Fecal Corticosterone Research Articles

Exposure to soiled bedding reduces abnormal repetitive behaviors in mice.

Hygiene management protocols in laboratory mouse husbandries worldwide most commonly employ soiled bedding-exposed sentinel mice to monitor the occurrence of infections in mouse colonies. Using this approach, sentinel mice repeatedly receive a mixture of used bedding, supplied by a variety of cages of a defined hygienic unit for a period of several months. Hereby, microorganisms shed in the used bedding can infect the sentinel animals and can be detected in subsequent health monitoring procedures. However, murine excrements carry more than only microorganisms. Mouse feces and urine also contain a multitude of olfactory molecules, which the animals use to code information about social status and context. However, if and how the persistent and repeated experience with these odor cues affects the behavior of sentinel mice, has not yet been explored. To address this question, we conducted a longitudinal study for neurochemical output parameters related to an organism's responsiveness to challenging conditions, and for the exploratory assessment of a panel of home cage behaviors in soiled bedding and control female C57BL/6J mice. We found that the number of mice showing abnormal repetitive behaviors, including barbering and bar mouthing, was lower in the soiled bedding group. While neutrophil/lymphocyte ratios and fecal corticosterone metabolites did not differ between groups, the within-group variance of the neutrophil/lymphocyte ratio was reduced in the soiled bedding group. These results show that the occurrence of abnormal repetitive behaviors is lower in sentinel than in control mice and suggest a beneficial effect of soiled bedding on the welfare of laboratory mice and on outcome variability.

The effects of transport of 18-day old hatching eggs on physiology and behaviour of slow growing broiler chicken

Incubation and hatching commonly takes places at hatcheries, separate from the grow-out facilities where broiler chicks are raised. This means that chicks are sorted and transported immediately after hatch, during which time they typically do not have access to feed and water, and are subjected to transport stress. Recently, innovative housing systems are being developed in which fertilised eggs are transported on embryonic day 18 (E18) from the hatchery to the grow-out facility, where they hatch on day 21. In chicken, the hypothalamic–pituitary–adrenal (HPA)-axis becomes functional around embryonic day 14–16. It is therefore conceivable that transport of eggs at E18 may lead to a stress response in the chick embryo. Exposure to prenatal stress may affect the coping capacity of the individual and negatively impact its further development. We investigated whether prolonged transport on E18 has effects on the development of a slow growing broiler chicken strain (Hubbard JA257). E18 eggs were transported for either 41 min (short transport, ST) or 219 min (long transport, LT). Transportation significantly increased embryonic heart rate after ST. This increase continued during an intermediate measure at 120 min. The increased embryonic HR then remained high at measurement immediately following LT. We did not find effects of prolonged transport on behavioural parameters measured in the juvenile chicken in the tonic immobility and open field test. Concentrations of feather corticosterone as well as faecal corticosterone metabolites did not differ on postnatal day 36. We showed that transport leads to an autonomic stress response in chicken embryos at E18, but that this elevation had no further effects on other indicators of prenatal stress. Nevertheless, our results emphasise that transport of incubated eggs should be as refined as possible to minimise the exposure to stress.

Generalizability, Robustness and Replicability When Evaluating Wellbeing of Laboratory Mice with Various Methods.

An essential basis for objectively improving the status of animals during in vivo research is the ability to measure the wellbeing of animals in a reliable and scientific manner. Several non-invasive methods such as assessing body weight, burrowing activity, nesting behavior, a distress score and fecal corticosterone metabolites were evaluated in healthy mice and after three surgical interventions or during the progression of four gastrointestinal diseases. The performance of each method in differentiating between healthy and diseased animals was assessed using receiver operating characteristic curves. The ability to differentiate between these two states differed between distinct surgical interventions and distinct gastrointestinal diseases. Thus, the generalizability of these methods for assessing animal wellbeing was low. However, the robustness of these methods when assessing wellbeing in one gastrointestinal disease was high since the same methods were often capable of differentiating between healthy and diseased animals independent of applied drugs. Moreover, the replicability when assessing two distinct cohorts with an identical surgical intervention was also high. These data suggest that scientists can reach valid conclusions about animal wellbeing when using these methods within one specific animal model. This might be important when optimizing methodological aspects for improving animal wellbeing. The lack of generalizability, however, suggests that comparing animal models by using single methods might lead to incorrect conclusions. Thus, these data support the concept of using a combination of several methods when assessing animal welfare.

