Article Brianna Gaskill, PhD Postdoctoral Research Scientist, Charles River Laboratories

Aggression in Laboratory Mice: Potential Influences and How to Manage It

Mouse aggression is a hot topic stable and are more vigorously and is the first question asked defended compared to wild when I give a talk on mouse territories where it is more behavior. “My mice are killing difficult to patrol and defend each other, what do I do?” vast areas10. Other aspects There are two obvious of the environment, such as reasons why this behavior is temperature, influence the so troubling to anyone who rigidity of territory boundaries11. works with laboratory mice. Dominant mice owning a ter- The first is concern for the ritory are likely to tolerate an animals. Excessive aggression outsider when temperatures leads to wounding, pain, are low and both mice benefit and inevitably suffering. thermally from huddling11. The common solution to Similar results have been alleviating unwanted aggression found in laboratory mice; less is through solitary housing, aggression occurs in cooler which is likely to cause other include food, a water source, temperatures12. Unfortunately, sources of stress for mice1, or access to breeding females. laboratory temperatures already which are social animals. The spatial and temporal induce cold stress13-14, which Second, is concern for the distributions of these resources can effect various physiologi- science. Aggression, pain, strongly influence the devel- cal systems altering scientific and social isolation can alter opment of territoriality even results15-18. Thus cooling physiological parameters, such within the same species. If laboratories further is not as circadian rhythm, glucocor- resources are in short supply a solution to reducing or ticoid levels, and immune or spread out, this will increase alleviating aggression. function2-6, creating variability the amount of space needed to The natural history of the wild and issues of scientific validity. support an individual or group7. house mouse also provides The best solution for aggres- For instance, territory size can insight into motivations and sion is prevention instead of be as large as 80,000 m2 for needs of the domesticated reaction. However, before mice in wheat fields where laboratory mouse. Vulnerability aggression can be prevented, resources are patchy or scarce9 to predation has shaped the we need to know what the and as small as a few square behavior and life history purpose of the behavior is, and meters in mice living commen- strategies of mice19. They are what causes and influences it. sally with people where high nocturnal, showing peaks of quality resources are frequently activity at dawn and dusk20-22, Aggression is a natural found10. Because of the stabil- and avoid brightly lit and open behavior related to territories ity of resources found in close spaces19,23. Mice live in complex and the resources they proximity to humans, commen- and variable social systems. contain7-8. Resources could sal territories remain relatively

22 WINTER 2014 | THE ENRICHMENT RECORD They generally live in loose kin interactions between territory before this age. groups called ‘demes’ which holders (dominant mice) and A physical structure or cover include a male, 1-2 breeding intruders (subordinates) consist is necessary for territory females (which are usually of a frontal attack by the owner formation11,33. Aspects of related), subadults, and pups10- and the intruder fleeing. Chasing physical structures in the 11. One male owns a territory ceases once line of sight is environment are utilized by but other adults, including fe- broken or the intruder has left wild mice to ambush intruders males, will defend it fiercely11. the boundaries of the territory11. through holes, choke-points, Groups of bachelors can be Since exiting a territory or and elevated platforms11. found when dispersion is re- breaking line of sight is difficult Research on the provision of stricted or there are no avail- to achieve within the confines retreat spaces in laboratories able territories to claim11. These of a standard laboratory cage, has produced mixed results. animals tend chasing duration may be Cardboard tubes have been to be subordinate to animals exacerbated in the laboratory. found to reduce wire-gnawing holding territories and Interactions between two stereotypies34 but the authors relatively more active during territory holders are more likely could not directly attribute the day when territory holders to result in injurious fighting11, the reduction to the fact that are asleep11. but this is unlikely to happen in the mice utilized the tube as the laboratory, where territories a retreat space since the tube As a social animal, mice exhibit are limited to cages. Inter-male was also used as nest build- many types of behaviors that dominance hierarchies are ing substrate. Nest boxes and maintain social structure. found, again when territories hard plastic shelters are very Affiliative behaviors, such as are restricted11. Van Loo et al.28 popular, easily cleaned, and grooming others, function found that group size influ- make the cage seem “enriched”. to strengthen social bonds. enced agonistic interactions and These structures generally Mediated aggression is an wounding more than housing increase male aggressive agonistic interaction which density. In that particular study, interactions24,35 but have solidifies dominance hierarchies conducted with one inbred also been found to increase but avoids fighting. An example strain, groups of 5 or 8 mice longevity35. Providing a shelter of this is a dominant mouse had more wounds than groups increased aggression, indicating mounting a subordinate, of 328. Dominance hierarchies that mice perceived this item as eliciting a submissive behavior in large groups of mice are less a coveted resource that should from the subordinate. This type stable29, thus resulting in more be defended24. Howerton et al,24 of aggression can account for status fluctuations and likely found that shelters increased up to 15% of daily activity24. escalated aggression. Kinship the incidence of escalated, If a subordinate responds to a and familiarity have also been injurious aggression and threat with aggressive behavior found to reduce aggressive destabilized the dominance instead of submission, the mice social interactions30-31. Aggres- hierarchy within the cage. This engage in escalated aggression. sion toward strangers has been study and only a few others36-37 This particular type of aggres- documented as early as 32-36 have utilized home cage sion can result in bite wounds, days of age32. Therefore, it may behavioral monitoring instead castration, or death if a mouse be beneficial to keep siblings of separating, disturbing, or does not display an appropriate together throughout life or placing mice in an unfamiliar subordinate signal to end the combine unrelated groups 24-27 fight . In the wild, aggressive continued on page 24

