Fecal Corticosterone Metabolites Research Articles

Stress and Predation Impacts on North American Quail Translocations

Translocations have been used in attempts to bolster or restore native quail populations for >150 years, often with little success. However, with some northeastern United States quail populations undetectable or extirpated, and others across the United States on the extreme decline, translocation as a tool for quail population restoration is becoming increasingly popular. Two factors contributing to translocation failure are physiological stress and predation. Chronic stress associated with translocations can result in weight loss, reduced immune system function, suppressed reproduction, and an altered fight-or-flight response. These stress-induced responses increase vulnerability to predation, the primary cause of quail mortality. Here, we review the relationship between quail translocations, stress, and predation, and recommend future research and best practices to mitigate the impacts of stress and predation on translocated quail. To improve future translocation outcomes, more research is needed on stress mitigation throughout the translocation process (capture, handling, transport, and release). While capture and handling are unavoidably stressful, there is greater potential to reduce stress levels during holding and transport. Recent validation of fecal corticosterone metabolites as a non-invasive method to quantify stress in quail offers a useful tool for testing stress reduction protocols. Preliminary experimental results regarding nutritional supplements and stress levels are inconclusive, but enrichment during temporary holding and access to travel rations may help improve survival in long-distance (>800 km) translocations. We also recommend predator control at release sites, particularly for raccoons (Procyon lotor) and other mesomammals.

Corticosterone Metabolite Concentration Is Not Related to Problem Solving in the Fawn-Footed Mosaic-Tailed Rat Melomys Cervinipes.

Animals can respond physiologically, such as by adjusting glucocorticoid hormone concentrations, to sudden environmental challenges. These physiological changes can then affect behavioural and cognitive responses. While the relationships between adrenocortical activity and behaviour and cognition are well documented, results are equivocal, suggesting species-specific responses. We investigated whether adrenocortical activity, measured using corticosterone metabolite concentration, was related to problem solving in an Australian rodent, the fawn-footed mosaic-tailed rat (Melomys cervinipes). Mosaic-tailed rats live in complex environments that are prone to disturbance, suggesting a potential need to solve novel problems, and have been found to show relationships between physiology and other behaviours. We measured problem solving using five food-baited puzzles (matchbox and cylinder in the home cage, and activity board with pillars to push, tiles to slide and levers to lift in an open field), and an escape-motivated obstruction task in a light/dark box. Faecal samples were collected from individuals during routine cage cleaning. Adrenocortical activity was evaluated non-invasively by measuring faecal corticosterone metabolites using an enzyme immunoassay, which was biochemically and biologically validated. Despite varying over time, adrenocortical activity was not significantly related to problem solving success or time spent interacting for any task. However, as adrenocortical activity is reflective of multiple physiological processes, including stress and metabolism, future studies should consider how other measures of physiology are also linked to problem solving.

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.

CRF-R1 Antagonist Treatment Exacerbates Circadian Corticosterone Secretion under Chronic Stress, but Preserves HPA Feedback Sensitivity.

Despite promising initial reports, corticotropin-releasing factor receptor type-1 (CRF-R1) antagonists have mostly failed to display efficacy in clinical trials for anxiety or depression. Rather than broad-spectrum antidepressant/anxiolytic-like drugs, they may represent an ‘antistress’ solution for single stressful situations or for patients with chronic stress conditions. However, the impact of prolonged CRF-R1 antagonist treatments on the hypothalamic–pituitary–adrenal (HPA) axis under chronic stress conditions remained to be characterized. Hence, our study investigated whether a chronic CRF-R1 antagonist (crinecerfont, formerly known as SSR125543, 20 mg·kg−1·day−1 ip, 5 weeks) would alter HPA axis basal circadian activity and negative feedback sensitivity in mice exposed to either control or chronic stress conditions (unpredictable chronic mild stress, UCMS, 7 weeks), through measures of fecal corticosterone metabolites, plasma corticosterone, and dexamethasone suppression test. Despite preserving HPA axis parameters in control non-stressed mice, the 5-week crinercerfont treatment improved the negative feedback sensitivity in chronically stressed mice, but paradoxically exacerbated their basal corticosterone secretion nearly all along the circadian cycle. The capacity of chronic CRF-R1 antagonists to improve the HPA negative feedback in UCMS argues in favor of a potential therapeutic benefit against stress-related conditions. However, the treatment-related overactivation of HPA circadian activity in UCMS raise questions about possible physiological outcomes with long-standing treatments under ongoing chronic stress.

