Voluntary Wheel Running as an Effective Intervention in the Management of Excessive Food Usage in CD-1 Mice (Mus musculus)

Abstract

Some mice demonstrate excessive food-grinding behaviors in which food pellets are broken down into crumbs (orts). This is considered abnormal behavior and is undesirable in a research environment, as it is thought to potentially be a stereotypic behavior suggestive of a negative welfare state in these animals. Further, food grinding often necessitates more frequent food and bedding changes. Research outcomes may also be affected if investigators do not exclude food losses due to grinding when measuring food consumption. We hypothesized some mice may excessively grind food in part to expend energy and access to a running wheel would contribute to a reduction in food grinding. Total daily food usage (the combined weight of food consumption and ort production) was measured for 40 d in CD-1 mice that exhibited food grinding. Median daily food usage was compared 10 d before, 20 d during, and 10 d after access to a running wheel. Additional cages of similar food-grinding mice that did not have access to a running wheel were monitored during the same period for comparison. A significant reduction in food usage was observed in 8 out of the 20 d in which mice had access to a running wheel compared with controls (P < 0.05). This reduction was significantly less than the median daily food usage before and after the running wheels were available (P < 0.01). Food usage significantly increased sharply in the 3 d following removal of the running wheel compared with controls during the same period (P < 0.05). A positive correlation between relative humidity and median daily food usage was observed (P < 0.05). Despite fluctuations in relative humidity, providing a running wheel effectively reduced excessive food-grinding behavior.

Introduction

Laboratory mice and other captive rodents often grind food pellets into small fragments or crumbs. This food material, commonly referred to as ‘orts’, is then left unconsumed to accumulate on the cage floor. Excessive food grinding in mice is undesirable from the perspectives of facility management, research data collection, and animal welfare. Ort production can substantially increase cage substrate levels (see Figure 1), necessitating more frequent bedding or cage changes compared with standard change intervals within a facility. Food pellets may also need to be replenished within the cage more often to replace pellets lost to ort production. It is unknown if mice consume orts,²³ but if they do it has not been observed within our facilities. Replenishing the food reservoir within the cage more frequently due to food grinding results in increased facility costs due to food turnover and staff effort. The impact of food grinding can also affect scientific data if researchers do not account for orts when measuring food consumption in study subjects, thereby inflating estimates of food intake and assimilation efficiency.^4,25 Some amount of food grinding and ort production may be normal, nutritionally motivated behavior in mice. Indeed, other captive rodents such as voles and lemmings been reported to sort and discard the fibrous parts of food pellets, preferring to consume the higher energy-yielding components.²² Previous studies have also observed a reduction in food grinding in mice when seeds and other high-fat foodstuffs are available.²³ However, excessive food-grinding behavior is speculated to be compulsive in nature and potentially representative of a stereotypy^8,15,23 or a stereotypic movement disorder that may negatively impact animal welfare.^11,28 Mice likely grind food pellets as a means of alleviating some level of distress. Identifying an effective strategy to reduce excessive food-grinding behavior in mice within the research environment is therefore desirable.

Figure 1.

(A–C) Images of mouse cages that contained contact bedding (A) without orts and (B, C) with orts. (A) Image was captured from a cage inhabited for 1 d by mice not known to exhibit food-grinding behavior. Contact bedding (BioFresh) is visible at the bottom of the cage and contains small amounts of fecal material. (Note: animals, environmental enrichment other than contact bedding, and the cage lid holding food and a water bottle were removed before image capture.) (B) Image is of a cage inhabited for 1 d by mice known to exhibit excessive food-grinding behavior. Orts are mixed with the same volume of contact bedding as in image A; an InnoDome (4.8 cm in height) is present to demonstrate loss of cage height available to mice due to excessive ort production. (C) Image depicts a close-up view of orts mixed with contact bedding from a cage of mice known to exhibit food-grinding behavior.