WINTER 2014 | THE ENRICHMENT RECORD 23 Aggression in Laboratory Mice continued from page 23

setting. When aggression is Urinary pheromones are used it escalates to injurious fighting. measured after disturbing or to determine the “maleness”of One study did find that pups stressing the mice or altering rivals, mark territory boundaries, weaned early (14 days the environment, this may and can elicit aggression43-44. of age) had more wounds than potentially be measuring differ- Importantly, nest sites are kept pups weaned at 21 days47. ent types of aggression or moti- clean of urine and feces and This particular study, unlike vations, making comparisons to perhaps free of these aggres- normal husbandry, isolated normal husbandry difficult24. sion eliciting pheromones 31,45. mice for 4 weeks before regrouping them with their Nesting material is preferred Another potential influence on wean group. Therefore, these by mice over nest boxes38 and mouse aggression is weaning isolation-induced results may mice are willing to work to gain and the early life experiences not be directly applicable to access to it39-40. Different leading up to it. In the wild, aggression seen in continuously materials can be manipulated weaning is a gradual process. housed mice. and combined to create a Pups begin to eat solid food flexible structure that decreases between 10-17 days of age and Aggression is a naturally cold stress and provides both nursing is significantly reduced occurring behavior of mice that structural and occupational after 21 days10,46. Maternal can provide benefit to those enrichment for mice. Nesting interactions, however, can that display it in the wild11. material and its transfer at cage continue up to 4 weeks after Many factors appear to influ- change has been found to pups are born. Even after pups ence these negative social reduce aggressive interactions31,41. are weaned, they are likely to interactions between mice, It is important to note that one remain in the natal nest until making mitigation in the study did report increased sexual maturity, continuing to laboratory more complicated. aggression when mice were interact with adult females and Based on the current literature provided with compressed the dominant male. Curley et and the natural history and cotton nesting material42. al.46 found that pups weaned motivations of mice, a few However, the mice in that study at 28 days were more likely to management strategies may did not spend much time in engage in longer bouts of social help keep escalated aggres- contact with the material and interaction than pups weaned at sion in the laboratory to a usage was not described. 21 days of age. They also found minimum41. First combine small The mechanism behind the that pups were increasingly stable groups at 3-4 weeks of mitigation of aggression by mounted by their dam as they age, so they are familiar with nests has not been specifically neared weaning46. This is likely one another prior to puberty. tested. Van Loo et al31 postulate the adult demonstrating their Avoid providing mice, males in that specific pheromones dominance over the pup, while particular, with items or enrich- deposited within the nest may exposing the pup to social cues. ments that can be monopolized be the reason for this Whether pups need this and guarded by dominant mice, behavioral response compared exposure prior to separation such as rigid shelters. Last, to behaviors seen after the to properly react to certain transferring nesting material transfer of soiled bedding. social cues has not been formally at cage change may maintain This is not surprising since tested. If it is necessary, it’s some olfactory cues related scent and pheromones play possible that mice without those to identification without such an instrumental role in experiences do not understand transferring aggression-eliciting rodent communication43-44. how to stop aggression before pheromones.