Analysis of Pain and Analgesia Protocols in Acute Cerulein-Induced Pancreatitis in Male C57BL/6 Mice.

Pancreatitis is known to be painful in humans and companion animals. However, the extent of pain in experimental mouse models of acute pancreatitis is unknown. Consequently, the severity classification of acute pancreatitis in mice is controversially discussed and standardized pain management is missing. In this study, we investigated acute Cerulein-induced pancreatitis with pain-specific and well-being orientated parameters to detect its impact on mice. Male C57BL/6J male mice were injected with Cerulein; animals that received saline injections served as control group. The animals were observed for weight change and water intake. To assess pain, behaviors like stretch-and-press and reduced rearing, the Mouse Grimace Scale, and von Frey hypersensitivity were assessed. Fecal corticosterone metabolites and burrowing behavior were assessed to detect changes in the animal’s well-being. Pancreatitis severity was evaluated with amylase and lipase in the blood and pancreas histology. To investigate whether different analgesics can alleviate signs of pain, and if they influence pancreas inflammation, animals received Buprenorphine, Paracetamol in combination with Tramadol, or Metamizole in the drinking water. The calculated intake of these analgesics via drinking reached values stated to be efficient for pain alleviation. While pancreatitis did not seem to be painful, we detected acute pain from Cerulein injections that could not be alleviated by analgesics. The number of inflammatory cells in the pancreas did not differ with the analgesic administered. In conclusion: (1) Cerulein injections appear to be acutely painful but pain could not be alleviated by the tested analgesics, (2) acute pancreatitis induced by our protocol did not induce obvious signs of pain, (3) analgesic substances had no detectable influence on inflammation. Nevertheless, protocols inducing more severe or even chronic pancreatitis might evoke more pain and analgesic treatment might become imperative. Considering our results, we recommend the use of Buprenorphine via drinking water in these protocols. Further studies to search for efficient analgesics that can alleviate the acute pain induced by Cerulein injections are needed.

Distress Analysis of Mice with Cervical Arteriovenous Fistulas.

Simple SummaryFunctional hemodialysis access is essential for the survival of patients with end-stage renal disease. Although various guidelines recommend autologous arteriovenous fistula as the first choice for hemodialysis, it is still the Achilles heel for patients. Several in vivo models have been used to study and improve the mechanisms of vascular remodeling of arteriovenous fistula. However, some models have the disadvantage of having anatomical features or a hemodynamic profile different from that of the arteriovenous fistula in humans. In the presented cervical arteriovenous fistula model, these disadvantages were eliminated. It resembles the human physiology and is an ideal animal model for arteriovenous fistula research. Moreover, in order to understand the impact of this model on animal welfare, the distress of this new animal model was analyzed. Body weight, faecal corticosterone metabolites, burrowing activity, nesting behaviour and distress scores were analysed after fistula creation and during the following three weeks. The physiological, behavioural, and neuroendocrine assessments all indicated that this model causes only moderate distress to the animals. This not only meets the need for animal ethics but also improves the quality of scientific research. Therefore, this cervical model is suitable for arteriovenous fistula research and should be used more frequently in the future.The welfare of laboratory animals is a consistent concern for researchers. Its evaluation not only fosters ethical responsibility and addresses legal requirements, but also provides a solid basis for a high quality of research. Recently, a new cervical arteriovenous model was created in mice to understand the pathophysiology of arteriovenous fistula, which is the most commonly used access for hemodialysis. This study evaluates the distress caused by this new animal model. Ten male C57B6/J mice with cervical arteriovenous fistula were observed for 21 days. Non-invasive parameters, such as body weight, faecal corticosterone metabolites, burrowing activity, nesting activity and distress scores were evaluated at each time point. Six out of ten created arteriovenous fistula matured within the observation time as defined by an increased diameter. The body weight of all animals was reduced after surgery but recovered within five days. In addition, the distress score was significantly increased during the early time point but not at the late time point after arteriovenous fistula creation. Neither burrowing activity nor nesting behaviour were significantly reduced after surgical intervention. Moreover, faecal corticosterone metabolite concentrations did not significantly increase. Therefore, the cervical murine arteriovenous fistula model induced moderate distress in mice and revealed an appropriate maturation rate of the fistulas.

