Cage Cleaning Research Articles

Longitudinal Study of Changes in Ammonia, Carbon Dioxide, Humidity and Temperature in Individually Ventilated Cages Housing Female and Male C57BL/6N Mice during Consecutive Cycles of Weekly and Bi-Weekly Cage Changes.

Housing conditions are essential for ensuring animal welfare and high-quality research outcomes. In this study, we continuously monitored air quality-specifically ammonia, carbon dioxide, relative humidity, and temperature-in Individually Ventilated Cages (IVCs) housing five female or male C57BL/6N mice. The cages were cleaned either weekly or bi-weekly, and the data were collected as the mice aged from 100 to 348 days. The survival rate remained above 96%, with body weight increasing by 35-52% during the study period. The ammonia levels rose throughout the cleaning cycle, but averaged below 25 ppm. However, in the older, heavier mice with bi-weekly cage cleaning, the ammonia levels reached between 25 and 75 ppm, particularly in the males. While circadian rhythms influenced the ammonia concentration only to a small extent, the carbon dioxide levels varied between 800 and 3000 ppm, increasing by 30-50% at night and by 1000 ppm with body weight. Humidity also correlated primarily with the circadian rhythms (10% higher at night) and, to a lesser extent, with body weight, reaching ≥70% in the middle-aged mice. The temperature variations remained minimal, within a 1 °C range. We conclude that air quality assessments in IVCs should be conducted during animals' active periods, and both housing density and biomass must be considered to optimise welfare.

Abstract 3391: Effects of repeated psychological stress on NNK-induced lung cancer development in young female mice

Abstract Lung cancer is the second most common cancer in both men and women in the US and the leading cause of cancer death. Among younger US women, lung cancer incidence rates have in recent years become higher than rates seen in men, despite comparable histories of tobacco use. The possible contribution of stress to higher lung cancer risk in young women has received little research attention despite preclinical evidence that exposure to stress-related neuroendocrine responses can cause DNA damage. The present study used a well-established lung cancer model in young female mice (in vivo exposure to NNK) along with a mouse stress model to explore their independent and combined effects on lung cancer development. In addition, we explored one possible biological pathway by chronic administration of a beta-adrenergic receptor blocker (propranolol). We also tested the use of in vivo imaging for repeated monitoring of tumor development over time. Cohort 1 (n=60) had 4 groups: Home cage control, NNK treatment alone, repeated stress (RS) alone and NNK+RS. Cohort 2 (n=60) had the same 4 groups with the addition of administration of propranolol in drinking water. Cohort 3 (n=48, imaging cohort) had 3 groups: Home cage control, NNK alone and NNK+RS. FVB/N (4-6 wk) were purchased from a commercial supplier and acclimated with gentle handling for two weeks. The repeated stress protocol began week-3 and continued for the duration of the study. It combined two well-established rodent models: social disruption stress via changes in cage mates at the time of cage cleaning (2x/week) and a 90-minute restraint stress immediately prior to cage change (1x/week). NNK or saline treatment began on week-4 (IP; 2x/week; 3 mg/injection) and continued for 4 weeks. 21 weeks after stress initiation, the mice in cohorts 1 and 2 were sacrificed, lungs removed, inflated and tumors counted and sized. Mice in cohort 3 were imaged by Hounsfield-calibrated micro-CT (μCT) at baseline, 14, 19 and 23 weeks. The entire lung space, excluding the heart, was defined as a volumetric region of interests (VOIs). The VOIs were subjected to thresholding to segment the lungs into air space and tissue space compartments. The volume of tissue space of each animal was ratioed against its own baseline tissue space volume. Results from Cohort 1 revealed significantly greater numbers and overall size of lung tumors in stressed mice that received NNK. No significant effects were seen in the absence of NNK. Results from Cohort 2 revealed no significant differences between stressed and unstressed groups treated with propranolol. Cohort 3 revealed a pattern of results with stressed mice generally showing the highest lung tumor volume levels (Hounsfield Units), which reached significance at 19 weeks in preliminary analyses. Conclusion: In this study, we found that repeated stress increased lung tumor burden in female mice treated with NNK, and that a beta-adrenergic pathway may be involved. Citation Format: Frank Jenkins, Laura P. Stabile, Jessie R. Nedrow, Kathryn Day, Joseph D. Latoche, Patrick M. Tarwater, Jessica Manculich, Dana H. Bovbjerg. Effects of repeated psychological stress on NNK-induced lung cancer development in young female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3391.