24 WINTER 2014 | THE ENRICHMENT RECORD References 17 Gaskill, B. N. et al. Impact of nesting mate- 34 Würbel, H., Chapman, R. & Rutland, C. rial on mouse body temperature and physiology. Effect of feed and environmental enrichment 1 Gonder, J. C. & Laber, K. A renewed look at Physiol. Behav. 110, 87-95, doi:10.1016/j.phys- on development of stereotypic wire-gnawing in laboratory rodent housing and management. beh.2012.12.018 (2013). laboratory mice. Appl. Anim. Behav. Sci. 60, Ilar Journal 48, 29-36 (2007). 69-81 (1998). 18 Rudaya, A. Y., Steiner, A. A., Robbins, J. R., 2 Pham, T. M. et al. Housing environment 35 Swetter, B. J., Karpiak, C. P. & Cannon, J. T. Dragic, A. S. & Romanovsky, A. A. Thermoregula- influences the need for pain relief during post- Separating the effects of shelter from tory responses to lipopolysaccharide in the mouse: operative recovery in mice. Physiol. Behav. 99, additional cage enhancements for group-housed dependence on the dose and ambient tempera- 663-668, doi:10.1016/j.physbeh.2010.01.038 BALB/cJ mice. Neurosci. Lett. 495, 205-209, ture. American Journal Of Physiology-Regulatory (2010). doi:10.1016/j.neulet.2011.03.067 (2011). Integrative And Comparative Physiology 289, 3 Spani, D., Arras, M., Konig, B. & Rulicke, T. R1244-R1252, doi:10.1152/ajpregau.00370.2005 36 Nevison, C. M., Hurst, J. L. & Barnard, C. J. Higher heart rate of laboratory mice housed (2005). Strain-specific effects of cage enrichment in individually vs in pairs. Lab. Anim. 37, 54-62 male laboratory mice (Mus musculus). 19 Jennings, M. et al. Refining rodent husbandry: (2003). Anim. Welf. 8, 361-379 (1999). The mouse—Report of the Rodent Refinement 4 Keeney, A. J. & Hogg, S. Behavioural Working Party. Lab. Anim. 32, 233-259 (1998). 37 Eskola, S. & Kaliste-Korhonen, E. Aspen consequences of repeated social defeat in the wood-wool is preferred as a resting place, 20 Gordon, C. J., Becker, P. & Ali, J. S. Behavioral mouse: preliminary evaluation of a potential but does not affect intracage fighting of male thermoregulatory responses of single- and group- animal model of depression. Behav. Pharmacol. BALB/c and C57BL/6J mice. Lab. Anim. 33, housed mice. Physiol. Behav. 65, 255-262 (1998). 10, 753-764 (1999). 108-121, doi:10.1258/002367799780578273 5 Miczek, K. A. Aggressive and social stress 21 Rowe, F. in Biology of the house mouse (ed R. (1999). J. Berry) 575-590 (Academic Press, 1981). responses in genetically modified mice: 38 Van de Weerd, H. A., Van Loo, P. L. P., from horizontal to vertical strategy. 22 Southern, H. N. House mice. Vol. 3 (Oxford Van Zutphen, L. F. M., Koolhaas, J. M. Psychopharmacology 147, 17-19, doi:10.1007/ University Press, 1954). & Baumans, V. Strength of preference for s002130051132 (1999). nesting material as environmental enrichment 23 Gray, J. A. The psychology of fear and stress. for laboratory mice. Appl. Anim. Behav. Sci. 55, 6 Barnard, C. J., Behnke, J. M. & Sewell, J. 2nd edn, (Cambridge University Press, 1991). Environmental enrichment, immunocompetence, 369-382 (1998). and resistance to Babesia microti in male mice. 24 Howerton, C. L., Garner, J. P. & Mench, J. A. 39 Gaskill, B. N. et al. Heat or insulation: Physiol. Behav. 60, 1223-1231 (1996). Effects of running wheel-igloo enrichment on Behavioral titration of mouse preference for aggression, hierarchy linearity, and stereotypy in 7 Macdonald, D. W. The ecology of canrivore warmth or access to a nest. PLoS ONE 7, e32799 group-housed male CD-1 (ICR) mice. Appl. Anim. (2012). social behavior. Nature 301, 379-384 (1983). Behav. Sci. 115, 90-103 (2008). 40 Roper, T. J. Nesting material as a reinforcer 8 Gray, S. J., Jensen, S. P. & Hurst, J. L. Effects 25 Mouse Ethogram, www.mousebehavior.org of resource distribution on activity and territory for female mice. Anim. Behav. 