry intensity and FCMs. When accounting for individual identity, we found a significant positive effect from increasing tourism intensity, although this relationship was not significant in the full dataset. Conclusions Our results highlight three conclusions for the conservation of capercaillie: (1) high-quality habitats reduce stress levels; (2) tourism-free refuge areas are important, and (3) individuality appears to be a strong determinant of response to environmental factors. Implications Generalising management recommendations from observations for single individuals to an entire population may be inappropriate.

Hidden but Potentially Stressed: A Non-Invasive Technique to Quantify Fecal Glucocorticoid Levels in a Fossorial Amphisbaenian Reptile.

To understand wildlife responses to the changing environment, it is useful to examine their physiological responses and particularly their endocrine status. Here, we validated an enzyme immunoassay (EIA) to non-invasively quantify fecal corticosterone metabolites (FCM) in the fossorial amphisbaenian reptile Trogonophis wiegmanni from North Africa. We supplemented animals assigned to the treatment group with corticosterone dissolved in oil applied non-invasively on the skin for several days, while control groups received the oil-alone solution. Fresh feces were collected at the end of the supplementation period, and FCM levels were quantified by an EIA. Basal FCM levels were similar for both treatments and increased at the end of the test, but FCM increased significantly more in corticosterone-treated animals. A further examination of FCM levels in a wild population of this amphisbaenian did not find overall sexual, size or seasonal differences but showed a high range of variation among individuals. This suggests that different uncontrolled intrinsic or local environmental variables might increase the circulating glucocorticoid levels of different individuals. Our results confirmed the suitability of EIA for analyzing physiological changes in FCM in this amphisbaenian species. This technique may be useful for understanding and remediating the little-explored potential stressors of the soil environment that may negatively affect the health state of fossorial reptiles.

Is negative density-dependent reproduction regulated by density-induced stress in root voles? Two field experiments.

Density dependence in reproduction plays an important role in stabilizing population dynamics via immediate negative feedback from population density to reproductive output. Although previous studies have shown that negative density‐dependent reproduction is associated with strong spacing behavior and social interaction between individuals, the proximal mechanism for generating negative density‐dependent reproduction remains unclear. In this study, we investigated the effects of density‐induced stress on reproduction in root voles. Enclosed founder populations were established by introducing 6 (low density) and 30 (high density) adults per sex into per enclosure (four enclosures per density in total) during the breeding season from April to July 2012 and from May to August 2015. Fecal corticosterone metabolite (FCM) levels, reproductive traits (recruitment rate and the proportion of reproductively active individuals), and founder population numbers were measured following repeated live trapping in both years. The number of founders was negatively associated with recruitment rates and the proportion of reproductively active individuals, displaying a negative density‐dependent reproduction. FCM level was positively associated with the number of founders. The number of founder females directly affected the proportion of reproductive females, and directly and indirectly through their FCM levels affected the recruitment rate; the effect of the number of male founders on the proportion of reproductive males was mediated by their FCM level. Our results showed that density‐induced stress negatively affected reproductive traits and that density‐induced stress is one ecological factor generating negative density‐dependent reproduction.

The impact of tethered recording techniques on activity and sleep patterns in rats

Electrophysiological recordings in animals constitute frequently applied techniques to study neuronal function. In this context, several authors described tethered recordings as a semi-restraint situation with negative implications for animal welfare and suggested radiotelemetric setups as a refinement measure. Thus, we here investigated the hypothesis that tethered recordings exert measurable effects on behavioral and sleep patterns in Sprague–Dawley rats. Animals were kept in monitoring glass cages either with or without a head connection to a recording cable. Saccharin preference, nest building, serum corticosterone and fecal corticosterone metabolite levels were in a comparable range in both groups. The proportion of vigilance states was not affected by the cable connection. Minor group differences were detected in bout lengths distributions, with a trend for longer NREM and WAKE stages in animals with a cable connection. However, a relevant effect was not further confirmed by an analysis of the number of sleep/wake and wake/sleep transitions. The analysis of activity levels did not reveal group differences. However, prolonged exposure to the tethered condition resulted in an intra-group increase of activity. In conclusion, the comparison between freely moving vs tethered rats did not reveal major group differences. Our findings indicate that telemetric recordings only offer small advantages vs cabled set ups, though this may differ in other experimental studies where for example anxiety- or drug-induced effects are analyzed.