Food grinding has been associated with low temperature, lack of environmental enrichment, nutritional preferences, and genetics.^4,10,15,23 Within the past 5 to 10 y, we have observed an increase in the number of mouse cages at our institution exhibiting evidence of excessive food-grinding behavior. Efforts to control this behavior with environmental enrichment (e.g., additional nesting material and gnaw blocks) or changes in food material and composition have yielded limited anecdotal success. We then hypothesized that grinding behavior may be related to a lack of opportunity for energy expenditure. Standard research housing environments for mice, including individually ventilated caging, contact bedding, and ad libitum access to food, create sedentary conditions where mice progressively gain weight during adulthood.¹⁷ Mice housed in such environments are thought to expend less energy through daily spontaneous physical activity compared with mice housed in larger caging.^2,24 Although it is not currently feasible to house mice in larger cages at our institution, enhancing access to voluntary exercise is possible using commercially available shelter devices equipped with a tilted running wheel attachment. Voluntary wheel running has reportedly been effective in satisfying play and metabolic drives in mice and thus provides an outlet for rewarding, rather than stereotypic, behaviors.^1,16

Another factor we hypothesized may contribute to food grinding is humidity. Many animal facilities at our institution are located in older buildings with limited means of controlling humidity through the automated building management system. As a result, some animal housing rooms experience daily and seasonal fluctuations in relative humidity based on changes in weather and other environmental factors. At least in some cases, animal husbandry staff have reported that the degree of food grinding among mice inclined to perform this behavior increases or decreases to a relative degree along seasonal or weather pattern changes. Such patterns, however, have not been confirmed or reported in the literature to the authors’ knowledge. The Guide for the Care and Use of Laboratory Animals asserts that an acceptable range of relative humidity is between 30% and 70% for most mammals, but the Guide also acknowledges that it can be difficult to control humidity in some climates.¹⁴ In cases where controlling relative humidity is difficult, animals should be monitored for clinical signs of low humidity such as excessively flaky skin.¹⁴ Even in animal facilities equipped with a narrowly defined set temperature range and humidity, other environmental conditions may still be highly susceptible to seasonal or circannual effects that influence physiologic and behavioral parameters in mice.⁹

It has been established that endotherms exhibiting a smaller body size have higher energy levels and a faster metabolism due to their body’s high surface area-to-low volume ratio.²⁶ In adherence to the Bergmann Rule, mice likely experience higher energy needs than most larger endothermic mammals.¹⁸ These biologic patterns may contribute to an understanding of humidity’s effect on excessive grinding behavior. Changes in relative humidity may influence exercise-related behaviors in response to daily energy expenditure requirements for young adult mice.

This study sought to assess the impact of exercise on food usage in mice known to excessively grind food through the addition of a tilted running wheel in the cage. Daily food usage (i.e., the total feed consumed and ground by the mice in the cage) was measured directly; orts that accumulated at the bottom of the cage were not quantified to avoid excessive handling of the animals. Cages of socially housed CD-1 mice exhibiting food-grinding behavior were identified to participate in a 40-d study evaluating daily food usage before, during, and after a tilted running wheel was made available. Cages of food grinders that did not receive a running wheel were included as controls to evaluate the impact of humidity during the study. Additional cages of age-matched CD-1 mice not known to exhibit food-grinding behavior were also evaluated to establish a baseline for normal food usage; these animals, like those in the control group, were not provided access to a running wheel.

Materials and Methods

Animals and housing.

Mice were housed in an animal facility at the University of Oklahoma Health Sciences Center (OUHSC) managed by the Division of Comparative Medicine (DCM). OUHSC’s animal care program is accredited by AAALAC International, and all animal work was approved by OUHSC’s Institutional Animal Care and Use Committee and performed in alignment with DCM’s standard husbandry practices. Female CD-1 mice (Charles River Laboratories, Wilmington, MA) arrived at 6 wk of age and were allowed to acclimate for at least 2 wk before the initiation of experiments. Mice were housed in groups of 5 per cage. CD-1 mice were chosen for this study because several cages were observed by DCM husbandry staff to exhibit spontaneous food-grinding behaviors during acclimation. Mice were purchased for use in training workshops by OUHSC’s Animal Welfare Assurance office, but no training activities commenced until mice completed this study.