Repeated Contrast-Enhanced Micro-CT Examinations Decrease Animal Welfare and Influence Tumor Physiology.

Computed tomography (CT) imaging is considered relatively safe and is often used in preclinical research to study physiological processes. However, the sum of low-dose radiation, anesthesia, and animal handling might impact animal welfare and physiological parameters. This is particularly relevant for longitudinal studies with repeated CT examinations. Therefore, we investigated the influence of repeated native and contrast-enhanced (CE) CT on animal welfare and tumor physiology in regorafenib-treated and nontreated tumor-bearing mice. Mice bearing 4T1 breast cancer were divided into 5 groups: (1) no imaging, (2) isoflurane anesthesia only, (3) 4 mGy CT, (4) 50 mGy CT, and (5) CE-CT (iomeprol). In addition, half of each group was treated with the multikinase inhibitor regorafenib. Mice were imaged 3 times within 1 week under isoflurane anesthesia. Behavioral alterations were investigated by score sheet evaluation, rotarod test, heart rate measurements, and fecal corticosterone metabolite analysis. Tumor growth was measured daily with a caliper. Tumors were excised at the end of the experiment and histologically examined for blood vessel density, perfusion, and cell proliferation. According to the score sheet, animals showed a higher burden after anesthesia administration and in addition with CT imaging ( P < 0.001). Motor coordination was not affected by native CT, but significantly decreased after CE-CT in combination with the tumor therapy ( P < 0.001). Whereas tumor growth and blood vessel density were not influenced by anesthesia or imaging, CT-scanned animals had a higher tumor perfusion ( P < 0.001) and a lower tumor cell proliferation ( P < 0.001) for both radiation doses. The most significant difference was observed between the control and CE-CT groups. Repeated (CE-) CT imaging of anesthetized animals can lead to an impairment of animal motor coordination and, thus, welfare. Furthermore, these standard CT protocols seem to be capable of inducing alterations in tumor physiology when applied repetitively. These potential effects of native and CE-CT should be carefully considered in preclinical oncological research.

Behavioral phenotyping of young Scn1a haploinsufficient mice

Dravet syndrome is a rare, severe, infancy-onset epileptic encephalopathy associated with a high premature mortality. In most patients, Dravet syndrome is caused by a heterozygous loss-of-function mutation in the SCN1A gene encoding the alpha 1 subunit of the sodium channel. Of the variety of SCN1A variants identified in patients with Dravet syndrome, SCN1A missense mutations occur in one-third of cases. The novel Scn1a-A1783V mouse model of Dravet syndrome carries the human Ala1783Val missense variant. Recently, the behavioral phenotype of Scn1a-A1783V haploinsufficient adult mice has been characterized, which may provide a valuable basis for assessment of novel therapeutic approaches. However, there is still limited information on the developmental course of behavioral alterations in the Scn1a-A1783V mouse model, which is of particular relevance for conclusions about face validity and severity classification of the model. Based on reference data from young wildtype mice, we analyzed selected behavioral parameters and fecal corticosterone metabolites in the Scn1a-A1783V mouse model during post-weaning development. Differences in the preference for a sweet saccharin solution between Dravet mice and wildtype mice were observed once mice reached sexual maturity. Nest building behavior was already influenced by the Scn1a genotype during prepubescence. Sexually mature Dravet mice showed a significantly reduced burrowing performance as compared to their wildtype littermates. In the open-field test, pronounced hyperactivity and increased thigmotactic behavior were evident in prepubescent and sexually mature Dravet mice. Analysis of Irwin scores revealed several genotype-dependent changes in handling-associated parameters during the course of adolescence. The information obtained provides insight into the age-dependence of behavioral patterns in the novel Scn1a-A1783V mouse model of Dravet syndrome. In addition, the dataset confirms the suitability of the applied behavioral composite measure scheme for evidence-based assessment of cumulative severity in genetic mouse lines.