Gradual Restraint Habituation for Awake Functional Magnetic Resonance Imaging Combined With a Sparse Imaging Paradigm Reduces Motion Artifacts and Stress Levels in Rodents.

Functional magnetic resonance imaging, as a non-invasive technique, offers unique opportunities to assess brain function and connectivity under a broad range of applications, ranging from passive sensory stimulation to high-level cognitive abilities, in awake animals. This approach is confounded, however, by the fact that physical restraint and loud unpredictable acoustic noise must inevitably accompany fMRI recordings. These factors induce marked stress in rodents, and stress-related elevations of corticosterone levels are known to alter information processing and cognition in the rodent. Here, we propose a habituation strategy that spans specific stages of adaptation to restraint, MRI noise, and confinement stress in awake rats and circumvents the need for surgical head restraint. This habituation protocol results in stress levels during awake fMRI that do not differ from pre-handling levels and enables stable image acquisition with very low motion artifacts. For this, rats were gradually trained over a period of three weeks and eighteen training sessions. Stress levels were assessed by analysis of fecal corticosterone metabolite levels and breathing rates. We observed significant drops in stress levels to below pre-handling levels at the end of the habituation procedure. During fMRI in awake rats, after the conclusion of habituation and using a non-invasive head-fixation device, breathing was stable and head motion artifacts were minimal. A task-based fMRI experiment, using acoustic stimulation, conducted 2 days after the end of habituation, resulted in precise whole brain mapping of BOLD signals in the brain, with clear delineation of the expected auditory-related structures. The active discrimination by the animals of the acoustic stimuli from the backdrop of scanner noise was corroborated by significant increases in BOLD signals in the thalamus and reticular formation. Taken together, these data show that effective habituation to awake fMRI can be achieved by gradual and incremental acclimatization to the experimental conditions. Subsequent BOLD recordings, even during superimposed acoustic stimulation, reflect low stress-levels, low motion and a corresponding high-quality image acquisition. Furthermore, BOLD signals obtained during fMRI indicate that effective habituation facilitates selective attention to sensory stimuli that can in turn support the discrimination of cognitive processes in the absence of stress confounds.

Long-Tailed Pygmy Rice Rats Modify Their Behavioural Response and Faecal Corticosterone Metabolites in Response to Culpeo Fox but Not to Lesser Grison.

Simple SummaryPrey species must fine-tune their antipredator responses to survive, but also to achieve a positive net energy balance which will enhance biological fitness. Given this, we investigated if Oligoryzomys longicaudatus would adapt their behavioural and physiological antipredator responses depending on their relative presence in the predator’s diet. By exposing this rodent species to culpeo fox and lesser grison faeces, we found that O. longicaudatus behavioural and physiological strategies were modulated depending on the predator’s diet. Specifically, rodents would trigger their antipredator responses in the presence of the most dangerous predator, the culpeo, which consumes a higher proportion of O. longicaudatus compared to the lesser grison. Our findings could be of importance for the development of more humane and efficient strategies to control rodent populations.Even though behavioural and physiological reactions to predation risk exhibited by prey species have received considerable attention in scientific journals, there are still many questions still unsolved. Our aim was to broaden the knowledge on one specific question: do long-tailed pygmy rice rats adapt their behavioural and physiological antipredator strategies depending on the predator species? For this question, we live-trapped in a temperate forest in Southern Chile long-tailed pygmy rice rats (Oligoryzomys longicaudatus), which were exposed to three predator odour phases (Phase 0: preliminary, no predator cues; Phase 1: one plot with culpeo fox faeces (Lycalopex culpaeus), one plot with lesser grison (Galictis cuja) faeces and one plot acting as a control with no odour; Phase 2: post treatment, no predator cues). We measured the behavioural response by the capture ratio. To assess the physiological stress response, we collected fresh faecal samples to quantify faecal corticosterone metabolites (FCM). Our results showed that O. longicaudatus increased both the capture ratio and FCM levels in the presence of culpeo cues. Culpeo foxes have higher densities in the study area than G. cuja and exhibit a higher activity pattern overlap with O. longicaudatus. Moreover, it has been also been reported in other regions that L. culpaeus consumption of O. longicaudatus is more frequent compared to G. cuja diet. The increase in capturability could be because traps can be regarded as a shelter in high-risk settings, but it can also be explained by the predator inspection behaviour. The increase in FCM concentrations during culpeo treatment can be linked to the adaptive mobilisation of energy to execute antipredator responses to increase survival chances.