Mice were housed on a single-sided rack (Tecniplast IVC DGM80 Rack, West Chester, PA) with the motor positioned next to the rack. Solid-bottom polycarbonate IVC cages (19.68 × 37.46 × 13.33 cm; Tecniplast Sealsafe Plus GM500, West Chester, PA) were used with 50 air changes per hour. Mice were housed on BioFresh contact bedding (1/3-cm pelleted cellulose, BioFresh, Lab Supply, Fort Worth, TX). Drinking water was sourced from the local municipality, purified by reverse osmosis, then treated with UV light, and acidified (pH 2.8 to 3.2) before being placed in water bottles. Mice were fed an irradiated laboratory rodent diet (PicoLab Rodent Diet 5053, St. Louis, MO), provided ad libitum. Cages of mice that did not exhibit any food-grinding behavior received 2 types of supplemental enrichment in accordance with the facility’s standard protocol: 8 g of specialty shredded paper (Bed-r’Nest, Lab Supply, Fort Worth, TX), and a 4.8-cm (height) × 9.6-cm (diameter) reusable plastic dome (InnoDome, Bio-Serv, Flemington, NJ). In addition, cages of mice exhibiting food-grinding behavior also received a 3.75-cm³ wood gnawing block (Bio-Serv, Lab Supply, Fort Worth, TX) in alignment with the facility’s enhanced enrichment protocol for mice exhibiting stereotypic behavior. Cages were changed every 21 d or more often as needed when excessive ort production was observed. Figure 1 provides images of cages without orts (Figure 1A) and with orts (Figure 1B, C) generated by study mice and mixed with contact bedding. The housing room was maintained on a 10:14-h light:dark cycle, with illumination level at 250 to 300 lux 1 m above the floor.

Excluded agents, as assessed by PCR testing of dirty bedding samples and exhaust dust from the IVC rack, were Sendai virus, hantavirus, pneumonia virus of mice, mouse hepatitis virus, mouse parvoviruses, Theiler murine encephalomyelitis virus, reovirus, lymphocytic choriomeningitis virus, ectromelia virus, epizootic diarrhea of infant mice, polyoma virus, murine adenovirus, murine cytomegalovirus, Encephalitozoon cuniculi, Filbacterium rodentium, Clostridium piliforme, Mycoplasma pulmonis, and endo- and ectoparasites.

Study design.

Female CD-1 mice were 8 to 10 wk of age at the start of the study. All cages were housed in the same room and on the same IVC rack for the duration of the study. A total of 15 cages (75 mice) exhibiting food-grinding behavior were selected for this study. Two of the 15 cages were assigned to the control group; the remaining 13 cages were assigned to the interventional group. Figure 2 depicts a visual representation of the study.

Figure 2.

(A, B) A visual summary of the overall study design. (A) For the primary study, young adult female CD-1 mice were socially housed (5 animals/cage) upon arrival at the facility. During an initial acclimation period, 15 cages (75 mice) were observed exhibiting food-grinding behavior; 13 cages were assigned to the interventional group, and the remaining 2 cages were assigned to the control group. All cages were monitored daily for food usage throughout the 40-d study (D1 to D40). During D1 to D10, all cages received the same type and amount of contact bedding and enrichment. On D11, a tilted running wheel was provided to the cages assigned to the interventional group to encourage voluntary wheel running. On D31, the tilted running wheels were removed from the cages assigned to the interventional group. The cages assigned to the control group did not receive a running wheel of any kind for the duration of the 40-d study. (B) Shortly after the conclusion of this primary study, a secondary study was performed to determine a baseline of food usage among female CD-1 mice that did not grind food. Eight cages (40 mice) of CD-1 mice of similar age, sex, and housing conditions that were not observed exhibiting food-grinding behavior were assigned to this baseline group. All cages were monitored daily for food usage for 10 d. Like the control group, cages assigned to the baseline group did not receive a running wheel of any kind throughout the study. Throughout both the primary and secondary studies, room temperature and relative humidity were monitored daily.

All cages were monitored daily for food usage. Specifically, the mass (255 g) of the empty wire cage top feeder (Tecniplast GM500 Full Length Lid, West Chester, PA) was subtracted from the mass of the feeder containing unconsumed or unground pellets at the same time point each day. Once the food usage was measured, the feeder was then replenished with the standard base level (732 g) of pellets and returned to the cage. Daily food usage, representing the sum of food consumed as well as any food ground into orts, was monitored in this way at least 5 d per wk throughout the 40-d study (D1 to D40). During D1 to D10, all 15 cages received the same amount of contact bedding plus the 3 standard types of environmental enrichment as described above. On D11, a 13.6-cm (diameter) disc-shaped tilted running wheel (InnoWheel; Bio-Serv, Flemington, NJ) was attached to the InnoDome in cages assigned to the interventional group. The running wheel remained in each interventional cage for 20 d. On D31, running wheels were removed and food usage continued to be monitored until D40. Mice housed in 2 cages assigned to the control group did not receive a tilted running wheel.