Tactile stimulation of young WAG/Rij rats prevents development of depression but not absence epilepsy.

Investigations in Wistar Albino Glaxo from Rijswijk (WAG/Rij) rats that are susceptible to genetic absence epilepsy have demonstrated that environmental modifications affect absence seizures. Previously, we showed that neonatal tactile stimulations produce disease-modifying effect on genetically determined absence epilepsy and associated depression in Wag/Rij rats. The study presented here examined the effect of TS during late ontogenesis (adolescence and young adulthood) on epilepsy and depression outcomes in this genetically epileptic rat strain. On postnatal day (PND) 38, male WAG/Rij rats randomly were assigned to either the tactile stimulation (TS), handled or control group (unhandled) with 8 animals in each group. Following a 7-day adaptation period to their new surroundings, the animals were submitted to tactile stimulation from PND 45 to PND 90, five days per week, for 5 min daily. The tactile-stimulated rat was removed from its cage, placed on the experimenter's lap, and had its neck and back gently stroked by the researcher. The handled rats were taken to another cage and left alone for 5 min daily from PND 45 to PND 90. The control rats were left undisturbed in their home cage, except for regular cage cleaning. After PND 90, all rats were left undisturbed until behavioral testing and EEG recording. When the animals were 7 months old, they were subjected to the sucrose consumption test (SCT) and the forced swimming test (FST). Electroencephalogram (EEG) recordings were made at 8 months of age in order to measure electroencephalographic seizure activity, thus, the spike-wave discharges (SWDs). Tactile-stimulated rats showed increased sucrose consumption and number of approaches to the sucrose solution in the SCT when compared with the handled and control rats. In the FST, rats in TS group showed lower immobility time and greater immobility latency, active swimming time and diving frequency than the handled and control rats. The duration and the number of seizures were not different amongst the groups. The data obtained suggest that TS in young rats is able to prevent depression in WAG/Rij rats.

Home-cage behavior is impacted by stress exposure in rats.

Being social animals, rats exhibit a range of social behaviors that help them build social bonds and maintain group cohesion. Behavior is influenced by multiple factors, including stress exposure, and the expression of the impact of stress on both social and non-social behaviors may also be affected by the living conditions of rats. In this study, we explored the physiological and behavioral effects of chronic unpredictable stress on group-housed rats in the PhenoWorld (PhW), a socially and physically enriched environment closer to real-life conditions. Two independent experiments were performed: one in the control condition (PhW control, n = 8) and one in the stress condition (PhW stress, n = 8). Control animals remained undisturbed except for cage cleaning and daily handling procedures. Stress group animals were all exposed to chronic unpredictable stress. Data confirm that stress exposure triggers anxiety-like behavior in the PhW. In terms of home-cage behaviors, we found that stress affects social behaviors (by decreased playing and increased huddling behaviors) and non-social behaviors (as shown by the decrease in rearing and walking behaviors). These results are of relevance to expand our knowledge on the influence of stress on social and non-social behaviors, which are of importance to understand better species-typical behaviors.

Sea transfer and net pen cleaning induce changes in stress-related gene expression in commercial Atlantic salmon (Salmo salar) gill tissue