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.

Assessing the severity of laparotomy and partial hepatectomy in male rats-A multimodal approach.

This study assessed the postoperative severity after three different visceral surgical interventions in rats by using objective parameters pertaining to various disciplines. The objective was to evaluate whether the degree of severity increases with the invasiveness of the intervention and whether this is in accordance with the EU Directive 2010/63. 136 adult male WistarHan rats were assigned to three groups: Sham-laparotomy (Sham) [7 days post-surgical survival time]; 50% partial hepatectomy (PH); 70% PH [PH groups with 1, 3, or 7 days post-surgical survival times]. Post-surgical severity assessment was performed via several multimodal assessment tools: I) model-specific score sheet focusing on body weight, general condition, spontaneous behavior, and the animals' willingness to move as well as on wound healing; II) Open Field tests evaluating the total distance and velocity an animal moved within 10 minutes and its rearing behavior during the test; III) telemetric data analyzing heart rate and blood pressure; and IV) analysis of blood (AST, ALT, and hemogram) and fecal samples (fecal corticosterone metabolites). Significant differences among the experimental groups and models were observed. We demonstrated that the Open Field test can detect significant changes in severity levels. Sham-laparotomy and removal of 50% of the liver mass were associated with comparable severity (mild-moderate); the severity parameters returned to baseline levels within seven days. Removal of 70% of the liver tissue seemed to be associated with a moderate severity grade and entailed a longer recovery period (>7 days) for complete regeneration. We recommend the use of Open Field tests as part of multimodal objective severity assessment.

Diagnostic Ability of Methods Depicting Distress of Tumor-Bearing Mice.

Simple SummaryExperiments on animals can provide important information for improving the life expectancy and life quality of patients. At the same time, the welfare of these animals is a growing public concern. Therefore, many laws and international guidelines were established with the goal of minimizing the harm inflicted on these animals. A prerequisite of improving animal welfare is to correctly measure how much distress the experiments cause to these animals. However, it is often unknown as to which methods are appropriate to assess distress. Mice bearing subcutaneous tumors are the most frequently used animal model to study the therapeutic effects of drugs. We evaluated if body weight, faecal corticosterone metabolites concentration, burrowing activity and a distress score were capable of differentiating between mice before cancer cell injection and mice bearing large tumors. We observed that only adjusted body weight change and faecal corticosterone metabolites concentration were capable of measuring distress caused by large subcutaneous tumors. Therefore, these two methods are appropriate to assess the welfare of mice with subcutaneous tumors. This knowledge provides a solid basis to optimize animal welfare in future studies. For example, both methods can define the ideal time point when an experiment should end by finding a good compromise between minimal distress for the animals and maximal knowledge gain for mankind.Subcutaneous tumor models in mice are the most commonly used experimental animal models in cancer research. To improve animal welfare and the quality of scientific studies, the distress of experimental animals needs to be minimized. For this purpose, one must assess the diagnostic ability of readout parameters to evaluate distress. In this study, we evaluated different noninvasive readout parameters such as body weight change, adjusted body weight change, faecal corticosterone metabolites concentration, burrowing activity and a distress score by utilising receiver operating characteristic curves. Eighteen immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used for this study; half were subcutaneously injected with A-375 cells (human malignant melanoma cells) that resulted in large tumors. The remaining mice were inoculated with SCL-2 cells (cutaneous squamous cell carcinoma cells), which resulted in small tumors. The adjusted body weight and faecal corticosterone metabolites concentration had a high diagnostic ability in distinguishing between mice before cancer cell injection and mice bearing large tumors. All other readout parameters had a low diagnostic ability. These results suggest that adjusted body weight and faecal corticosterone metabolites are useful to depict the distress of mice bearing large subcutaneous tumors.