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.

Effects of methylmercury and food stress on migratory activity in song sparrows, Melospiza melodia

Avian migration is a challenging life stage susceptible to the adverse effects of stressors, including contaminants like methylmercury (MeHg). Although birds often experience stressors and contaminants concurrently in the wild, no study to date has investigated how simultaneous exposure to MeHg and food stress affects migratory behavior. Our objectives were to determine if MeHg or food stress exposure during summer, alone or combined, has carry-over effects on autumn migratory activity, and if hormone levels (corticosterone, thyroxine) and body condition were related to these effects. We tested how exposure to dietary MeHg and/or food stress (unpredictable temporary food removal) affected migratory behavior in captive song sparrows, Melospiza melodia. Nocturnal activity was influenced by a 3-way interaction between MeHg × stress × nights of the study, indicating that activity changed over time in different ways depending on prior treatments. Thyroxine was not affected by treatment or sampling date. During the migratory season, fecal corticosterone metabolite concentrations increased in birds co-exposed to MeHg and food stress compared to controls, suggesting an additive carry-over effect. As well, during the period of behavioral recording, body condition increased with time in unstressed birds, but not in stressed birds. Fecal corticosterone metabolite concentrations were positively correlated to duration of nocturnal activity, but thyroxine levels and body condition were not. The differences in nocturnal activity between groups suggest that food stress and MeHg exposure on breeding grounds could have direct and indirect carry-over effects that have the potential to affect the fall migration journey.

Effect of Different Ambient Temperatures on Reproductive Outcome and Stress Level of Lactating Females in Two Mouse Strains.

Simple SummaryThe optimal temperature for laboratory mice has been under discussion for some time. Current standard temperature is 20 °C–24 °C but it has been suggested to elevate the standard to 30 °C, which is the thermoneutral zone for mice. In this study, the effect of different cage temperatures (20 °C, 25 °C, 30 °C) on reproduction and stress hormone metabolite excretion was evaluated in lactating females of two commonly used mouse strains. Pup loss was higher, and weights of mothers and pups were reduced at 30 °C compared to the lower temperatures. In addition, pups showed increased tail length at weaning under the high temperature (30 °C). There was no difference in stress hormone metabolite excretion in mice between temperature groups. We could not show any detrimental effects of the lower or higher cage temperature on stress hormone metabolite excretion, but found decreased reproductive outcome under the higher temperature.Ambient temperature is an important non-biotic environmental factor influencing immunological and oncological parameters in laboratory mice. It is under discussion which temperature is more appropriate and whether the commonly used room temperature in rodent facilities of about 21 °C represents a chronic cold stress or the 30 °C of the thermoneutral zone constitutes heat stress for the animals. In this study, we selected the physiological challenging period of lactation to investigate the influence of a cage temperature of 20 °C, 25 °C, and 30 °C, respectively, on reproductive performance and stress hormone levels in two frequently used mouse strains. We found that B6D2F1 hybrid mothers weaned more pups compared to C57BL/6N mothers, and that the number of weaned pups was reduced when mothers of both strains were kept at 30 °C. Furthermore, at 30 °C, mothers and pups showed reduced body weight at weaning and offspring had longer tails. Despite pronounced temperature effects on reproductive parameters, we did not find any temperature effects on adrenocortical activity in breeding and control mice. Independent of the ambient temperature, however, we found that females raising pups showed elevated levels of faecal corticosterone metabolites (FCMs) compared to controls. Peak levels of stress hormone metabolites were measured around birth and during the third week of lactation. Our results provide no evidence of an advantage for keeping lactating mice in ambient temperatures near the thermoneutral zone. In contrast, we found that a 30 °C cage temperature during lactation reduced body mass in females and their offspring and declined female reproductive performance.