Stress is a major concern in aquaculture production and more knowledge is needed on physiological responses towards different operational events. Few studies have been performed on fish reared in an actual commercial setting. Transferring salmon from hatchery to sea involves handling, crowding, pumping, transport as well as adjusting to a new environment. This case study investigates the relative expression of selected stress related-genes in farmed Atlantic salmon (Salmo salar) post-smolts that were relocated from land-based breeding tanks with few environmental stressors to sea phase with numerous environmental stressors and major management operations e.g. net pen cleaning. Gill tissue for analysis (n = 60) was harvested at four distinct time points: before, during, and 3 and 6 weeks after sea transfer. RT-qPCR was performed on a panel of 12 genes involved in different cellular pathways (alox5, cyp1α, hif1α, il4/13a, muc2, muc5, muc18, nrf2, pcna, phb, p38 and tnfα). While the transport process itself did not appear to induce notable stress levels, metabolic gene markers showed significant changes in expression after transfer to sea, implying cellular adaptations to sea phase. The occurrence of net pen cleaning induced a strong upregulation of pro-inflammatory markers (alox5, tnfα) and mucins (muc2, muc5 and muc18), suggesting their gene products to be relevant during this operational event. As p38 expression was significantly elevated during transport and after cage cleaning, we cautiously propose p38 as an interesting stress marker for future exploration. The study provides insight into the lives of farmed Atlantic salmon and demonstrates that timing of major operations is crucial to avoid accumulation of stress.

Prevalence and Risk Factors of Avian Chlamydiosis Detected by Polymerase Chain Reaction in Psittacine Birds in Thailand.

This study surveyed avian chlamydiosis, with the aim to estimate the prevalence and potential risk factors associated with Chlamydia psittaci infection in psittacine birds kept as domestic pets in Thailand. Oropharyngeal swabs were collected from 120 psittacine birds that were randomly selected from hospitals in the central (Bangkok) and northeastern regions (Khon Kaen) of Thailand between 2019 and 2021. The oropharyngeal swabs were subject to polymerase chain reaction testing to detect the C psittaci ompA gene. The prevalence of C psittaci was 2.5% (3/ 120, 95% confidence interval = 0.3-5.3). Of the 3 positive birds, 1 was a Forpus parrot (Forpus species)(CP43TH) and 1 was an African grey parrot (Psittacus erithacus)(CP49TH) from Bangkok; both were juvenile birds with clinical signs of disease. The third positive bird (CP12TH) was a subclinical adult sun conure (Aratinga solstitialis) from Khon Kaen. Two sequences of samples that were previously identified in human psittacosis cases (accession numbers MK032053.1 and HM450409.1) were also examined. Since there was a low number of infected birds, potential associations between C psittaci infection and various environmental variables (eg, cage cleaning, synanthropic birds, quarantine of new birds, and overcrowding) were assessed by Fisher exact tests. This study provides estimates of the prevalence and potential risk factors associated with C psittaci infection in psittacine birds from central (Bangkok) and the northeastern regions (Khon Kaen) of Thailand. The detection of C psittaci in captive psittacine birds demonstrates that there is a possibility for bird-to-bird transmission as well as some zoonotic potential for the human caretakers of these birds. Furthermore, larger-scale studies should be conducted to confirm these findings.

Rats move nesting materials to create different functional areas: Short report.

Here we document how rats separate their living space into different functional regions. Five groups of four female Sprague Dawley rats were housed in caging systems that consisted of two standard cages connected by a tube. Both cages were provided with the same amount of bedding and nesting materials, but only one contained food and water. Nesting cover and weight of each cage were measured once a week for five weeks during cage cleaning. We found that the cages with food and water had 9% less nesting material coverage but had gained 90% more weight when compared with cages where food and water were absent. These results indicate that, when provided with separate spaces, rats move nesting materials away from the cage containing food and water sources, but preferentially excrete in the cage with water and food.

Establishing an inexpensive, space efficient colony of Bemisia tabaci MEAM1 utilizing modelling and feedback control principles.

A stable, synchronized colony of whitefly (Bemisia tabaci MEAM1 Gennadius) was established in a single ~30 cu.ft. reach‐in incubator and supported on cabbage host plants which were grown in a 2 × 2′ mesh cage without the need for a greenhouse or dedicated growth rooms. The colony maintenance, including cage cleaning and rotation of plants, was reduced to less than 10 h per week and executed by minimally experienced researchers. In our hands, this method was approximately 10‐fold less expensive in personnel and materials than current typical implementations. A predator‐prey model of whitefly colony maintenance that included whitefly proliferation and host plant health was developed to better understand and avoid colony collapse. This quantitative model can be applied to inform decisions such as inoculum planning and is a mathematical framework to assess insect control strategies. Extensive measurements of colony input and output (such as image analysis of leaf area and whitefly population size) were performed to define basic ‘feedback control’ parameters to gain reproducibility of this inherently unstable scaled‐down whitefly colony. Quantitative transfer of ~100 whiteflies repeatedly produced more than 5000 adult whiteflies over a 6‐week, two‐generation period. Larger scale experimentation could be easily accommodated by transferring adult whiteflies from the maintenance colony with a low flow vacuum capture device. This approach to colony maintenance would be useful to programs that lack extensive plant growth room or greenhouse access and require a “clean” implementation that will not contaminate an axenic tissue culture laboratory.