Changes in maternal fecal corticosterone metabolites across lactation and in response to chronic stress

Maternal exposure to stressors during lactation has previously been demonstrated to impact various aspects of milk synthesis and to have long-term physiological effects on offspring. Much of the current literature investigating the effects of stress during lactation has used acute stressors, and the studies investigating the effects of chronic stressors largely focus on neurological changes. Further, temporal variation in glucocorticoids across lactation in response to stressors has rarely been assessed. The present work uses a novel male intruder paradigm to model the effects of chronic stress on maternal fecal corticosterone metabolites (FCMs) in Sprague-Dawley rats across lactation. FCM levels were elevated in chronically-stressed mothers relative to the control group. Further, FCMs in the stress group were time-dependent either due to repeated exposure to the stressor or lactation stage. Together, this work demonstrates the efficacy of this established paradigm in increasing circulating glucocorticoids in lactating rats. These results highlight the need for repeated temporal sampling, as glucocorticoid levels in response to a chronic stressor may change across lactation.

Additional Assessment of Fecal Corticosterone Metabolites Improves Visual Rating in the Evaluation of Stress Responses of Laboratory Rats

Simple SummaryAssessment of animal welfare is an important aspect of preclinical studies to minimize suffering and burden and to improve scientific data. In a standard preclinical setup, such an assessment is normally done via so-called score sheets, which are part of the official documentation and approval of a preclinical study. These score sheets contain different categories, including objective parameters such as animals’ body weight, as well as more subjective criteria such as general status, behavior, and appearance, by which the animal is assessed and given a score reflecting the burden. However, very little is known about whether this mainly visual-based and subjective evaluation of the animals’ welfare reliably reflects the status of the animal and correlates well with more objective parameters used for assessment of animal welfare. To this end, the current study investigates the concordance of parameters obtained via standardized score sheets and fecal corticosterone metabolites in a preclinical neuroscientific setup. Determination of fecal corticosterone metabolites as response parameter of adrenocortical activity is thereby a well-validated parameter often used to determine animals’ stress levels. Our data reveal that specific but subjective scores did not mirror the stress response assessed via fecal corticosterone metabolites in the same animals.Since animal experiments cannot be completely avoided, the pain, suffering, and distress of laboratory animals must be minimized. To this end, a major prerequisite is reliable assessment of pain and distress. Usually, evaluation of animal welfare is done by visual inspection and score sheets. However, relatively little is known about whether standardized, but subjective, score sheets are able to reliably reflect the status of the animals. The current study aimed to compare visual assessment scores and changes in body weight with concentrations of fecal corticosterone metabolites (FCMs) in a neuroscientific experimental setup. Additionally, effects of refinement procedures were investigated. Eight male adult Sprague-Dawley rats underwent several experimental interventions, including electroencephalograph electrode implantation and subsequent recording, positron emission tomography (PET), and sleep deprivation (SD) by motorized activity wheels. Additional 16 rats were either used as controls without any treatment or to evaluate refinement strategies. Stress responses were determined on a daily basis by means of measuring FCMs, body weight, and evaluation of the animals’ welfare by standardized score sheets. Surgery provoked a significant elevation of FCM levels for up to five days. Increases in FCMs due to PET procedures or SD in activity wheels were also highly significant, while visual assessment scores did not indicate elevated stress levels and body weights remained constant. Visual assessment scores correlate with neither changes in body weight nor increases in FCM levels. Habituation procedures to activity wheels used for SD had no impact on corticosterone release. Our results revealed that actual score sheets for visual assessment of animal welfare did not mirror physiological stress responses assessed by FCM measurements. Moreover, small changes in body weight did not correlate with FCM concentration either. In conclusion, as visual assessment is a method allowing immediate interventions on suffering animals to alleviate burden, timely stress assessment in experimental rodents via score sheets should be ideally complemented by validated objective measures (e.g., fecal FCM measured by well-established assays for reliable detection of FCMs). This will complete a comprehensive appraisal of the animals’ welfare status in a retrospective manner and refine stressor procedures in the long run.