Physiological and behavioural assessments of stress levels in owls housed at owl cafes

AbstractOwl cafes, where customers view and interact with owls, have become popular in Japan. There are multiple aspects of the environment which may be stressful to nocturnal owls, including lighting, tethering and frequent interactions with humans but, to date, welfare has not been investigated. This preliminary study examines the effects of owl cafes and customers on the physiological stress (faecal corticosterone levels [FCL]) and behaviour of the owls. Seven eagle owls (Bubo bubo) and two African scops owls (Ptilopsis leucotis), in two cafes, were studied over an eight-day period. Cafe A (n = 5) was closed for one day per week, whilst cafe B (n = 4) was open every day. In cafe A there was higher FCL in owls during open days than closed days suggesting that the conditions on open days increase stress in owls. Eight of nine owls showed evidence of some aversion or avoidance of humans, whilst no affiliative behaviours were observed. The number of visitor interactions was not associated with the level of physiological stress; and when comparing owls, there was a tendency for individual owls with a higher percentage of aversive responses to customers to also have higher FCL. Close human interactions of a negative nature, that result in aversion, may be a significant stressor, but further research is required.

3D-printed lightweight dorsal skin fold chambers from PEEK reduce chamber-related animal distress

The dorsal skinfold chamber is one of the most important in vivo models for repetitive longitudinal assessment of microcirculation and inflammation. This study aimed to refine this model by introducing a new lightweight chamber made from polyetheretherketone (PEEK). Body weight, burrowing activity, distress, faecal corticosterone metabolites and the tilting angle of the chambers were analysed in mice carrying either a standard titanium chamber or a PEEK chamber. Data was obtained before chamber preparation and over a postoperative period of three weeks. In the early postoperative phase, reduced body weight and increased faecal corticosterone metabolites were found in mice with titanium chambers. Chamber tilting and tilting-related complications were reduced in mice with PEEK chambers. The distress score was significantly increased in both groups after chamber preparation, but only returned to preoperative values in mice with PEEK chambers. In summary, we have shown that light chambers reduce animal distress and may extend the maximum dorsal skinfold chamber observation time. Chambers made of PEEK are particularly suitable for this purpose: They are autoclavable, sufficiently stable to withstand rodent bites, inexpensive, and widely available through 3D printing.

Comparative Severity Assessment of Genetic, Stress-Based, and Pharmacological Mouse Models of Depression.

The use of animals in neurosciences is pivotal to gaining insights into complex functions and dysfunctions of behavior. For example, various forms of physical and/or psychological stress are inherent to various animal models for psychiatric disorders, e.g., depression. Regarding animal welfare, it would be mandatory to use models that inflict the least amount of stress necessary to address the underlying scientific question. This study compared the severity of different approaches to induce depression in mice: mutagenesis in GluA1 knockout, immobilization stress, and stress-induction via stress hormone treatment. While genetic alterations potentially represent a lifelong burden, the temporary intervention only affects the animals for a limited time. Therefore, we used home cage-based behavioral and physiological parameters, including nest building, burrowing, body weight, and fecal corticosterone metabolites, to determine the well-being of male and female mice. In addition, we performed an evidence-based estimate of severity using a composite score for relative severity assessment (RELSA) with this data. We found that even though restraint stress and supplementation of corticosterone in the diet both aimed at depression-related precipitating stress effects, the latter affected the well-being much stronger, especially in females. Restraint leads to less noticeable well-being impairments but causes depression-associated anhedonic behavior. Mice of both sexes recovered well from the stress treatment. GluA1 KO and their littermates showed diminished well-being, comparable to the immobilization experiments. However, since this is a lifelong condition, this burden is not reversible and potentially accumulative. In line with the 3Rs (Replacement, Reduction, and Refinement), the process of choosing the most suitable model should ideally include an evidence-based severity assessment to be able to opt for the least severe alternative, which still induces the desired effect. Promoting refinement, in our study, this would be the restraint stress.

