Brianna N. Gaskill

Heat or insulation: behavioral titration of mouse preference for warmth or access to a nest.

In laboratories, mice are housed at 20–24°C, which is below their lower critical temperature (≈30°C). This increased thermal stress has the potential to alter scientific outcomes. Nesting material should allow for improved behavioral thermoregulation and thus alleviate this thermal stress. Nesting behavior should change with temperature and material, and the choice between nesting or thermotaxis (movement in response to temperature) should also depend on the balance of these factors, such that mice titrate nesting material against temperature. Naïve CD-1, BALB/c, and C57BL/6 mice (36 male and 36 female/strain in groups of 3) were housed in a set of 2 connected cages, each maintained at a different temperature using a water bath. One cage in each set was 20°C (Nesting cage; NC) while the other was one of 6 temperatures (Temperature cage; TC: 20, 23, 26, 29, 32, or 35°C). The NC contained one of 6 nesting provisions (0, 2, 4, 6, 8, or 10g), changed daily. Food intake and nest scores were measured in both cages. As the difference in temperature between paired cages increased, feed consumption in NC increased. Nesting provision altered differences in nest scores between the 2 paired temperatures. Nest scores in NC increased with increasing provision. In addition, temperature pairings altered the difference in nest scores with the smallest difference between locations at 26°C and 29°C. Mice transferred material from NC to TC but the likelihood of transfer decreased with increasing provision. Overall, mice of different strains and sexes prefer temperatures between 26–29°C and the shift from thermotaxis to nest building is seen between 6 and 10 g of material. Our results suggest that under normal laboratory temperatures, mice should be provided with no less than 6 grams of nesting material, but up to 10 grams may be needed to alleviate thermal distress under typical temperatures.

Impact of nesting material on mouse body temperature and physiology.

In laboratories, mice are housed at 20-24 °C, which is below their lower critical temperature (≈30 °C). Thus, mice are potentially cold stressed, which can alter metabolism, immune function, and reproduction. These physiological changes reflect impaired wellbeing, and affect scientific outcomes. We hypothesized that nesting material would allow mice to alleviate cold stress by controlling their thermal microenvironment, thus insulating them, reducing heat loss and thermogenic processes. Naïve C57BL/6, CD-1, and BALB/c mice (24 male and 24 female/strain in groups of 3) were housed in standard cages at 20 °C either with or without 8 g nesting material for 4 weeks. Core body temperature was followed using intraperitoneal radio telemetry. The thermal properties of the nests were assessed using a thermal imaging camera, and related to nest quality. Higher scoring nests were negatively correlated with the mean radiated temperature and were thus more insulating. No effects of nesting material on body temperature were found. CD-1 mice with nesting material had higher end body weights than controls. No effect was seen in the other two strains. Mice with the telemetry implant had larger spleens than controls, possibly indicating an immune response to the implant or low level infection from the surgery. BALB/c mice express less mRNA for the UCP1 protein than mice without nesting material. This indicates that BALB/cs with nesting material do not utilize their brown fat to create heat as readily as controls. Nests can alleviate thermal discomfort by decreasing the amount of radiated heat and reduce the need for non-shivering thermogenesis. However, different strains appear to use different behavioral (through different primary modes of behavioral thermoregulation) and physiological strategies (utilizing thermogenesis to different degrees) to maintain a constant body temperature under cool standard laboratory ambient temperatures.

Nest building as an indicator of health and welfare in laboratory mice.

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

Energy Reallocation to Breeding Performance through Improved Nest Building in Laboratory Mice.

Mice are housed at temperatures (20-26°C) that increase their basal metabolic rates and impose high energy demands to maintain core temperatures. Therefore, energy must be reallocated from other biological processes to increase heat production to offset heat loss. Supplying laboratory mice with nesting material may provide sufficient insulation to reduce heat loss and improve both feed conversion and breeding performance. Naïve C57BL/6, BALB/c, and CD-1breeding pairs were provided with bedding alone, or bedding supplemented with either 8g of Enviro-Dri, 8g of Nestlets, for 6 months. Mice provided with either nesting material built more dome-like nests than controls. Nesting material improved feed efficiency per pup weaned as well as pup weaning weight. The breeding index (pups weaned/dam/week) was higher when either nesting material was provided. Thus, the sparing of energy for thermoregulation of mice given additional nesting material may have been responsible for the improved breeding and growth of offspring.

Introducing Therioepistemology: the study of how knowledge is gained from animal research

This focus issue of Lab Animal coincides with a tipping point in biomedical research. For the first time, the scale of the reproducibility and translatability crisis is widely understood beyond the small cadre of researchers who have been studying it and the pharmaceutical and biotech companies who have been living it. Here we argue that an emerging literature, including the papers in this focus issue, has begun to congeal around a set of recurring themes, which themselves represent a paradigm shift. This paradigm shift can be characterized at the micro level as a shift from asking “what have we controlled for in this model?” to asking “what have we chosen to ignore in this model, and at what cost?” At the macro level, it is a shift from viewing animals as tools (the furry test tube), to viewing them as patients in an equivalent human medical study. We feel that we are witnessing the birth of a new discipline, which we term Therioepistemology, or the study of how knowledge is gained from animal research. In this paper, we outline six questions that serve as a heuristic for critically evaluating animal-based biomedical research from a therioepistemological perspective. These six questions sketch out the broad reaches of this new discipline, though they may change or be added to as this field evolves. Ultimately, by formalizing therioepistemology as a discipline, we can begin to discuss best practices that will improve the reproducibility and translatability of animal-based research, with concomitant benefits in terms of human health and animal well-being.