Non-Invasive Monitoring of Adrenocortical Activity in Three Sympatric Desert Gerbil Species.

Simple SummaryIn this era, characterized by remarkable anthropogenic impacts on wildlife, it is crucial to monitor the health of wild animal populations while minimizing the interference to them. To this end, for a better understanding of the eco-physiology of wild animals, the adrenocortical activity can be non-invasively evaluated by measuring glucocorticoid metabolites excreted in feces. However, to ensure that the endocrine information is reliable, the experimental assays should be first validated and the causes for the major variability among individuals should be considered. Here we validated an enzyme immunoassay for measuring fecal corticosterone metabolites (FCM) in three wild gerbil species and emphasized the differences among them. These are endangered species, which play a key role in psammophilic communities, and provide a model system for various aspects in ecology. Thus, this work constitutes the first step toward using the FCMs of these species as indicators for individual and community stress.The study of the endocrine status can be useful to understand wildlife responses to the changing environment. Here, we validated an enzyme immunoassay (EIA) to non-invasively monitor adrenocortical activity by measuring fecal corticosterone metabolites (FCM) in three sympatric gerbil species (Gerbillus andersoni, G. gerbillus and G. pyramidum) from the Northwestern Negev Desert’s sands (Israel). Animals included into treatment groups were injected with adrenocorticotropic hormone (ACTH) to stimulate adrenocortical activity, while control groups received a saline solution. Feces were collected at different intervals and FCM were quantified by an EIA. Basal FCM levels were similar in the three species. The ACTH effect was evidenced, but the time of FCM peak concentrations appearance differed between the species (6–24 h post-injection). Furthermore, FCM peak values were observed sooner in G. andersoni females than in males (6 h and 18 h post-injection, respectively). G. andersoni and G. gerbillus males in control groups also increased FCM levels (18 h and 48 h post-injection, respectively). Despite the small sample sizes, our results confirmed the EIA suitability for analyzing FCM in these species as a reliable indicator of the adrenocortical activity. This study also revealed that close species, and individuals within a species, can respond differently to the same stressor.

A complex regulating pattern induced by the effects of predation and parasites on root vole (Microtus oeconomus) populations during the breeding season

Abstract Predators and parasites have synergistic effects on the phenotype of the species they share as prey and host. Experimental studies that incorporate the interactions between predation and parasitism are, however, scarce in small-mammal populations. Our previous work has shown that the combined effects of predation and coccidian infection reduce overwinter survival and population density in root voles (Microtus oeconomus). Here, we examined the separate and combined effects of these two drivers on the population growth of root voles during the breeding season. We carried out a two-level factorial experiment, in which we manipulated predator exclusion and the removal of parasites in enclosures and measured survival, fecal corticosterone metabolite (FCM) concentration, recruitment, and population density. An expected synergistic effect of predators and parasites on vole population was not found, due to no effect of parasites on FCM level and recruitment rate during the period of the experiment. Instead, we found phase-related effects of predation on demography. Predation reduced the survival rate of voles in spring, which was intensified by parasite infection. Predation risk reduced recruitment rate in early summer by elevating FCM levels. Consequently, both direct and indirect effects of predation lowered population density during the experimental period. In addition, for populations free of predators, the peak density that occurred in early autumn elevated FCM level of adult voles, which reduced recruitment rates and halted population growth. Moreover, predation, parasites, and density affected the quality of the offspring. Our study suggests that multiple regulation processes influence population fluctuations during the breeding season. We conclude that a population experiencing stress acts as a common interface through which interactions between intrinsic and extrinsic factors can be important determinants of fluctuations. We propose a new hypothesis of integrative stress effects to explain small-mammal population fluctuations.