Male and Female C57BL/6 Mice Respond Differently to Awake Magnetic Resonance Imaging Habituation.

Traditionally, preclinical magnetic resonance imaging (MRI) has been performed in anesthetized animals. However, anesthesia has been shown to perturb normal brain function and physiology. Such effects limit our ability to detect subtle physiological alterations in disease models and treatment studies, thus hampering discovery and compromising generality of findings. Therefore, methods for awake animal MRI are needed to study the rodent brain in its natural physiological state, free of anesthetics. Current setups for awake animal MRI rely on restraining systems to avoid animal movement during scanning. To reduce restraint stress, animals are habituated to the scanner environment prior to MRI data collection. To date, however, most awake MRI studies employ male rodents only. This is a fundamental limitation as results obtained may be pertinent only to half of the population. We characterized training and habituation responses of male and female mice to provide improved, sex-dependent training procedures for awake mouse MRI. We recorded heart rate, monitored behavioral responses (body weight and fecal boli weight) and fecal corticosterone levels (FCM) as indicators of wellbeing and stress during a 14-day progressive habituation protocol. In addition, we also assessed discomfort levels and anxiety using the mouse grimace scale (MGS) and light/dark test (LDT), respectively. All scores were compared between both groups. We found that heart rate was significantly decreased after 10 and 11 days of training for both males and females, respectively. However, the specific time course for this decrease was significantly different between males and females, and females exhibited higher anxiety levels during habituation and 14 days after habituation than males. Lastly, we also found that mean FCM levels for both groups were decreased after 11 days of MRI habituation. The present work shows that mice can be successfully trained for extended MRI sessions which is necessary for many (particularly non-fMRI) studies. Importantly, we find that males and females differ in their response to awake MRI habituation, which should be considered in future awake MRI studies that aim to include male and female mice.

Impact of Indigestible Materials on the Efficiency of Fecal Corticosterone Immunoassay Testing in Pituophis Species.

Simple SummaryEvaluating stress in animals is important for improving animal welfare and husbandry. However, it has been challenging to establish reliable and noninvasive methods for quantifying stress. Steroid hormones released during prolonged periods of stress are metabolized by the liver and excreted in feces. In snakes, corticosterone is the primary hormone of stress and is often measured in fecal samples collected from these animals. Assays can be used to measure the metabolites of corticosterone from feces, but are limited by compounding factors (season, reproductive status, diet, etc.) that influence the ability to confidently conclude the results. Our overall objective is to standardize these methods by first improving the extraction methodology. We found that 75% of the fecal sample contained indigestible materials (hair, teeth, bone, etc.) and, therefore, interferes with the extraction process. After removing the indigestible material, we found that we had a 95% improvement in overall yield. These findings alleviate one limitation to using fecal samples to measure stress in animals. Measuring fecal glucocorticoid metabolites (FGM) has recently become a sought-after method for assessing stress in animals. While there are many benefits to this methodology, there are also recognized limitations, including the apprehensive interpretation of results. While many factors can influence FGM levels, we aimed to standardize and improve these methods in snakes. Fecal samples were collected from Pituophis species and FGMs were extracted by two different sample collection methods: (1) fecal sample containing undigested materials and (2) fecal samples with undigested materials removed. These extracts were then used to quantify FGM concentrations using a corticosterone EIA kit. The results indicated that the samples with the undigestible materials removed had a 95% increase in overall yield (p < 0.01). Since the collected fecal samples contain 75% undigestible materials by weight, these results support our hypothesis that removing these materials will improve extraction methods for a more reliable measurement of corticosterone. This is the first step towards standardizing the methods for assessing stress by measuring fecal glucocorticoid metabolites in snakes.

