The Use of Cage Enrichment to Reduce Male Mouse Aggression Neil Ambrose & David B. Morton Published online: 04 Jun 2010.

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3 JOURNAL OF APPLIED ANIMAL WELFARE SCIENCE, 3(2), Copyright 2000, Lawrence Erlbaum Associates, Inc. The Use of Cage Enrichment to Reduce Male Mouse Aggression Neil Ambrose and David B. Morton Centre for Biomedical Ethics The Medical School, University of Birmingham The complete cleaning of cages has been shown to reduce the level of intermale aggression in mice. This study investigated the effects of the addition of enrichment objects on postcage-cleaning aggression in male BALB/c mice. Enrichment objects were found to significantly reduce aggressive interactions during this period for up to 7 weeks and can make an overall economic saving to husbandry costs. Research animals spend the majority of their time in cages, and frequent cleaning is an unavoidable consequence of keeping these nonhuman animals in captivity. During the period immediately following cage cleaning, the level of aggression in male mice can rise to high levels (Gray & Hurst, 1995). If the levels of aggression reach a point at which there is a risk of severe injury, the cage is usually split and aggressive are animals kept singly, with the welfare problem of permanent solitary confinement of social animals. The factors influencing aggression in male mice are numerous. The first is the familiarity of olfactory cues (Hurst, 1990, 1993; Hurst, Fang, & Barnard, 1993; Jones & Nowell, 1973; Mugford, 1973). It has been shown that complete cage cleaning (new cage and new substrate) can reduce aggression in male CFLP mice, compared with partial cage cleaning, and that dominant animals are more likely to be aggressive to both familiar and strange animals when surrounded by familiar odors (Gray & Hurst, 1995). Other aggression modulators are the familiarity of visual and vibrissal (tactile) cues. As with olfactory cues, the increasing familiarity of the environment leads to an increasing level of aggression, with the dominant animal in his home cage displaying the greatest number and shortest latency of aggressive acts (Jones & Nowell, 1973). Armstrong, Clark, and Peterson (1998) Requests for reprints should be sent to David B. Morton, Centre for Biomedical Ethics, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

4 118 AMBROSE AND MORTON showed that cornhusk enrichment provided subordinate mice with more flexible escape routes, thus avoiding aggressive interactions. Takingthesefactorsintoconsideration,theaimofthisstudywastoseeifthelevel of aggression after a total cage change could be improved by the addition of enrichment objects(ambrose& Morton, 1997). Indeed, Armstrong et al.(1998) found that the addition of cornhusk nesting material led to a decrease in the infliction of wounds by the dominant mouse. Enrichment objects should both decrease the level of familiarity of the cage and give the mice more objects with which to interact. The major hurdle to the general introduction of enrichment objects is cost. The disposable objects used in the short-term study detailed in this article (wooden block, cardboard box, and cardboard tube) cost approximately 36 pence(u.s. $0.50) per cage, and thus the effect of changing the cage but returning the soiled enrichment objects back into the clean cage was investigated. The long-term effect of the use of an nondestructible object (glass water bottle) that can be cleaned and reused numerous times was also measured to evaluate the significance of olfactory cues: The glass bottle cannot be physically altered by the mice only its olfactory profile when the bottle is cleaned. Short-Term Study MATERIALS AND METHODS A total of 66, 7-week-old, male SPF BALB/c mice were obtained from the University of Birmingham breeding colony. The animals were housed in 11 groups of 6 mice each. The groups were allowed to acclimate to their new surroundings for 2 weeks before the experiment began. This group size was chosen as the animals were between 2,630 g throughout the course of the study, close to the maximum stocking levels recommended by the Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (Home Office, 1989). The animals were housed in polypropylene M3 ( cm) cages (North Kent Plastics, Rochester, UK) on sawdust substrate with tissue paper bedding. All cages were kept on the same ventilated rack. Food (Rat and Mouse Maintenance diet, Special Diet Services, UK) and tap water were provided ad libitum. All animals were maintained on a 12:12 light dark cycle (lights were on from 7:30 a.m. 7:30 p.m.), at a temperature of ºC and a relative humidity of 40 60%. The animals were observed daily for signs of aggression, such as blood in the sawdust or wounds on the tail, flank, or ear. At no time did the animals suffer any injury more severe than slight tail bites. During the study, the animals had their cages changed twice a week. Water was topped up on the first cage clean, but clean water bottles were given on the second. Cages were changed in the late afternoon