Validation of a noninvasive technique to quantify stress in northern bobwhite (Colinus virginianus).

Examination of the endocrine system through non-invasive fecal sampling may improve population management more than using demographic indicators alone. By addressing the physiological mechanisms that are influencing fitness, management actions can be proactively developed to alleviate stressors. Proactive determination of vulnerable populations is critical for species of concern, such as the Northern Bobwhite (Colinus virginianus), which have suffered decades of population decline. We validated an assay to noninvasively measure the adrenocortical response of captive reared bobwhite through fecal corticosterone metabolites (FCM). All individuals received three sequential 48-hour treatments in which samples were collected every 4 hours, including a reference period, an adrenocorticotropic hormone (ACTH) challenge and a biological stressor (exposure to a hunting dog). Reference FCM values had a mean concentration of 16.75 pg/mg (95% CrI: 13.68, 19.91) with adrenocortical activity increasing by 73% for the duration of the ACTH challenge (29.00 pg/mg; CrI: 25.01, 33.78). FCM concentrations remained similar to that of the reference levels during the biological stressor (16.56 pg/mg; CrI: 13.33, 19.92). Our study validates the use of feces to detect changes in FCM levels in our subject species but also demonstrates the complexity of FCM and the importance of both physiological and biological validation prior to field implementation.

When food access is challenging: evidence of wood mice ability to balance energy budget under predation risk and physiological stress reactions

Prey species must balance the energetic cost of locomotion, foraging effort, and predation risk. In our work, we assessed how the wood mice (Apodemus sylvaticus) balance these costs by manipulating food access difficulty and predation risk. Live trapping was conducted in Madrid (Spain) where 80 Sherman traps were set in four different plots. To assess how wood mice manage their energy budget, all traps were first subjected to a control period followed by four treatments of 3-day duration in which food access was experimentally manipulated (free access, straw balls, straw balls wrapped in metal wire, opened plastic bottles, closed bottles). Predation risk was simulated by exposing half of the traps to fox feces. To quantify food intake, we weighed the remnants of food left by each captured mouse. Furthermore, we collected mouse fecal samples from traps to evaluate the physiological stress response by quantifying fecal corticosterone metabolites (FCM). Results showed that despite mice generally avoiding traps treated with fox feces, predation risk did not modulate food intake or FCM levels. By contrast, the experimental manipulation of food access determined the amount eaten and increased FCM levels, probably owing to the different degrees of difficulty and the energy required to obtain the food. Moreover, recaptured individuals ate more, indicating that experience critically determines mice ability to reduce the costs of accessing food. By analyzing the joint variation between mice intake and FCM levels depending on food restriction treatments, we found that mice were able to modulate their energetic expenditure depending on food access difficulty probably in order to achieve a positive marginal value of energy. For wild animals, to optimally balance the energetic budget is critical to maximize fitness. However, foraging decisions are influenced by numerous internal and external factors which are poorly understood. We studied free-ranging mice behavioral and physiological stress response to different food restriction treatments, testing also the influence of predation risk and experience. We found that predation risk modulated mice capturability but not the feeding behavior or the stress response. We also discovered that mice seem to be able to adjust energy allocation when a novel resource appear, and that energy budget optimization may be mediated via glucocorticoid release and individuals experience.

Effect of Ground Transportation on Adrenocortical Activity in Prepuberal Female Mice from Five Different Genetic Backgrounds.