Effects of separated pair housing of female C57BL/6JRj mice on well-being

In laboratory animal facilities, it is a common code of practice to house female mice in groups. However, some experimental conditions require to house them individually, even though social isolation may impair their well-being. Therefore, we introduced a separated pair housing system and investigated whether it can refine single housing of adult female C57BL/6JRj mice. Individually ventilated cages (IVC) were divided by perforated transparent walls to separate two mice within a cage. The cage divider allowed visual, acoustic, and olfactory contact between the mice but prevented interindividual body-contact or food sharing. Short- and long-term effects of the separated pair housing system on the well-being of the mice were compared with single and group housing using a range of behavioral and physiological parameters: Nest building behavior was assessed based on the complexity of nests, the burrowing performance was measured by the amount of food pellets removed from a bottle, and trait anxiety-related behavior was tested in the free exploratory paradigm. For the evaluation of the ease of handling, interaction with the experimenter's hand was monitored. Social interaction with unknown conspecifics and locomotor activity were investigated in a test arena. Moreover, body weight and stress hormone (metabolites) were measured in feces and hair. After the mice spent a day under the respective housing conditions, concentrations of fecal corticosterone metabolites were higher in separated pair-housed mice, and they built nests of a higher complexity when compared to single-housed mice. The latter effect was still observable eight weeks later. In week 8, separated pair-housed mice showed less locomotor activity in the social interaction arena compared to mice from the other housing systems, i.e., single and group housing. Regardless of the time of testing, pair housing improved the burrowing performance. Separated pair-housed mice were more difficult to catch than group-housed mice. Hair corticosterone, progesterone, and dehydroepiandrosterone concentrations changed with increasing age independently of the housing system. There were no effects of the housing systems on trait anxiety-related behavior in the free exploratory paradigm, voluntary interaction with the experimenter’s hand, and body weight. Overall, the transfer to the separated pair housing system caused short-term stress responses in female C57BL/6JRj mice. Long-term effects of separated pair housing were ambiguous. On one hand, separated pair housing increased nesting and burrowing behavior and may therefore be beneficial compared to single housing. But on the other hand, locomotor activity decreased. The study underlined that the effects of the housing conditions on physiological and behavioral parameters should be considered when analyzing and reporting animal experiments.

When left is right: The effects of paw preference training on behaviour in mice

Spontaneous limb preferences exist in numerous species. To investigate the underlying mechanisms of these preferences, different methods, such as training, have been developed to shift preferences artificially. However, studies that systematically examine the effects of shifting preferences on behaviour and physiology are largely missing. Therefore, the aim of this study was to assess the impact of shifting paw preferences via training on spontaneous home cage behaviour, as well as anxiety-like behaviour and exploratory locomotion (Elevated plus maze test, Dark light test, Open field test, Free exploration test), learning performance (Labyrinth-maze) and stress hormones (fecal corticosterone metabolites) in laboratory mice (Mus musculus f. domestica). For this, we assessed spontaneous paw preferences of C57BL/6J females (Nambilateral = 23, Nleft = 23, Nright = 25). Subsequently, half of the individuals from each category were trained once a week for four weeks in a food-reaching task to use either their left or right paw, respectively, resulting in six groups: AL, AR, LL, LR, RL, RR. After training, a battery of behavioural tests was performed and spontaneous preferences were assessed again. Our results indicate that most mice were successfully trained and the effect of training was present days after training. However, a significant difference of preferences between RL and LL mice during training suggests a rather low training success of RL mice. Additionally, preferences of L mice differed from those of A and R mice after training, indicating differential long-term effects of training in these groups. Furthermore, left paw training led to higher levels of self-grooming, possibly as a displacement behaviour, and more time spent in the light compartment of the Dark light test. However, overall, there was no systematic influence of training on behavioural measures and stress hormones. Different explanations for this lack of influence, such as the link between training and hemispheric functioning or the intensity and ecological relevance of the training, are discussed.