Shortly after the conclusion of this 40-d study, we identified an additional 8 cages of CD-1 mice that did not exhibit food-grinding behaviors. These mice were assigned to a baseline group. We monitored food usage for 10 d in these animals, which were of identical age and sex as the interventional and control groups. Nongrinding mice represented animals for which consumption served as the primary variable for food usage. The mice housed in the 8 cages assigned to the baseline group did not have access to a tilted running wheel.

Throughout this period, all cages were changed at least once every 3 wk. At each change, animals were transferred to a sanitized cage containing fresh contact bedding and freshly sanitized enrichment materials (including the running wheels for cages assigned to the interventional group during D11 to D30). In accordance with standard husbandry practices for this facility, some cages were changed more frequently, as necessary, and sometimes daily, when orts accumulated at the bottom of the cage.

Temperature and relative humidity in the animal housing room were monitored daily. Humidity was measured by a digital hygrometer and reported as the relative percentage of water vapor in the room air relative to the total amount of vapor that the same room air may contain at a given temperature.²⁷

Statistical analysis.

Data were recorded into spreadsheets for recordkeeping (Excel, Microsoft, Redmond WA). Daily food usage was measured for the interventional and control groups of grinding mice for each phase of the 40-d study: preintervention (D1 to D10), intervention (D11 to D30), and postintervention (D31 to D40). Similarly, food usage was measured for the 8 cages of nongrinding mice during a subsequent 10-d monitoring period to represent baseline food consumption. Because food usage is not normally distributed, results were reported as daily median values (g/d). Median daily food usage among cages in the interventional group was compared with those of the control group using a one-sample Wilcoxon test. Median daily food usage during the intervention phase (D11 to D30) was compared with usage during the preintervention (D1 to D10) and postintervention (D31 to D40) phases using the Friedman test followed by Dunn post hoc tests to determine differences between specific groups. Linear regression was used to analyze the relationship between relative humidity (%) and food usage (g). Statistical tests were selected based on the normality of the data and the use of a repeated measures design within cages of each study set. All analyses were performed in Prism, version 10.1.2 (GraphPad Software, Boston, MA). The significance level (α) for all tests was 0.05.

Results

Mice.

All cages and animals initially assigned to this study were included in the final analyses. All animals remained with their cohorts (5/cage) as assigned to the interventional or control groups (for those cages demonstrating grinding behavior) or to the baseline group (for cages demonstrating no grinding behavior) throughout the assigned monitoring periods. Mice were 8 to 10 wk of age at the start of the study, and no more than 25 wk of age at the time of study completion.

Food usage.

Among the 8 cages of nongrinding mice studied over a 10-d period, the baseline median daily food usage (representing consumption as the sole variable) was 22.5 g/d (range 20 to 31 g/d). Among the 13 cages assigned to the interventional group, median daily food usage was 55 g/d (range 50 to 100 g/d) during D1 to D10. While running wheels were accessible to the mice (D11 to D30), median daily food usage declined to 34.5 g/d (range 26 to 715 g/d). During this period, 8 of the 20 d exhibited significantly less (P < 0.05) median daily food usage compared with control cages, as shown in Figures 3A, B and 4A, B. Food usage while mice in the interventional group had access to the running wheel was significantly less (P < 0.01) than that observed before the running wheels were introduced (D1 to D10) and after the wheels were removed (D31 to D40).

Figure 3.

(A, B) Longitudinal graphs of daily median food usage in cages of mice exhibiting food-grinding behavior throughout the 40-d study. Median daily food usage is depicted in grams on the Y-axis. Separate lines indicate individual variation among cages; open circles indicate daily values of food usage in grams. Red data points and lines indicate the portion of the experiment in which wheels are not present; blue data points and lines indicate when a wheel was present in the intervention group.

Figure 4.

(A) Data for food usage over time in the interventional group are presented as a proportion of food usage in the control group. Daily food usage of each interventional cage was normalized to the average daily food usage among the control cages by dividing each food usage data point in the interventional group by the average of the controls on each day. The one-sample Wilcoxon test was then used to compare the median daily food usage of interventional cages to the theoretical value of 1. (B) The magnitude of the difference between daily food usage in the interventional group compared is also compared with the control group, and medians are compared with a theoretical value of 0 g. When running wheels were introduced (D11 to D30), 8/20 d exhibited significantly less food usage than control cages based on a one-sample Wilcoxon test with Bonferroni’s correction for multiple comparisons. *, P < 0.05 and †, P < 0.01. Food usage is significantly lower when a wheel is present. Dotted line at 1 indicates the level of food usage in the control cages.