Simple SummaryFor research purposes, mice are often transported between institutions, which may elicit stress, thereby influencing results. We determined adrenocortical activity by measuring fecal corticosterone metabolites (FCMs), as a stress marker, in prepuberal mice from five genetic backgrounds, namely C57BL/6J, C57BL/6NCrl, FVB/NCrl, Crl:CD1(ICR), and BALB/cAnCrl. Only C57BL/6N showed significantly higher FCM levels the day after transport, but baseline levels were attained within four days.Specific experimental protocols necessitate transportation, a potentially stressful event that could confound results. We determined adrenocortical activity by measuring fecal corticosterone metabolites (FCMs), as a stress marker, in prepuberal (three-week old) female C57BL/6J, C57BL/6NCrl, FVB/NCrl, Crl:CD1(ICR), and BALB/cAnCrl mice. On each transport day, five female cage mates per genetic background were weaned and transported in stable groups via truck from the breeding to the research facility. Fecal pellets were collected on Days 0, 1, and 4. Mice were superovulated for embryo production to determine if repeated fecal collection impacts this procedure. The average duration of transportation over 600 km and from packing to unpacking of mice was 7.24 and 22.62 h, respectively. FCM levels increased from Day 0 to Day 1 and decreased on Day 4 in all genetic backgrounds except in FVB/NCrl, but only B6N showed significantly higher FCM levels on Day 1. Furthermore, embryo production was not affected by repeated feces collection. The results show that weaning and immediate transport of prepuberal mice from the breeding to the research facility led to temporal and genetic background-dependent increases of adrenocortical activity in four of the five genetic backgrounds investigated, which returned to baseline levels within four days.

Limited effects of early life manipulations on sex-specific gene expression and behavior in adulthood

Exposure to childhood adversity is associated with increased vulnerability to stress-related disorders in adulthood which has been replicated in rodent stress models, whereas environmental enrichment has been suggested to have beneficial effects. However, the exact neurobiological mechanisms underlying these environment influences on adult brain and behavior are not well understood. Therefore, we investigated the long-term effects of maternal separation (MS) or environmental enrichment (EE) in male and female CD1 mice. We found clear sex-specific effects, but limited influence of environmental manipulations, on adult behavior, fecal corticosterone metabolite (FCM) levels and stress- and plasticity related gene expression in discrete brain regions. In detail, adult females displayed higher locomotor activity and FCM levels compared to males and EE resulted in attenuation in both measures, but only in females. There were no sex- or postnatal manipulation-dependent differences in anxiety-related behaviors in either sex. Gene expression analyses revealed that adult males showed higher Fkbp5 mRNA levels in hippocampus, hypothalamus and raphe nuclei, and higher hippocampal Nos1 levels. Interestingly, MS elevated Nos1 levels in hippocampus but reduced Fkbp5 expression in hypothalamus of males. Finally, we also found higher Maoa expression in the hypothalamus of adult females, however no differences were observed in the expression levels of Bdnf, Crhr1, Nr3c1 and Htr1a. Our findings further contribute to sex-dependent differences in behavior, corticosterone and gene expression and reveal that the effects of postnatal manipulations on these parameters in outbred CD1 mice are limited.

Assessing space use in meadow voles: the relationship to reproduction and the stress axis

Abstract Voles are key mammals in understanding how social interactions can affect large-scale population processes. Previous studies have shown that at high population densities, meadow voles (Microtus pennsylvanicus) have a lower proportion of breeding animals, higher average corticosterone levels, and can be limited by female territorial spacing. Based on this, we compared corticosterone levels and spatial use between breeding and nonbreeding free-ranging adult meadow voles within populations. We measured intrasexual spatial overlap to examine if breeding females minimize occupying the same areas as other females, and noninvasively assessed corticosterone levels using fecal corticosterone metabolites (FCMs). We found that female meadow voles have much lower intrasexual spatial overlap than males, even though both sexes have similar range sizes, and that females have generally higher FCM levels than males. However, breeding and nonbreeding females did not differ from one another in spatial use or in FCM levels. Conversely, reproductive classes of males differed greatly in all measures: nonbreeding males had FCM levels that were two times higher than those of breeding males, occupied a smaller range, and had lower spatial overlap, indicating they were moving less widely than breeding males. We additionally validated an enzyme immunoassay for noninvasively measuring FCMs in meadow voles. The assay was successful in detecting an increase in corticosterone stimulated by adrenocorticotropic hormone injection; however, dexamethasone did not induce negative feedback. FCMs reflect circulating corticosterone levels approximately 5 h prior. These results highlight differences in FCMs and spacing in meadow voles related to sex and reproductive status, and reflect the respective strategies males and females employ during the breeding season.