Comparação da cicatrização experimental de feridas em codornas usando dois tipos de curativos hemostáticos comerciais

The use of powdered hemostatic products and propolis-based preparations has become increasingly common to control bleeding and aid healing. This study evaluated two hemostatic dressings, one based on propolis (Alveolex®) and the other on ferric subsulfate powder (Quick Blood Stopper®), on experimental wound in quails. The experiment was conducted with 36 quails assigned to three groups: control, propolis-based hemostatic dressing and ferric subsulfate-based hemostatic dressing, on two types of wounds, superficial (skin and subcutaneous) and deep (skin, subcutaneous and muscle). Pre- (day-7) and post-procedure (days 2 and 10) examinations showed a tendency to reduce hematocrit values on day 2 and a tendency to increase fibrinogen levels and total leukocyte count on the same day. Visual assessment of wound healing indicated that birds using Quick Blood Stopper® showed signs of poor healing, while the group treated with Alveolex® had uncomplicated healing within the expected time. The groups treated with hemostatic based on ferric subsulfate showed a tendency to increase the values of fecal corticosterone metabolites on day 10, suggesting an increased level of stress in these animals. During the surgical procedure, both hemostatic products controlled bleeding, but Alveolex®was more efficient and easy to handle.

Heterospecific competitors and seasonality can affect host physiology and behavior: key factors in disease transmission

AbstractEcological and environmental factors can influence the transmission of infectious diseases. They can accomplish this via effects on host susceptibility and exposure to infection, which are governed by host physiology and behavior, respectively. To better inform disease control, more information is needed about how extrinsic factors affect physiological and behavioral processes that determine transmission. We investigated how heterospecific competitors and seasonality may influence host susceptibility and intraspecific contact rates using a directly transmitted disease system, the North American deer mouse (Peromyscus maniculatus)—Sin Nombre hantavirus (SNV) system. In grasslands of western Montana, USA, deer mice compete with dominant voles (Microtus spp.) and shrews (Sorex spp.) and experience a seasonal temperate climate. Higher SNV transmission occurs primarily during spring/summer, when changes in physiology and behavior may serve as influential contributors. We hypothesized that (1) voles, and to a lesser extent shrews, will induce chronic stress, suppress immunity, and may change contact rates of deer mice; and (2) during spring/summer, deer mice may experience chronic stress, suppressed immunity, and higher contact rates, which may help explain the reported seasonality in SNV transmission. Over two years, we trapped small mammals at four grids in western Montana. Deer mice were sampled for feces and blood and evaluated for scar numbers, demography, and body condition scores (BCSs). We evaluated stress physiology with fecal corticosterone metabolites (FCMs), neutrophil/lymphocyte (N/L) ratios and BCSs, immunity with white blood cell (WBC) counts, and contact rates with scar numbers. We found that shrew density was negatively associated with stress response FCMs, suggestive of chronic stress. Additionally, although complex interactions existed, shrew and vole densities were negatively associated with BCSs, but differentially with scar numbers. N/L ratios were higher in spring/summer, whereas WBC counts were lower in summer, suggestive of chronic stress and suppressed immunity, respectively. Our results suggest that (1) heterospecific competitors may differentially influence disease transmission via stress physiology and contact rates, and that (2) chronic stress, suppressed immunity, and higher contact rates may help explain why higher SNV transmission has been previously reported during spring/summer in Montana. Our findings may extend to other directly transmitted disease systems.

