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As experienced authors, statisticians, editors, and scientists, we present the following comments to highlight some usages or omissions that are common in research manuscripts. Consideration of these comments will improve practices of data analysis and reporting.

To determine how housing density and ambient temperature interact to influence the physiology and behavior of mice, we systematically varied housing density (1 to 5 mice per cage) and ambient temperature (22, 26, or 30 °C) and measured effects on body weight, food intake, diurnal patterns of locomotor activity and core temperature, fecal corticosterone, and serum cytokine and adipokine panels. Temperatures inside cages housing 5 mice were 1 to 2 °C higher than the ambient temperature. As the housing density decreased, in-cage temperatures began to fall at a density of 2 or 3 mice per cage and did not differ from ambient temperature at 1 mouse per cage. Ambient temperature, but not housing density, significantly affected food intake. Although neither ambient temperature nor housing density affected core temperature or activity, hyperthermia and behavioral activation occurred during the 12-h period after cage change. Fecal concentrations of corticosterone metabolites and serum cytokines, chemokines, insulin, and leptin were not influenced by cage density and were only sporadically influenced by ambient temperature. Our data document that the number of mice housed per cage influences the intracage environmental conditions and that ambient temperature influences food intake even when temperatures are within or near recommended or thermoneutral ranges. We conclude that investigators should be cautious when changing the number of mice housed in a cage over the course of a study, because doing so significantly alters the cage environment to which remaining mice are exposed.

Although human subjects are widely used to study sleep and sleep disorders, animals have been invaluable in developing our understanding of the physiology of sleep and underlying mechanisms of sleep disorders. Environmental stimuli are likely to modify sleep in both animals and people, suggesting that environmental stability must be controlled carefully by both husbandry and research staff to allow collection of valid results with minimal numbers of animals. However, few studies have measured the effects of cage condition on sleep parameters in mice. Current guidelines recommend social housing and environmental enrichment for standard rodent housing. Environmental factors such as these create potential confounds in studies for which facets of sleep are outcome measures. We therefore sought to determine whether cage changes, group housing, or single housing with a shelter altered measures of sleep in C57BL/6J mice. The resulting data indicate that 1) cage changing disrupts sleep for approximately 3 h; 2) group housing is associated with shorter bouts of rapid-eye-movement sleep (REMS) and less slow-wave sleep (SWS) during the light phase and with more REMS during the dark phase; and 3) mice housed with a shelter spend less time awake and more time in SWS, with longer bouts of SWS during the dark phase. In additional, both group housing and housing with a shelter were associated with less locomotor activity than occurred in individually housed mice without a shelter. These findings provide evidence for long-held beliefs that housing conditions must be controlled carefully in studies that require assessment of sleep.

Many physiological and molecular processes are strongly rhythmic and profoundly influenced by sleep. The continuing effort of biological, medical, and veterinary science to understand the temporal organization of cellular, physiological, behavioral and cognitive function holds great promise for the improvement of the welfare of animals and human beings. As a result, attending veterinarians and IACUC are often charged with the responsibility of evaluating experiments on such rhythms or the effects of sleep (or its deprivation) in vertebrate animals. To produce interpretable data, animals used in such research must often be maintained in carefully controlled (often constant) conditions with minimal disruption. The lighting environment must be strictly controlled, frequent changes of cages and bedding are undesirable, and daily visual checks are often not possible. Thus deviations from the standard housing procedures specified in the Guide for the Care and Use of Laboratory Animals are often necessary. This report reviews requirements for experiments on biological rhythms and sleep and discusses how scientific considerations can be reconciled with the recommendations of the Guide.

The goal of this study was to identify objective criteria that would reliably predict imminent death in aged mice. Male and female ICR mice (age, 8 mo) were subcutaneously implanted with an identification chip for remote measurement of body temperature. Mice then were weighed and monitored regularly until spontaneous death occurred or until euthanasia was administered for humane reasons. Clinical signs that signaled implementation of euthanasia included inability to walk, lack of response to manipulation, large or ulcerated tumors, seizures, and palpable hypothermia. In mice that died spontaneously, gradual weight loss was the most frequent and earliest sign of imminent death. Hypothermia developed during the 2 wk prior to death. Slow or labored breathing were observed in about half of the mice before death. A composite score of temperature x weight can be used to provide an objective benchmark to signal increased observation or euthanasia of individual mice. Such assessment may allow the collection of terminal tissue samples without markedly altering longevity data, although application of this criterion may not be appropriate for all studies of longevity. Timely euthanasia of mice based on validated markers of imminent death can allow implementation of endpoints that alleviate terminal distress in aged mice, may not significantly affect longevity data, and can permit timely collection of biologic samples.

Mite infestation of mice remains a persistent problem for many institutions, leading to numerous health problems and creating unknown and unwanted variables for research. In this study, mice with mite infestation demonstrated significantly higher levels of inflammatory cytokines, both at draining lymph nodes (axillary) and systemically, as compared with mice without mites. In addition, histologic evaluation revealed significant inflammation in mite-infested mice. Inflammatory changes were still present in the skin of mice at 6 to 8 wk after treatment, despite absence of detectable infestation at that time. Because these significant and lasting local and systemic changes have the potential to alter research findings, eradication of mites infestations should be an important goal for all institutions.

A widely used in vivo technique in mice and other species is the surgical implantation of transmitters for telemetric monitoring of core body temperature, locomotor activity, and other variables. However, these devices are quite large relative to the size of the mouse abdomen. We report here on the results of several related studies that we conducted to evaluate refinement strategies relevant to implantation of abdominal devices in mice. First, we evaluated survival from surgery as a function of strain and body weight and found that both parameters influence the proportion of mice that survive. Second, we assessed the effect of several interventions on postsurgical recovery of food and water intakes, core temperature, and locomotor activity. Some of the interventions were associated with increased mortality (atipamezole) or were otherwise detrimental (the abdominal lubricant carboxymethylcellulose), whereas others had little or no effect on recovery (thermal support). These findings indicate that interventions presumed to promote recovery from surgery that are based on data from other species may not always have the anticipated positive effect in mice. This study therefore underscores the need to carefully assess the effect of modifications in experimental procedures to avoid causing unexpected complications in mice.

Fenbendazole is commonly used in laboratory animal medicine as an anthelmintic for elimination of pinworms. It is generally regarded as a safe drug with minimal side effects. In our facility, 2 breeding colonies of rats were treated with fenbendazole to eliminate pinworms. Analysis of the breeding records revealed that feeding Sprague-Dawley rats a diet containing fenbendazole on a continuous basis for 7 consecutive weeks was associated with a significant reduction in litter size. Although the mechanism underlying this effect is unknown, the finding prompts caution when using fenbendazole to treat valuable breeding colonies or strains that are poor breeders.