Physiological links with behavior and fitness: The acute adrenocortical response predicts trappability but not survival in male and female deermice

The “Cort-Fitness” hypothesis predicts a negative relationship between baseline glucocorticoids (GCs) and fitness, although evidence for this hypothesis remains mixed. Such ambiguity could partially exist because blood GCs, typically used in field studies, can fluctuate too rapidly to measure accurately, while the relationship between GCs and trappability is often neglected. Here, by addressing these factors, we examined relationships between GC measures and survival of North American deermice (Peromyscus maniculatus; hereafter deermice) as a model system. To do this, we used more stable GC measures, including the integrated measures of baseline and stress response fecal corticosterone metabolites (FCMs), and downstream measures of neutrophil/lymphocyte ratio (N/L ratio), and body condition score (BCS), to characterize their relationships with survival and trappability. Over two years, deermice were live-trapped monthly, evaluated for BCS, and sampled for feces and blood. Stress response FCMs were evaluated only at first capture. Mark-recapture models, with GC measures as predictors of either survival or trappability, were compared to identify top models. We found that stress response FCMs negatively predicted trappability, and weaker evidence that BCS positively predicted survival. Although the latter provides some support for the “Cort-Fitness” hypothesis, there was no support when using integrated measures. Instead, our findings suggest that deermice with a lower adrenocortical response (i.e. stress response FCMs) were more likely to be captured. Therefore, GC-trappability relationships must be investigated in field studies to avoid linking the wrong GC profile to fitness, and physiological measures other than blood GCs may be useful for detecting GC-fitness patterns.

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.

Effects of dietary polyunsaturated fatty acids on corticosterone concentrations and spatial learning in rats

Dietary intake of polyunsaturated fatty acids (PUFAs) is crucial for neuronal functions, can positively affect cognition, and reduce glucocorticoid (e.g. corticosterone) concentrations in response to stress. We investigated the effects of walnut oil high in PUFAs on spatial cognition and fecal corticosterone metabolite (FCM) concentrations under non-stressed conditions in rats. Unexpectedly, PUFA-supplemented rats had higher FCM concentrations and elevated concentrations generally impaired learning in the subsequent T-maze task. Statistically adjusting for individual FCM concentrations, however, revealed that learning performance was improved in PUFA-supplemented rats. The results suggest that glucocorticoids can modulate the effects of PUFAs on spatial learning under normal (non-stressed) conditions and call for consideration of basal physiological conditions in spatial learning tasks.

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.

Ultrasonic Vocalization Analysis as a Novel Metric to Assess Cage Enrichment in Rats.

Laboratory rodent housing conditions vary significantly across laboratories and facilities. Variation in housing can be associated with animal stress leading to study variability and the subsequent inability to replicate experimental findings. Optimization and standardization of animal housing are necessary to promote animal welfare and data consistency, thereby reducing the number of animals necessary to detect treatment effects. While interest in environmental enrichment is increasing, many studies do not examine the behavior of animals in the home cage, neglecting important aspects of enrichment. To determine how increased vertical home cage area affects animal welfare, double-decker cages (enriched), which allow rats to upright stand, were compared with standard single-level cages, which impede the ability to upright stand. Home cage welfare was assessed by analyzing ultrasonic vocalizations, fecal corticosterone, upright standing, and fighting. Ultrasonic vocalization was further explored by analyses of call type as defined by a 14 call-type schematic. Rats housed in enriched cages spent more time fighting, produced fewer 50 kHz calls, and had higher levels of fecal corticosterone. Rats in standard cages attempted to upright stand more often but remained upright for a shorter amount of time due to the height limitation imposed by standard cages. In addition, standard cages restrict some naturalistic behaviors such as upright standing and reduce fighting, which may be attributable to their single-tier organization and floor space. Enriched cages permit rats to engage in normal ethological behavior but also increase fighting. This study demonstrates that housing conditions have a meaningful impact on multiple measures of animal affect. When considering study design, researchers should be aware of how housing conditions affect animal subjects.