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.

Compounds from plantar foot sweat, nesting material, and urine show strain patterns associated with agonistic and affiliative behaviors in group housed male mice, Mus musculus.

Excessive home cage aggression often results in severe injury and subsequent premature euthanasia of male laboratory mice. Aggression can be reduced by transferring used nesting material during cage cleaning, which is thought to contain aggression appeasing odors from the plantar sweat glands. However, neither the composition of plantar sweat nor the deposits on used nesting material have been evaluated. The aims of this study were to (1) identify and quantify volatile compounds deposited in the nest site and (2) determine if nest and sweat compounds correlate with social behavior. Home cage aggression and affiliative behavior were evaluated in 3 strains: SJL, C57BL/6N, and A/J. Individual social rank was assessed via the tube test, because ranking may influence compound levels. Sweat and urine from the dominant and subordinate mouse in each cage, plus cage level nest samples were analyzed for volatile compound content using gas chromatography-mass spectrometry. Behavior data and odors from the nest, sweat, and urine were statistically analyzed with separate principal component analyses (PCA). Significant components, from each sample analysis, and strain were run in mixed models to test if odors were associated with behavior. Aggressive and affiliative behaviors were primarily impacted by strain. However, compound PCs were also impacted by strain, showing that strain accounts for any relationship between odors and behavior. C57BL/6N cages displayed the most allo-grooming behavior and had high scores on sweat PC1. SJL cages displayed the most aggression, with high scores on urine PC2 and low scores on nest PC1. These data show that certain compounds in nesting material, urine, and sweat display strain specific patterns which match strain specific behavior patterns. These results provide preliminary information about the connection between home cage compounds and behavior. Salient compounds will be candidates for future controlled studies to determine their direct effect on mouse social behavior.

Chronic adolescent stress causes sustained impairment of cognitive flexibility and hippocampal synaptic strength in female rats

Females that experience chronic stress during development, particularly adolescence, are the most vulnerable group to stress-induced disease. While considerable attention has been devoted to stress-induced manifestation of anxiety, depression, and PTSD, evidence indicates that a history of chronic stress is also a risk factor for cognitive decline and dementia – with females again in a higher risk group. This interplay between sex and stress history indicates specific mechanisms drive neural dysfunction across the lifespan. The presence of sex and stress steroid receptors in the hippocampus provides a point of influence for these variables to drive changes in cognitive function. Here, we used a rodent model of chronic adolescent stress (CAS) to determine the extent to which CAS modifies glutamatergic signaling resulting in cognitive dysfunction. Male and female Wistar rats born in-house remained non-stressed (NS), unmanipulated aside from standard cage cleaning, or were exposed to either physical restraint (60 min) or social defeat (CAS) each day (6 trials each), along with social isolation, throughout the adolescent period (PND 35–47). Cognition was assessed in adult (PND 80–130) male and female rats (n = 10–12) using the Barnes Maze task and the Attention Set-Shift task. Whole hippocampi were extracted from a second cohort of male and female rats (NS and CAS; n = 9–10) and processed for RNA sequencing. Brain tissue from the first cohort (n = 6) was processed for density of glutamatergic synaptic markers (GluA1, NMDA1a, and synaptophysin) or whole-cell patch clamping (n = 4) to determine glutamatergic activity in the hippocampus. Females with a history of chronic stress had shorter latencies to locate the goal box than NS controls during acquisition learning but showed an increased latency to locate the new goal box during reversal learning. This reversal deficit persisted across domains as females with a history of stress required more trials to reach criterion during the reversal phases of the Attention Set-Shift task compared to controls. Ovariectomy resulted in greater performance variability overall during reversal learning with CAS females showing worse performance. Males showed no effects of CAS history on learning or memory performance. Bioinformatic prediction using gene ontology categorization indicated that in females, postsynaptic membrane gene clusters, specifically genes related to glutamatergic synapse remodeling, were enriched with a history of stress. Structural analysis indicated that CAS did not alter glutamate receptor density in females. However, functionally, CAS females had a decreased AMPA/NMDA-dependent current ratio compared to controls indicating a weakening in synaptic strength in the hippocampus. Males showed only a slight change in density of NMDA1a labeling in the CA3 region with a history of stress. The data observed here suggest that females are at risk for impaired cognitive flexibility following a history of adolescent stress, possibly driven by changes in glutamatergic signaling.