An exploratory investigation of glucocorticoids, personality and survival rates in wild and rehabilitated hedgehogs (Erinaceus europaeus) in Denmark

BackgroundThe European population of hedgehogs (Erinaceus europaeus) is declining. It is therefore essential to optimise conservation initiatives such as the rehabilitation of sick, injured and orphaned hedgehogs. Wild animals placed in captivity may be prone to chronic stress, potentially causing negative health effects. Therefore, the effects of these rehabilitation efforts should consequently be evaluated. Furthermore, hand-raising orphaned hedgehogs is a laborious and costly task, and it is therefore relevant to document whether they have equal post release survival rates compared to their wild conspecifics.The objectives of this research were therefore to conduct an exploratory study of glucocorticoid levels in hedgehogs from different backgrounds and compare the post release survival of translocated, rehabilitated and wild, juvenile hedgehogs as well as the possible effect on survival of differences in shy or bold behaviour (personality) exhibited by individuals.ResultsWe measured glucocorticoid levels in 43 wild-caught (n = 18) and rehabilitated (n = 25) hedgehogs and compared the post release survival and spatial behaviour of 18 translocated juvenile hedgehogs (eight hand-raised and ten wild) until hibernation. The possible effect on survival of differences in shy or bold behaviour (personality) exhibited by 17 juvenile individuals (seven hand-raised and ten wild) was also examined.Rehabilitated individuals and females had higher levels of faecal corticosterone metabolites compared to wild individuals and males, respectively. Rehabilitated individuals showed higher levels of saliva corticosterone than wild. The personality tests labelled 13 individuals as shy and 11 as bold. Post release survival was 57% for rehabilitated and 50% for wild individuals. Neither background nor personality affected post release survival. Home range measures were 3.54 and 4.85 ha. Mean dispersal length from the release sites was 217 ± 100 m.ConclusionThe higher levels of corticosterone observed in rehabilitated compared to wild hedgehogs calls for consideration of the duration of admission to wildlife rehabilitation centres to reduce stress levels in the patients.Hand-raised juveniles appear to have the same prospects as wild, and personality does not seem to affect post release survival in hedgehogs, indicating that hand-raising of orphaned juvenile hedgehogs is a relevant contribution to the conservation of this species.

Routine handling does not lead to chronic stress in captive green anole (Anolis carolinensis)

AbstractRoutine handling has been shown to affect stress levels in a variety of animal species. This could result in a general decrease in welfare and may confound the results of scientific experiments or observations on captive study animals. In reptiles, there seems to be variation in the effects of handling on stress levels. Furthermore, most studies on reptiles only look at the effect of handling in the short term. In this study we quantified the physiological and behavioural impact of being held, twice daily, for 1 min at a time over a three-week period on the green anole (Anolis carolinensis). Measurements were collected at the end of the three-week repeated handling period. Our results showed no effect of repeated handling on body mass, tail-base width, heterophil to lymphocyte ratios (H/L ratios), behaviour and faecal corticosterone metabolite (FCM) levels for both males and females in the experimental treatments (‘handled’, ‘unhandled’). Our study animals did score very highly for several stress-indicating variables in the three weeks preceding the experiments — suggesting that they had experienced considerable stress during capture, transport and temporary housing in the pet store.

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.

Soil pollution by heavy metals correlates with levels of faecal glucocorticoid metabolites of a fossorial amphisbaenian reptile.

Soil degradation may have strong negative consequences for soil biodiversity, but these potential effects are understudied and poorly understood. Concentration of nesting seabirds may be a source of soil pollution by heavy metals, which are incorporated into the food chain and may have toxicological effects in vertebrates, especially in fossorial animals with low dispersal ability. We examined whether contamination by heavy metals, derived from seagull depositions, and other soil characteristics, may affect the levels of faecal glucocorticoid metabolites (as a potential indicator of physiological stress) of the fossorial amphisbaenian reptile Trogonophis wiegmanni. We found a relationship between soil pollution by heavy metals and increased levels of faecal corticosterone metabolite of the amphisbaenians that live buried in those soils. This can be due to the strong endocrine disruption effect of heavy metals. In addition, there was an independent effect of the soil texture, with amphisbaenians showing higher levels of faecal corticosterone metabolite in soils with less sand and more silt and clay, which are more energetically costly to dig. Long-term exposure to high glucocorticoid levels might have serious effects on health state and fitness of fossorial animals that may be unnoticed. Our study emphasizes that, to prevent future conservation problems, we need to perform periodic surveys on the physiological health state of the little-known subterranean biodiversity.