When the running wheels were removed from the cages (D31 to D40), median daily food usage initially increased dramatically (175 to 200 g/d) during D31 to D34. This difference was significantly different from that of control cages during the same time (P < 0.05). Usage then declined in the interventional cages to levels approximately equivalent to when mice had access to running wheels from D35 to D40. The median increase in daily food usage during the initial 3 d in the postinterventional period was significantly greater than that of the preintervention period (D1 to D10) and the overall postintervention period (D31 to D40). A summary of this data is captured in Figure 5A-D.

Figure 5.

(A–D) Daily median food usage in cages of mice exhibiting food-grinding behavior during preintervention (D1 to D10), intervention (D11 to D30), and postintervention (D31 to D40) study phases among (A, B) cages in the interventional group that received running wheels during D11 to D30 and (C, D) cages in the control group that did not receive running wheels. (A) Among the interventional group, median food usage when a wheel was present (D11 to D30, blue) was significantly lower than both preintervention (D1 to D10) and postintervention (D31 to D40) phases. †, P < 0.01. (B) Further, when D31 to D40 is divided for cages in the intervention group, the median increase in daily food usage during the initial 3 d of the postintervention phase was significantly greater (P < 0.01) than that of the preinterventional period (D1 to D10) and the overall postintervention period (D31 to D40). (C, D) Mice in control cages did not demonstrate a change in daily food usage over the 40-d study.

Impact of relative humidity.

During the study, the average relative humidity in the animal housing room was 35%, with a daily range of 16% to 56%. The average room temperature during this time was 22.7 °C (range 21.1 to 22.8 °C). Food usage was associated with relative humidity during the preintervention period (D1 to D10). Every 1% rise in relative humidity resulted in an increase of the median daily food usage by 5.2 g/d among the 13 cages in the interventional group before the running wheel was offered (D1 to D10), P < 0.05. When 15 cages that exhibited grinding behavior (the interventional group and the control group) were combined, the median daily food usage rose with every 1% rise in relative humidity by 4.7 g/d during D1 to D10, P < 0.05. Among the interventional group, median food usage declined to 0.7 g/d with every 1% rise in relative humidity when mice had access to a running wheel (D11 to D30), P < 0.05. By comparison, the median daily food usage was not significantly associated with relative humidity among the 8 cages in the baseline group that did not exhibit food-grinding behavior over a subsequent 10-d period (P = 0.14). A summary of this data is represented in Figure 6A–D.

Figure 6.

(A, B) Linear regression models displaying every 1% rise in relative room humidity had a greater effect on the daily median food usage during D1 to D10 for (A) the interventional cages (5.2 g) and (B) both interventional and control cages together (4.7 g). (C) Humidity was not significantly associated with food usage in the baseline cages that did not exhibit food-grinding behavior. (D) For every 1% rise in relatively room humidity, cages that had access to running wheels used an additional 0.7 g of food.

Discussion

This study was undertaken to determine if access to a running wheel could reduce excessive food grinding in mice. We have observed excessive food-grinding behaviors at our institution to be highest among young adult mice between 3 and 9 mo of age. Although mice from a variety of stocks and strains may exhibit this behavior, at OUHSC we have identified several batches of CD-1 and J:DO mice that seem to exhibit disproportionately high frequencies of food-grinding behavior compared with other stocks and strains (unpublished observation). We decided to focus on CD-1 female mice for the current study, because these animal subjects were available in the greatest numbers to monitor food usage for the desired study duration.

Voluntary wheel running is not considered a standard form of enrichment offered to mice at OUHSC. However, angled, low-profile running wheels are commercially available that can attach to shelters and easily fit within a standard mouse cage without negatively impacting floor space.¹⁴ Koteja and colleagues previously reported that lines of Hsd:ICR mice selected for high voluntary running wheel behavior did not vary significantly in food wasting (i.e., grinding or ort production) when compared with control lines.¹⁵ However, other studies have revealed voluntary wheel running satisfies normal species-specific drives in a self-rewarding and nonstressful manner.^1,13,16 We hypothesized that under normal housing conditions within our facility, voluntary wheel running could help replace excessive food grinding among mice inclined to this behavior.