Postweaning Grouping as a Strategy to Reduce Singly Housed Male Mice.

Rearing laboratory mice in groups is important since social isolation after weaning induces brain alterations, which entails behavioral abnormalities in adulthood. Age is an important factor when grouping males of different litters due to inter-male aggressiveness. The aim of this study was to determine whether newly weaned mice could safely be grouped with late juvenile or early and late pubescent mice, and whether cage cleaning, the number of the hosting group members and testosterone plasma levels have any influence. Newly weaned C57BL/6J, CD1, and SCID Beige male mice were systematically grouped with same strain late juvenile, early or late pubescent male mice in clean or dirty cages of 1, 2 or 3 hosting members. We also analyzed plasma testosterone levels at different postnatal days. Our result showed that only strain and hosting male's age influence agonistic behavior toward newly weaned mice. Thus, in order not to house a recently weaned male alone, we would recommend grouping it with late juvenile same strain mice in all studied strains. In the same way, CD1 and SCID Beige pubescent mice will admit a newly weaned mouse in their group. However, we would not recommend grouping newly weaned and pubescent C57BL/6J males.

High laboratory mouse pre-weaning mortality associated with litter overlap, advanced dam age, small and large litters.

High and variable pre-weaning mortality is a persistent problem in laboratory mouse breeding. Assuming a modest 15% mortality rate across mouse strains, means that approximately 1 million more pups are produced yearly in the EU to compensate for those which die. This paper presents the first large study under practical husbandry conditions to determine the risk factors associated with mouse pre-weaning mortality. We analysed historical records from 219,975 pups from two breeding facilities, collected as part of their management routine and including information on number of pups born and weaned per litter, parents’ age and identification, and dates of birth and death of all animals. Pups were counted once in their first week of life and at weaning, and once every one or two weeks, depending on the need for cage cleaning. Dead pups were recorded as soon as these were found during the daily cage screening (without opening the cage). It was hypothesized that litter overlap (i.e. the presence of older siblings in the cage when new pups are born), a recurrent social configuration in trio-housed mice, is associated with increased newborn mortality, along with advanced dam age, large litter size, and a high number and age of older siblings in the cage. The estimated probability of pup death was two to seven percentage points higher in cages with litter overlap compared to those without. Litter overlap was associated with an increase in death of the entire litter of five and six percentage points, which represent an increase of 19% and 103% compared to non-overlapped litters in the two breeding facilities, respectively. Increased number and age of older siblings, advanced dam age, small litter size (less than four pups born) and large litter size (over 11 pups born) were associated with increased probability of pup death.

Routine Cleaning of Poultry Cages as a Determinant of Underweight among Children Aged 6-59 Months in Indonesia