5 CAGE ENRICHMENT AND MALE MOUSE AGGRESSION 119 when extraneous disturbance was minimal. During the cage changing process, the animals were moved from the soiled cage directly into a clean cage. When soiled enrichment objects were carried over to a clean cage, they were moved prior to the animals. During the initial 2 weeks of acclimation, the animals were observed for 3 hr after cage cleaning to identify the period of maximum postcage-change aggression. Gray and Hurst (1995) showed that postcage aggression peaked in fewer than 15 min. Aggression in our study was also found to peak within the first 15 min; because levels of aggressionwerestillraisedat45min,itwasdecidedthatduringthestudyeachgroup would be videotaped for 1 hr immediately after the first cage clean of the week. The mice were subjected to five treatments (see Table 1). The enrichment objects were all of a disposable type (Bantin & Kingman Universal, Hull, UK) and consisted of a white cardboard mouse box ( cm), a softwood block (12 3 3cm), and a white cardboard tube (D = 3.5 cm, l = 7.5 cm). It should be noted that the enrichment objects were partially destroyed during the first week and in the treatment in which soiled enrichment objects were transferred to a clean cage (Week 3), the visual and tactile profiles of the objects would have changed. In each treatment involving enrichment objects, care was taken to arrange the objects in the same position and orientation (see Figure 1). Week TABLE 1 Experimental Treatments in Short-Term Study Treatment (Completely Clean Cage, Substrate, and Bedding) 1 No enrichment 2 New and novel enrichment added 3 Used enrichment transferred to clean cage 4 New but no longer novel enrichment added 5 No enrichment FIGURE 1 Enrichment object layout short-term study.

6 120 AMBROSE AND MORTON Analysis. The videotapes were viewed and a time budget analysis was carried out to determine the amount of time spent in aggressive (chasing, threatening, biting, and fighting) interactions (Gray & Hurst, 1995). A second observer viewed five randomly selected time periods, and these were found to differ from the original observations by less than 5%. Data were compared using a Friedman s test, with a post hoc k-comparisons Mann Whitney U test conducted on critical data points. Long-Term Study Sixty, 8-week-old, male BALB/c mice (SPF health status) were obtained from the University of Birmingham breeding colony. The animals were housed in groups of 5, with five enriched groups and seven nonenriched groups. As the animals weights were greater than 30 g at the end of the study, the group size was set at 5. Using a group size of 6 would have exceeded the maximum stocking density recommended by the Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (Home Office, 1989). All groups were allowed to acclimate to their new environment for 2 weeks before the start of the experiment. The animals were housed in caging identical to the animals in the short-term study. Animals were observed daily for signs of excessive aggression, and three nonenriched groups had to be split up and removed from the study as levels of aggression in these groups reached a point at which the mice were at risk of serious injury or death. During the study, all the animals had their cages changed twice a week, water was topped up on the first cage change, and clean fresh plastic water bottles were given on the second. Cages were changed in the late afternoon when extraneous disturbance was minimal. Enriched groups were provided with a single glass water bottle (11 6 cm, 3 mm glass thickness) per cage. Two glass enrichment bottles were assigned to each enriched group and the bottles exchanged during the first cage change of each week. Soiled glass bottles were cleaned in water with Hibi-Scrub used as a detergent, thoroughly rinsed in tap water, and air dried. In Week 0, both the enriched and nonenriched groups were subjected to the nonenriched treatment, with the enrichment object added to the enriched groups for Weeks 1 7. The study ran for 8 weeks, as this is the average time that male mice were kept in the laboratory in which the study was conducted. All the groups were videotaped during the 15 min after the first cage clean of the week. Analysis. The videotapes were viewed and a time budget analysis was carried out as in the short-term study. In this study, aggression (chasing, threatening, biting, and fighting) was determined. A second observer viewed five randomly se-

7 CAGE ENRICHMENT AND MALE MOUSE AGGRESSION 121 lected time periods, and these were found to differ from the original observations by less than 5%. A k-group regression analysis was conducted on the data from Weeks 1 7 with the control data (Week 0) compared using a Mann Whitney U test. Short-Term Study RESULTS Results for the short-term study can be seen in Figure 2 and statistical analysis in Table 2. The results of the Friedman test showed that aggression significantly changed over the 5 weeks of the study (p <.0001). The post hoc Mann Whitney U test showed that aggression was significantly reduced by the addition of novel enrichment. This reduction was expected as the provision of environmental enrichment for naive animals would decrease the familiarity of the cage and, therefore, lead to reductions in aggression. FIGURE 2 Duration of the short-term study (n = 11). TABLE 2 Statistical Analysis (k-corrected Mann-Whitney U Tests) of Aggression Results for Each of the Treatment Programs Short-Term Study Treatment Week 2: Novel Enrichment Week 4: Not Novel Enrichment Week 5: No Enrichment Week 1: No enrichment * ns Week 3: Used enrichment * * Note. = not tested. *p >.05/4.