21, 733-740 defence in house mice, Mus domesticus. Anim. 26 Grant, E. C. & Mackintosh, J. H. A comparison (1973). Behav. 63, 531-539 (2002). of the social postures of some common laboratory 41 Van Loo, P. L. P., Van Zutphen, L. F. M. & rodents. Behaviour 21, 246-259 (1963). 9 Chambers, l., Singleton, G. & Krebs, C. Baumans, V. Male management: coping with ag- Movements and social organisation of wild house 27 Van Abeelen, J. H. F. Mouse mutants studied gression problems in male laboratory mice mice (Mus domesticus) in the wheatlands of by means of ethological methods.1. Ethogram. Lab Anim (Lond) 37, 300-313 (2003). northwestern Australia. Journal of Mammology 81, Genetica 34, 79-& (1963). 42 Kaliste, E. K., Mering, S. M. & Huuskonen, 59-69 (2000). 28 Van Loo, P. L. P., Mol, J. A., Koolhaas, J. M., H. K. Environmental modification and agonistic 10 Latham, N. & Mason, G. From house mouse van Zutphen, B. F. M. & Baumans, V. Modulation behavior in NIH/S male mice: Nesting material to mouse house: The behavioural biology of of aggression in male mice: influence of group enhances fighting but shelters prevent it. free-living Mus musculus and its implications size and cage size. Physiol. Behav. 72, 675-683 Comp. Med. 56, 202-208 (2006). in the laboratory. Appl. Anim. Behav. Sci. 86, (2001). 43 Brown, R. E. in Social odours in mammals 261-289 (2004). Vol. I eds Richard E. Brown & David W. 29 Poole, T. B. & Morgan, H. D. R. Differences 11 Crowcroft, P. Mice all over. (G.T. Foulis and CO MacDonald) Ch. 9, 345-457 (Oxford University in aggressive behviour between male mice (Mus Press, 1985). LTD, 1966). musculus) in colonies of different sizes. Animal 12 Greenberg, G. The effects of ambient tempera- Behavior 21, 788-795 (1973). 44 Arakawa, H., Cruz, S. & Deak, T. From ture and population density on aggression in two models to mechanisms: Odorant communication 30 Kareem, A. M. & Barnard, C. J. as a key determinant of social behavior in inbred strains of mice, Mus musculus. Behaviour The importance of kinship and familiarity in social 42, 119-130 (1972). rodents during illness-associated states. interactions between mice. Anim. Behav. 30, Neurosci. Biobehav. Rev. 35, 1916-1928, 13 Gordon, C. J. Temperature regulation in labora- 594-601, doi:10.1016/s0003-3472(82)80073-0 doi:10.1016/j.neubiorev.2011.03.007 (2011). tory rodents. (Cambridge University Press, 1993). (1982). 45 Blom, H. J. M. et al. in Evaluation of housing 14 Cannon, B. & Nedergaard, J. Nonshivering 31 Van Loo, P. L. P., Kruitwagen, C. L. J. J., conditions for laboratory mice and rats (ed H. J. thermogenesis and its adequate measurement Van Zutphen, L. F. M., Koolhaas, J. M. & Baumans, M. Blom) 81-99 (Febodruk, 1993). in metabolic studies. J. Exp. Biol. 214, 242-253 V. Modulation of aggression in male mice: (2011). Influence of cage cleaning regime and scent 46 Curley, J. P. et al. The meaning of weaning: marks. Anim. Welf. 9, 281-295 (2000). Influence of the weaning period on behavioral 15 Kokolus, K. M. et al. Baseline tumor growth development in mice. Dev. Neurosci. 31, 318- and immune control in laboratory mice are sig- 32 Scott, J. P. Agonistic behavior of mice and rats: 331 (2009). nificantly influenced by subthermoneutral housing A review. Am. Zool. 6, 683-701 (1966). temperature. Proceedings of the National Academy 33 Mackintosh, J. Factors affecting the recognition 47 Kikusui, T., Takeuchi, Y. & Mori, Y. of Sciences, doi:10.1073/pnas.1304291110 of territory boundaries by mice (Mus musculus). Early weaning induces anxiety and aggres- (2013). Anim. Behav. 21, 464-470 (1973). sion in adult mice. Physiol. Behav. 81, 37-42, doi:10.1016/j.physbeh.2003.12.016 (2004). 16 Yamauchi, C., Fujita, S., Obara, T. & Ueda, T. Effects of room temperature on reproduction, body and organ weights, food and water intakes, and hematology in mice. Exp. Anim. 32, 1-11 (1983).

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