Objective: This study examined whether there is a significant relationship between routine cleaning of poultry cages and the incidence of underweight in children aged 6-59 months in Indonesia. Raising poultry has a significant relationship with the incidence of recurrent infections. Families who keep poultry have greater incidence of fevers and colds. This is a concern when the management of the poultry cage is poor, as the presence of poultry in the environment is one of the causes of recurrent infectious diseases with symptoms of cold or fever, and has a significant relationship with underweight. Method: This cross sectional study in Babakan Madang Subdistrict with participants of 612 mother and children under five, and 298 of them have poultry. Interviewing respondents using questionnaire and poultry management observations. Anthropometric measurements in children under five are weight and height. Descriptive analysis and the relationship of independent variables with the incidence of underweight using chi-square for bivariate analysis. Results: 10.8% of children aged 6-59 months were underweight. Significant relationships were found between underweight status and routine cage cleaning (p= 0.017; OR 3.205; 90%CI:1,268-8,101); family income (p = 0.050; OR 1,728; 90% CI:1,033-2,892); recurrent fever (p = 0.046; OR 1,763; 90% CI:1,042- 2981); and recurrent colds (p = 0.003; OR 2,259; 90%CI: 1,348-3,784). Conclusion: Routine cleaning of poultry cages at least once per day is the key to maintaining environmental cleanliness so that the poultry that are kept do not cause infectious diseases that lead to underweight among children.

Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice

Handling can stimulate stress and anxiety in laboratory animals that negatively impacts welfare and introduces a confounding factor in many areas of research. Picking up mice by the tail is a major source of handling stress that results in strong aversion to the handler, while mice familiarised with being picked up in a tunnel or cupped on the open hand show low stress and anxiety, and actively seek interaction with their handlers. Here we investigate the duration and frequency of handling required for effective familiarisation with these non-aversive handling methods, and test whether this is sufficient to prevent aversion and anxiety when animals then experience immobilisation and a mild procedure (subcutaneous injection). Very brief handling (2 s) was sufficient to familiarise mice with tunnel handling, even when experienced only during cage cleaning. Brief but more frequent handling was needed for familiarisation with cup handling, while pick up by tail induced strong aversion even when handling was brief and infrequent. Experience of repeated immobilisation and subcutaneous injection did not reverse the positive effects of tunnel handling. Our findings demonstrate that replacing tail with tunnel handling during routine cage cleaning and procedures provides a major refinement with little if any cost for familiarisation.

Group and Single Housing of Male Mice: Collected Experiences from Research Facilities in Sweden.

Simple SummaryThe mouse is the most commonly used mammal in scientific research, and housed in research facilities around the world. Mice are a social species, but when housing male mice together in a confined environment in the laboratory, aggression is often observed and can be problematic. Fighting or trying to avoid fighting can be stressful. Furthermore, fighting can lead to injuries which can sometimes be fatal. Mouse aggression is therefore a significant welfare problem and has implications on the 3Rs (Replacing, Reducing, and Refining animal use in scientific procedures and education). In this study, we used a survey and workshops to collect the experiences of animal technicians, veterinarians, and researchers at Swedish research animal facilities relating to mouse aggression and what methods of preventing aggression they practice. Both group housing and single-housing as a consequence of aggression was perceived as problematic and stressful for the animals. In line with current recommendations from the literature, participants perceived that aggression occurred less if mice were grouped with litter mates at an early age, that nesting material was transferred at cage cleaning, and disturbance was kept to a minimum. Experience from practice will play a valuable part in developing guidelines for group-housed male mice.Animals used for scientific purposes are protected by EU legislation. Social animals should be kept in stable groups that enable species-typical social behavior and provide individuals with social comfort. However, when group-housing male mice, aggression within the homecage is a common husbandry and welfare problem. Excessive fighting and injuries due to aggression can cause pain and stress, resulting in individuals being euthanized or housed individually. In addition, stress can alter physiological parameters, risking scientific validity and generating larger sample sizes. Mouse aggression, and the consequences thereof, thus opposes the 3R goals of Refining the methods to minimize potential pain and suffering and Reducing the number of animals used. Animal technicians, veterinarians, and scientists using animals have valuable information on how these problems are experienced and handled in practice. We assembled these experiences from laboratory animal facilities in Sweden, mapping problems observed and identifying strategies used to prevent mouse aggression. In line with current literature, less aggression was perceived if mice were grouped before sexual maturity, re-grouping avoided and nesting material transferred at cage cleaning. Preventing aggression will minimize pain and suffering and enable housing of stable groups, leading to more reliable scientific outcomes and is thus of high 3Rs relevance.