8 122 AMBROSE AND MORTON The reduction in aggression seen in Week 2 (new and novel enrichment) was reversed when the soiled enrichment was carried over into a clean cage (Week 3 treatment). Again, this was predicted because there was an increased number of olfactory, visual, and vibrissal cues, giving the cage greater familiarity than both Week 2 (new and novel enrichment) and Week 1 (no enrichment). It was also predicted that the enrichment would act as a focal point in the cage and cause animals to scent-mark in a more concentrated area (Hurst, 1987, 1993). Reintroducing the old enrichment back into the cage was shown to be an effect of soiling rather than habituation to the objects as when identical, but clean, objects were used in Week 4 (new, but not novel enrichment), the level of aggression was again significantly reduced. The return to a barren environment in the Week 5 treatment (nonenrichment) posed an interesting question as to whether the mice would see the environment as more or less familiar than Week 1 (i.e., would the mice remember the barren environment as familiar). The aggression results show no significant difference between Week 1 and Week 5, which suggests that the animals remembered the barren environment as familiar. Long-Term Study Results for the long-term study can be seen in Figure 3. The Week 0 treatment in which both enriched and nonenriched groups were housed in identical, barren cages resulted in no significant difference in levels of aggression between the two groups. Regression analysis of the nonenriched group showed a linear relation between aggression and time (time involved in aggressive interactions = week). A linear relation was also seen between aggression and time for the enriched group (time involved in aggressive interactions = FIGURE 3 Aggression results for the long-term study.

9 CAGE ENRICHMENT AND MALE MOUSE AGGRESSION week). K-group regression analysis showed that the two curves had significantly different intercepts (p <.001) and slopes (p <.05), indicating that there was a divergence between the curves. Short-Term Study DISCUSSION The results of this study reaffirm previous observations that decreasing the familiarity of the environment and providing flexible avenues of escape lead to reduced levels of aggression (Armstrong et al., 1998; Gray & Hurst, 1995; Jones & Nowell, 1973). The addition of disposable enrichment aids reduced aggression to levels lower than found in the standard cage. This is likely to be due to a decrease in familiarity of the cage, with respect to visual cues, although it is unlikely that this had any effect on the olfactory makeup of the cage. The study showed that with the addition of inexpensive enrichment aids, male mouse aggression could be easily reduced, possibly negating the need to split a group into solitary animals. Both Jones and Nowell (1973) and Gray and Hurst (1995) found that the greatest level of aggression was found in cages that had undergone incomplete cleaning, such as a renewal of substrate only. This study closely mirrored this situation when the cages were changed and soiled enrichment aids returned. This has important implications where disposable enrichment aids are used routinely. Attempts to reduce costs by keeping the aids for more than one cage-cleaning cycle negates the benefit attained by their use and may lead to an increase in injuries. Gray and Hurst (1995) found that the cage grills were particularly well marked due to sustained physical contact. This study found that mice explored the enrichment aids intensely and that all aids showed urine staining which may indicate that the enrichment aids received more than average attention and marking, leading to the high increases in aggression seen in the Week 3 treatment. A second reason for this may be seen in wild mice colonies in which the dominant animal forms a raised marking post and heavily marks this area (Hurst, 1987, 1993). In the enclosed space of the cage, the enrichment aids become the predominant features and may be used by the dominant animal instead of forming marking posts. Although Week 4 treatment (new, but no longer novel enrichment added) demonstrated that the increase in aggression seen in Week 3 treatment (used enrichment transferred to clean cage) was not caused by the animals becoming habituated to the enrichment aids, when the results were compared with those for Week 2 treatment (new and novel enrichment added) it did indicate that some habituation to the enrichment aids had occurred. Therefore, it is suggested that any enrichment aids used should be changed so the mice do not become familiar with