Our results demonstrate mice exhibiting food-grinding behaviors reduced their median daily food usage when they had access to a running wheel. Notably, we did not distinguish between food consumed by mice and food ground into orts in this study. We also did not attempt to monitor ort production or voluntary wheel running activity among individual animals. Doing so may have revealed greater insight into the nature of food grinding among socially housed mice, but would have also likely required more manipulation of cages and animals in a manner not reflective of routine husbandry interactions performed within our facility. We also suspect variation in food-grinding behavior in individual mice is likely to be high within cages of socially housed animals. Further, voluntary wheel running can directly impact food consumption⁶ and thereby interfere with our assessment of grinding and its impact on overall food usage. To help address this, we identified 8 cages of CD-1 mice that did not exhibit food-grinding behavior but were housed under similar conditions in the same location, and evaluated their median daily food usage over a 10-d period to establish a baseline representation of the typical rate of food consumption with minimal-to-no ort production. This revealed a baseline voluntary food intake among female CD-1 mice to be 22.5 g/d. This rate was notably lower than the 36 g/d intake recently described for male CD-1 mice of similar age at another institution.³ Establishing a baseline from nongrinding mice in our facility was therefore necessary and useful to our data analysis.

Once running wheels were removed from cages in the interventional group, the median daily food usage increased sharply. This suggests that in the absence of voluntary wheel running, mice that have previously exhibited food-grinding behaviors will return to this behavior. This was further demonstrated when median daily food usage rose sharply during the first 3 d of removing the running wheels (D31 to D34), as shown in Figures 3A, 4, and 5A. We also observed that relative room humidity was highest (56%) during this period, which may have partially contributed to the temporary spike in median daily food usage. However, a rebound effect in which mice increased food usage directly in response to the absence of the running wheel is more consistent with the results of our data analysis. The following decrease in both median daily food usage and humidity starting on D36 may suggest that relative humidity partially contributed to changes observed in median daily food usage during this time. Cordeira recently reported the mass of a very high-fat diet (VHFD) stored in the food hopper of standard mouse cages can vary based on changes in humidity over time⁷; although VHFD was not used in our study, these data support the notion that food quality, and how animals interact with food, may be influenced by relative humidity.

These results support the use of physically engaging types of enrichment devices to decrease the severity of food-grinding behavior. Although we did not explore this in depth in the current study, the spike in food usage we observed when we removed the running wheels from the interventional cages at D30 suggests that separation from the running wheel may interfere with its potential benefits on food grinding. At institutions where enrichment devices are routinely replaced at regular intervals to facilitate novelty, mice exhibiting food-grinding behaviors may be better served with continuous access to devices that facilitate voluntary exercise. Continuous access to a running wheel is unlikely to eliminate ort production in mice prone to grinding food. However, this study offers evidence in support of the use of a running wheel to reduce ort production.

Our study overlapped with a seasonal shift from warmer to colder weather in Oklahoma, which typically corresponds to a period of declining room humidity levels within our animal facilities. Notably, the decreased median daily food usage in cages with a running wheel during D11 to D30 despite relatively high room humidity supports our conclusion that voluntary wheel running helped suppress the effects of humidity on daily food usage in mice exhibiting food-grinding behavior. It is also worth noting that even though humidity was at virtually the same percentage during D16 as it was during D36 to D40, the presence of the running wheel during D16 to D19 had a more successful result in reducing grinding rates to a normal level of food consumption than simply low humidity alone.

The study was performed between September and November of 2022. Oklahoma City is located in a humid subtropical climate range, with mean temperatures between 14 and 22.2 °C and approximately 89 cm of precipitation annually.²¹ The time in which the study took place overlapped with an expected seasonal shift to colder external temperatures with lower relative humidity. The control group of mice exhibiting food-grinding behavior was monitored primarily to help us evaluate fluctuations in relative humidity within the housing room during the 40-d study. Although room temperature within the animal housing room remained within a relatively narrow range (21.1 to 22.8 °C) throughout the study, relative humidity fluctuated more widely (16% to 56%). During 14 d of the 40-d study, relative humidity fell below 30%, the minimum threshold recommended for animal housing rooms by the Guide.¹⁴ Half of these low-humidity days occurred during the study’s postinterventional period (D34 to D40).