Cage aggression in group-housed laboratory male mice: an international data crowdsourcing project

Aggression in group-housed laboratory mice is a serious animal welfare concern. Further understanding of the causes of mouse aggression could have a significant impact on a large number of laboratory animals. The NC3Rs led a crowdsourcing project to collect data on the prevalence and potential triggers of aggression in laboratory mice. The crowdsourcing approach collected data from multiple institutions and is the first time such an approach has been applied to a laboratory animal welfare problem. Technicians observed group-housed, male mice during daily routine cage checks and recorded all incidents of aggression-related injuries. In total, 44 facilities participated in the study and data was collected by 143 animal technicians. A total of 788 incidents of aggression-related injuries were reported across a sample population of 137,580 mice. The mean facility-level prevalence of aggression-related incidents reported across facilities was equivalent to 15 in 1,000 mice. Key factors influencing the prevalence of aggression included strain; number of mice per cage; how mice were selected into a cage; cage cleaning protocols; and transfer of nesting material. Practical recommendations have been provided to minimise aggressive behaviour in group-housed, male mice based upon the results of the study and taking into consideration the current published literature.

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.

Can live with ‘em, can live without ‘em: Pair housed male C57BL/6J mice show low aggression and increasing sociopositive interactions with age, but can adapt to single housing if separated

The basic question as to whether male laboratory mice should be singly or group housed represents a major animal welfare concern within current laboratory animal legislation and husbandry. To better understand the behavioural and physiological mechanisms underlying this issue, we conducted two longitudinal experiments using C57BL/6J mice. In the first experiment (N = 32), we explored social behaviour of pair housed males from weaning to adulthood. We took weekly measures of agonistic, socio-exploratory and affiliative behaviours within two different contexts, i.e. in the undisturbed home cage and immediately after cage cleaning. In the second experiment (N = 36), we investigated whether separation of male pairs into single housing at different ages (35, 56 or 77 days of age) affected welfare-related measures such as faecal corticosterone metabolites (FCMs) and anxiety-like behaviours. In the first experiment we found that levels of agonistic behaviour were higher after cage cleaning than in the undisturbed cage as expected, but did not significantly change with age in either context. Instead, affiliative behaviour increased with age in the undisturbed home cage. In the second experiment, social separation did not affect levels of FCMs or anxiety-like behaviours at any age point. Taken together, this study shows that pair housed male mice can maintain low levels of aggression across a long period of their life and perform increasing levels of sociopositive behaviours which may serve to promote stable social relations. At the same time, our results suggest that male mice can quickly adapt to separation into single housing at different ages, from adolescence to adulthood. These findings are in line with the behavioural ecology of wild male mice, which suggests that both solitary and group living represent two alternative strategies.

Retrospective Analysis of the Effects of Identification Procedures and Cage Changing by Using Data from Automated, Continuous Monitoring.

Many variables can influence animal behavior and physiology, potentially affecting scientific study outcomes. Laboratory and husbandry procedures-including handling, cage cleaning, injections, blood collection, and animal identification-may produce a multitude of effects. Previous studies have examined the effects of such procedures by making behavioral and physiologic measurements at specific time points; this approach can be disruptive and limits the frequency or duration of observations. Because these procedures can have both acute and long-term effects, the behavior and physiology of animals should be monitored continuously. We performed a retrospective data analysis on the effects of 2 routine procedures, animal identification and cage changing, on motion and breathing rates of mice continuously monitored in the home cage. Animal identification, specifically tail tattooing and ear tagging, as well as cage changing, produced distinct and reproducible postprocedural changes in spontaneous motion and breathing rate patterns. Behavioral and physiologic changes lasted approximately 2 d after tattooing or ear tagging and 2 to 4 d for cage changing. Furthermore, cage changes showed strain-, sex-, and time-of-day-dependent responses but not age-dependent differences. Finally, by reviewing data from a rodent model of multiple sclerosis as a retrospective case study, we documented that cage changing inadvertently affected experimental outcomes. In summary, we demonstrate how retrospective analysis of data collected continuously can provide high-throughput, meaningful, and longitudinal insights in to how animals respond to routine procedures.