10 124 AMBROSE AND MORTON the aids used. It should be noted that the results for Week 5 treatment show that even after 3 weeks in an enriched cage they do not find a return to a barren cage unfamiliar enough to modulate aggression. Long-Term Study The results of this study show that even a simple enrichment aid such as a glass water bottle can significantly reduce postcage-cleaning aggression compared with mice kept in a barren cage. The results also reinforce the Week 4 (new, but no longer novel enrichment added) result of the short-term study, that the animals become habituated to the enrichment: Alternatively, this increase in aggression could be caused by the growth of the animals. However, the addition of this single enrichment object was sufficient to keep aggression significantly lower than the nonenriched group throughout the whole study. As the glass bottles were nondestructible and could be deodorized by thorough cleaning, both the visual and vibrissal profiles remained the same throughout the course of the study, whereas olfactory cues were removed. As both treatments involved the removal of all olfactory cues, only the visual and vibrissal profiles are different between the experimental groups. If aggression was purely modulated by environmental familiarity, the aggression levels could be expected to converge as the study progressed, rather than the divergence that is seen in the results. This indicates that the complexities of the visual and vibrissal environments play a significant role in modulating aggression. Although the addition of enrichment aids can increase the cost of keeping animals, this study has demonstrated the efficacy of using reusable enrichment aids if they are thoroughly cleaned at cage-change time. The use of reusable enrichment aids, in combination with group-housed mice (i.e., only one cage to change compared with five or six if the mice are single-housed), would more than offset the cost of the enrichment aids. In addition, they would provide a more complex, and therefore more interesting, environment for the animals. CONCLUSION In conclusion, this study has shown that the addition of environmental enrichment can reduce the levels of aggression after cage cleaning. The short-term study mirrors the conclusions of both Armstrong et al. (1998; that enrichment leads to a decrease in aggression) and of Gray and Hurst (1995; that a complete cage clean leads to a greater reduction in aggression than a partial clean). Indeed, the return of soiled enrichment objects back into a clean cage leads to an increase in aggressive behaviors.

11 CAGE ENRICHMENT AND MALE MOUSE AGGRESSION 125 The long-term study showed that environmental familiarity is not the only factor involved in modulation of aggression and that the complexity of the cage also reduces aggression levels. Indeed, the glass bottle kept mean aggression levels below the Week 0 (no enrichment) levels for 5 weeks and significantly lower than the control group for the entire study. The levels of aggression in three of the nonenriched groups reached such levels (severe tail injuries, foot damage, and deep flank wounds) that the cages had to be removed from the study and the mice split up. In the enriched groups, none of the mice suffered more than slight tail or flank injuries. This alone is important as it fulfills one of the aims of the study, that of trying to reduce aggression to a level at which male mice can be housed in social groups rather than kept singly. ACKNOWLEDGMENT This work was financially supported by The University of Birmingham. REFERENCES Ambrose, N., & Morton, D. B. (1997). The effects of environmental enrichment on cage-cleaning aggression in male laboratory mice. B & K Science Now, 6, 13. Armstrong, K. R., Clark, T. R., & Peterson, M. R. (1998). Use of cornhusk nesting material to reduce aggression in caged mice. Contemporary Topics in Laboratory Science, 37(4), Gray, S., & Hurst, J. L. (1995). The effects of cage cleaning on aggression within groups of male laboratory mice. Animal Behaviour, 49, Home Office. (1989). Code of practice for the housing and care of animals used in scientific procedures. London: Her Majesty s Stationary Office. Hurst, J. L. (1987). The functions of urine marking in a free-living population of house mice, mus domesticus rutty. Animal Behaviour, 35, Hurst, J. L. (1990). Urine marking in populations of wild house mice, mus domesticus rutty, I. Communications between males. Animal Behaviour, 40, Hurst, J. L. (1993). The priming effects of urine substrate marks on interactions between male house mice, Mus musculus domesticus Schwarz & Schwarz. Animal Behaviour, 45, Hurst, J. L., Fang, J., & Barnard, C. J. (1993). The role of substrate odours in maintaining social tolerance between male house mice, Mus musculus domesticus. Animal Behaviour, 45, Jones, R. B., & Nowell, N. W. (1973). The effects of familiar visual and olfactory cues on the aggression behaviour of mice. Physiology and Behaviour, 10, Mugford, R. G. (1973). Inter male fighting affected by home-cage odours of male and female mice. Journal of Comparative and Physiological Psychology, 84,