Rat tickling: A systematic review of applications, outcomes, and moderators

Introduction Rats initially fear humans which can increase stress and impact study results. Additionally, studying positive affective states in rats has proved challenging. Rat tickling is a promising habituation technique that can also be used to model and measure positive affect. However, current studies use a variety of methods to achieve differential results. Our objective was to systematically identify, summarize, and evaluate the research on tickling in rats to provide direction for future investigation. Our specific aims were to summarize current methods used in tickling experiments, outcomes from tickling, and moderating factors. Methods We systematically evaluated all articles about tickling identified from PubMed, Scopus, Web of Science, and PsychInfo. Our inclusion criteria were publication in a peer-reviewed journal and collection of original, empirical data on rats using the handling method of tickling. One researcher extracted information from each article. Bias was assessed by 2 investigators using the SYRCLE bias assessment tool. Results We identified 32 articles (56 experiments) published in peer-reviewed journals about rat tickling for inclusion. A wide variety of strains, sexes, and ages of rats were included. The most common method used for tickling was cycling through 15 seconds of tickling and 15 seconds of rest for 2 minutes for 3 to 5 days. Experiments with a control for tickling (N = 22) showed that tickling increases positive vocalization, approach behavior, decreases anxiety measures, improves handling, and in some cases decreases stress hormones. Tickling juvenile, individually housed rats with a trait predisposition to respond more positively to tickling, results in the most positive outcomes. Methods to reduce bias were insufficiently reported. Conclusions We conclude that tickling is a promising method for improving rat welfare and investigating positive affect. However, the establishment of tickling best practices is essential as the outcomes from tickling can be moderated by several factors.

Aggression in group-housed laboratory mice: why can't we solve the problem?

Group housing is highly important for social animals. However, it can also give rise to aggression, one of the most serious welfare concerns in laboratory mouse husbandry. Severe fighting can lead to pain, injury and even death. In addition, working with animals that are severely socially stressed, wounded or singly-housed as a result of aggression may compromise scientific validity. Some general recommendations on how to minimize aggression exist, but the problem persists. Thus far, studies attempting to find solutions have mainly focused on social dominance and territorial behavior, but many other aspects of routine housing and husbandry that might influence aggressive behavior have been overlooked. The present way of housing laboratory mice is highly unnatural: mice are prevented from performing many species-typical behaviors and are routinely subjected to painful and aversive stimuli. Giving animals control over their environment is an important aspect of improving animal welfare and has been well-studied in the field of animal welfare science. How control over the environment influences aggression in laboratory mice, however, has not been closely examined. In this article, we challenge current ways of thinking and propose alternative perspectives that we hope will lead to an enhanced understanding of aggression in laboratory mice.

An automated maze task for assessing hippocampus-sensitive memory in mice

Highlights • Alternation procedures in rodents are highly sensitive to manipulations of the hippocampus.• However as they require hand testing, they are low throughput and stressful for the animal.• An automated maze was developed for assessing alternation performance in mice.• Alternation performance was shown to be impaired in mice with lesions to the hippocampus.

The effect of early life experience, environment, and genetic factors on spontaneous home-cage aggression-related wounding in male C57BL/6 mice

Aggression is a major welfare issue in mice, particularly when mice unfamiliar to each other are first placed in cages, as happens on receipt from a vendor, and following cage cleaning. Injuries from aggression are the second leading cause of unplanned euthanasia in mice, following ulcerative dermatitis. Commonly employed strategies for reducing aggression-related injury are largely anecdotal, and may even be counterproductive. Here we report a series of experiments testing potential explanations and interventions for post-shipping aggression-related injuries in C57BL/6 mice. First, we examined the effects of weaning: testing whether manipulating weaning age reduced aggression-related injuries, and if repeated mixing of weaned mice before shipping increased these injuries. Contrary to our predictions, repeated mixing did not increase post-shipping injurious aggression, and early weaning reduced aggression-related injuries. Second, we examined potential post-shipping interventions: testing whether lavender essential oil applied to the cage reduced aggression-related injuries, and whether a variety of enrichments decreased injurious aggression. Again, contrary to predictions, lavender increased wounding, and none of the enrichments reduced it. However, consistent with the effects of weaning age in the first experiment, cages with higher mean body weight showed elevated levels of aggression-related wounding. Finally, we tested whether C57BL/6 substrains and identification methods affected levels of intra-cage wounding from aggression. We found no effect of strain, but cages where mice were ear-notched for identification showed higher levels of wounding than cages where mice were tail-tattooed. Overall, these results emphasize the multifactorial nature of home-cage injurious aggression, and the importance of testing received wisdom when it comes to managing complex behavioral and welfare problems. In terms of practical recommendations to reduce aggressive wounding in the home cage, tail tattooing is recommended over ear notching and late weaning should be avoided.

Can seeds help mice with the daily grind

Some laboratory mice gnaw food pellets without ingesting much of the gnawed material, resulting in the production of waste material called ‘orts’. The fact that this food grinding behavior is not seen in all individuals of a particular strain suggests that it might be abnormal, and thus indicate a welfare concern. Furthermore, the increased rate of feed consumption and cage soiling is undesirable from a husbandry perspective. To try to determine possible motivations for the behavior, and identify potential treatments, outbred Crl:CD1(Icr) mice exhibiting food grinding were selected for one of three treatments placed in the feeder: no enrichment, a chewing device, or sunflower seeds. Both enrichment groups showed a significant decrease (P < 0.05) in ort production when compared with baseline measurements, but only mice provided with sunflower seeds maintained the decreased rate of food wastage after the treatment was withdrawn. A relationship between body weight and ort production was also found, in that cages with greater average body weights had lower levels of ort production. This suggests that a simple need to gnaw cannot alone explain food grinding, and that a nutritional motivation may also be involved.