Regular touchscreen training affects faecal corticosterone metabolites and anxiety-like behaviour in mice

Automated touchscreen techniques find increasing application for the assessment of cognitive function in rodents. However, hardly anything is known about the potential impact of touchscreen-based training and testing procedures on the animals under investigation. Addressing this question appears particularly important in light of the long and intensive training phases required for most of the operant tasks. Against this background, we here investigated the influence of regular touchscreen training on hormones and behaviour of mice. Faecal corticosterone metabolites (FCMs), reflecting corticosterone levels around the time of treatment, were significantly increased in touchscreen-trained mice, even one week after the training phase was already terminated. Such an effect was not detected on baseline FCMs. Thus, regular touchscreen training can be assumed to cause long-term effects on hypothalamus-pituitary-adrenal axis activity. Furthermore, anxiety-like behaviour was increased in touchscreen-trained mice two weeks after the end of the training phase. Traditionally, this would be interpreted as a negative influence of the training procedure on the animals’ affective state. Yet, we also provide two alternative explanations, taking the possibility into account that touchscreen training might have enriching properties.

Establishing models of corticosteroid patterns during the life history of killer whales (Orcinus orca) under human care

The use of corticosteroids and their metabolites as a physiologic measure of stress in wildlife species is increasing in both in and ex situ populations. However, factors such as season, circadian rhythm, sex and age are also known to influence corticosteroid production in wildlife. Our objective was to evaluate the influence of these variables on serum cortisol, corticosterone, aldosterone and their fecal metabolites in zoo-based killer whales (Orcinus orca). For evaluation of season, sex and age, we examined 30 animals (21 females, 9 males), and for circadian rhythm, we studied 18 animals (10 females, 8 males). Season did not influence corticosteroids (P ≥ 0.19). Circadian rhythm influenced all corticosteroids (P ≤ 0.012). Serum cortisol and corticosterone were highest in the morning and lower by mid-day and evening. Serum aldosterone was lowest during mid-day and highest in the evening. Excreted corticosteroid metabolites were higher in the morning and mid-day compared to evening (P ≤ 0.002). Serum cortisol was higher in males than females (P = 0.011). The inverse was observed for fecal corticosterone metabolites (P = 0.03). Serum corticosterone increased with age in all animals (P = 0.002), but serum cortisol increased with age in males only (P < 0.001). Finally, combining previously published data and these data reported herein, we developed models of corticosteroid patterns for zoo-based killer whales during life history events, including circadian variation, pregnancy and acute stress that may serve as a guide for evaluating stress physiology and animal welfare in this species.

Not all mice are alike: Mixed-strain housing alters social behaviour

The use of millions of mice in scientific studies worldwide emphasises the continuing need for a reduction of sample sizes, however, not at the expense of scientific validity. Split-plot designs have been suggested to enhance statistical power while allowing a reduction of animal numbers in comparison to traditional experimental designs. Recently, a promising approach of a split-plot design has been implemented and proven useful using mixed-strain housing of at least three different mouse strains. However, the impact of co-housing different strains of mice in one cage on animal welfare has still to be defined. This study aimed at comparing the effects of mixed-strain and same-strain housing of female C57BL/6J and DBA/2N mice on welfare and behaviour in two experimental phases. In a first phase, mice were housed in either mixed- or same-strain pairs. Home cage behaviour, activity rhythm, body weight, and faecal corticosterone metabolites were assessed. Furthermore, tests for anxiety-like and exploratory behaviour as well as spatial learning were performed. In a second phase, sociability was investigated in newly formed mixed-strain quartets. Mixed-strain housing did not induce alterations in anxiety, locomotion, learning, stereotypic behaviour, and stress hormone levels. However, changes in social behaviours and activity rhythm were observed. Increased agonistic and decreased socio-positive behaviours might point towards mild impacts on welfare in C57BL/6J mice under co-housing conditions. Altogether, scientific research may greatly benefit from co-housing mice of different strains within the same cages (e.g. for the realisation of a split-plot design), provided that strains are carefully selected for compatibility.