There is some debate regarding whether the clinical symptoms historically attributed to low humidity in humans (primarily eye- and airway-associated health conditions) should instead be attributed at least in part to indoor air pollutants.²⁷ Daily veterinary records indicated that all animals involved in this study remained clinically healthy despite fluctuations in room humidity. In general, as relative humidity and temperature increase the overall heat index increases, which in turn will make an animal feel warmer.²⁰ Mice respond to heat by either lowering their metabolism or raising their overall body temperature.^5,12 The Nutrient Requirement of Laboratory Animals¹⁹ states that “exposure to temperatures below the threshold of the thermonuclear zone increases energy requirements as animals are obliged to expend energy to maintain a constant body temperature.” If we assume the reverse is also true, then mice that are experiencing higher temperatures may have excess energy left unspent, which could result in increased grinding as relative humidity rises with increased temperatures.

We speculate that the observed correlation between relative humidity and increased food usage may have been due to the influence of humidity on the relative hardness or texture of food pellets in the hopper within the cage. Previous reports indicate food pellets with greater hardness qualities reduce food grinding or wastage when compared with softer pellets.¹⁰ Further, the mass of pellets is known to change over time based on relative humidity.⁷ Thus, it seems possible that when relative humidity is low, food pellets become drier and therefore more challenging for mice to grind throughout the day. Additional studies are needed to confirm the association between relative humidity and food grinding in mice and to elucidate potential causes.

Energy expenditure in mice is dependent in part on environmental conditions, including temperature and humidity. The lower critical temperature of mice is around 30 °C; however, the temperature and humidity levels within the cage may be substantially higher than those of the room in which the cages are housed.²⁵ Further, group housing density, age, and individual animal health status can also influence energy expenditure.²⁵ Because we did not monitor temperature and humidity at the cage level or assess body composition or energy expenditure among individual mice, we cannot definitively conclude that the correlation we observed between relative room humidity and median daily food usage is attributed to an elevated energy expenditure. However, the reduction of median daily food usage when running wheels were available despite fluctuations in relative humidity supports the theory that elevated food-grinding behavior may be attributed to energy expenditure needs.

A limitation of this study was the sample size; in particular, only 2 cages of mice were assigned to the control group. Given the variability in food usage observed between cages, this small sample may not be representative of a larger population. In the future, we plan to evaluate the impact of voluntary wheel running among food-grinding mice using larger sample sizes. It would also be beneficial to evaluate ort production and energy expenditure more directly to evaluate access to voluntary exercise and its impact on mice inclined to engage in food-grinding behavior. While individually housed animals may not behave the same as group-housed mice, including individually housed animals to measure and compare food-grinding and voluntary wheel running outcomes in a direct manner would be useful in future studies. The true relationship between humidity and food grinding should also be explored in more detail. Further, it would be desirable in the management of this behavior to know if food grinding declines with age in the same manner as metabolism. We suspect that as animals age and their metabolic rate slows, their energy expenditure needs, and thus food grinding, may also decline. Additional studies are needed to understand the differences in mouse stock or strain characteristics on energy expenditure and the effects of relative humidity on the cage environment. If certain characteristics of mice make them more susceptible to food-grinding behavior, it would also be useful to understand how humidity influences the rate of food grinding.

We observed a positive correlation between increased relative humidity and median daily food usage. Figure 6A–D demonstrates that even during periods of relatively high humidity (D20 and D28 to D32), mice with access to running wheels maintained relatively low median daily food usage compared with periods of higher humidity (D3 to D4 and D8 to D10) when the same mice did not have access to running wheels. Our results suggest that among CD-1 mice inclined to exhibit excessive food-grinding behavior, they are more likely to grind food when housed in an environment that does not otherwise accommodate energy needs. The addition of a tilted running wheel in the cage may reduce daily food usage by providing mice with a more productive outlet to meet energy expenditure needs. Further, room humidity may also play a role in the degree to which mice excessively grind food. However, lowering the relative humidity in an animal housing room is likely to be less effective in reducing food grinding than the addition of a running wheel in the cage. Thus, incorporating running wheels into environmental enrichment protocols may be a viable option to improve both animal welfare and facility efficiency when the research model allows voluntary wheel running.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

This word was internally funded through the Office of the Vice President for Research at the University of Oklahoma Health Sciences Center.