Minimising aggression in group-housed male mice
Recommendations for reducing aggression between group-housed male mice.
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Within a research environment, mice are exposed to husbandry conditions that can disrupt social stability and limit the ability of subordinate animals to escape from aggressors. Consequently, aggressive behaviour in laboratory mice can escalate into a serious welfare issue, resulting in stress, pain and even death. The stress caused by poor welfare alters animal physiology, influencing the scientific outcomes of experimental studies and resulting in potentially unreliable findings. A common approach to reducing aggression-related injuries is to singly-house the aggressor or injured mouse. However, individually housing social animals or animals that have become accustomed to group living is itself a welfare concern [1,2].
This resource provides recommendations for reducing aggression between group-housed male mice. It also offers guidance on monitoring aggression and taking action when aggression is evident. The recommendations are based on peer reviewed literature and expert guidance, including the findings of a NC3Rs crowdsourced study which obtained data from 137,580 male mice at 44 facilities. Although both male and female mice engage in aggressive encounters, aggression is more commonly reported and cited as a welfare concern in males. This resource focuses on aggression in male mice but some of the more general information may apply to both sexes.
Recommendations for reducing aggression
Aggression in group-housed laboratory mice is influenced by genetic, social and environmental factors. The following recommendations provide practical advice on factors known to influence aggressive behaviour in male mice.
Where it is possible to do so, choose strains that show a low prevalence of aggression.
Some strains of mice are more aggressive than others. Check what is known about aggression for the strain(s) you work with or plan to work with. Information on aggressive tendencies can be found online (e.g. www.mice.jax.org; in the scientific literature), or directly from your supplier.
Higher levels of aggression have been noted in male C3H, CBA, DBA, CD1 , male SJL  and male BALB/c  mice. A lower prevalence of aggression has been observed in 129S, BALB/c  and C57BL/6 mice [3,6].
If a choice of strain is possible for the scientific aims of your study, choose a strain showing a low prevalence of aggression. If using a more aggressive strain is necessary, it is important to know what strain-specific behaviours to expect. This knowledge can be used to inform housing and husbandry ; for example, it may be necessary to remove SJL/J males after mating to avoid aggression towards the female and pups .
Ask your supplier what steps they take to minimise the risk of aggression and discuss what more can be done; for example, request that animals are transported with littermates during the ordering process.
Transportation is a major life event for mice and wherever possible it should be avoided by instead transporting fresh embryos or cryopreserved embryos and gametes. The preparations and processes around transportation of live mice can influence the prevalence of aggressive behaviours after mice have reached their destination. Hence the source of mice can be associated with increased levels of aggression .
If excessive aggression is noted in mice purchased from a certain supplier, ask what steps they take to minimise social disruption and stress (e.g. transporting with littermates and habituating to transport boxes). If necessary, make specific requests for the transportation process.
Establish stable groups with littermates prior to sexual maturity and minimise social disruption. When allocating mice to experimental groups, do so without mixing unfamiliar mice.
Selecting cage mates from littermates is associated with a decreased prevalence of aggression and potentially higher welfare in male mice [3,9,10]. When housed in same-sex sibling groups, young animals can establish their dominance hierarchy early on, and once group stability is established excessive fighting is not necessary . Removal of the dominant individual will result in fighting to establish a new dominance hierarchy.
Introducing the scent of unfamiliar mice can also disrupt group stability, so ensure that steps are taken to avoid this when moving between cages. Introducing the scent of female mice into a group of males is a major trigger of aggression that should be avoided. House the sexes apart, clean males before females and be mindful of PPE, clothing or equipment that may have come into contact with female mice [8,11].
Selecting cage mates randomly is associated with an increased prevalence of aggression . If randomisation is required as part of the allocation process for experimental design, this can be done without mixing unfamiliar mice in a cage (see  for more details).
Spot clean daily and minimise the frequency of full cage cleans. Retain some clean and dry (i.e. used but not soiled) nesting material and transfer this during cage changes.
Mice communicate using scent (olfactory cues) and the presence of these scents are important for maintaining social stability in groups. Cage cleaning disturbs these scents and disrupts the social hierarchy. Spot cleaning when needed, rather than a weekly or fortnightly full cage change, is associated with a lower prevalence of aggression . When cage changes take place, the transfer of clean, dry nesting material (but not soiled bedding) decreases the prevalence of aggression [3,12].
Evaluate environmental enrichment to ensure it is improving welfare.
It is possible to reduce the prevalence of aggression in groups of mice by providing environmental enrichment that facilitates species-specific behaviours and improves animal welfare [13-15]. Increasing cage complexity (e.g. by providing partitions within the cage or providing multiple tunnels) can give subordinate mice the opportunity to move out of sight of an aggressor . Cage partitions have been shown to reduce aggression in male Balb/c mice . However, some studies have found mice can monopolise structural enrichment, such as cage furniture, leading to a higher incidence of aggressive behaviour. Therefore, environmental enrichment should be evaluated for the species, sex, strain, model and life stage of mice you intend to provide it for to ensure it is improving welfare .
Understanding sudden behavioural changes
When aggression is evident within a group of mice, the first step should be to consider each recommendation for reducing aggression. In addition to the other sources cited within the text, these practical steps are based on findings from crowdsourced data obtained from 44 UK facilities with a total of 137,580 male mice. If aggression levels suddenly increase and the recommendations for reducing aggression have been reviewed and implemented, asking the following questions may help to identify the cause of the change in behaviour.
Have there been any changes in the wider environment that may be causing stress to the animals and disrupting group stability? For example: lighting regime or intensity; temperature levels; ultrasonic sounds from equipment; aversive smells from cleaning solutions; the sight, sound of smell of other animals.
Have there been any within-cage changes that may be causing stress to the animals and disrupting group stability? These are largely covered in grouping, cleaning protocols and environmental enrichment. Environmental enrichment includes essential bedding and nesting material, and changes to the type or quantity should be evaluated. Changes in the diet that animals receive and how they are fed can also result in behavioural changes.
What individual factors might be causing some mice to have higher aggression levels? Animals that are stressed or in pain may be more likely to be aggressive. The method used to mark mice can increase aggression levels [16,17], as can breeding-related hormonal changes .
It may not be possible to completely eradicate fighting even when all the recommendations for reducing aggression are implemented. It is important to monitor aggression levels and the impact on mouse welfare, and protocols should be in place for this. Monitoring should be carried out in a consistent way with a clear definition of aggressive behaviour and validated welfare tools [18,19]. Home cage monitoring software can be employed for investigations into triggers of aggression and for continual monitoring of behaviour and welfare. Automated systems are particularly useful for monitoring behaviour during the dark phase of the light-dark cycle when mouse activity levels are higher and aggressive interactions are more prevalent . Signs of aggression can be observed from behaviour and physical evidence , such as:
- Submissive behaviour (e.g. crouching)
The mouse should be checked from head to tail, as aggression-related injuries can occur anywhere on the animal. However, a high concentration of aggression-related injuries are focused towards the rear :
- Tail (wounds)
- Rump and back (wounds and hair loss)
- Urogenital region (wounds)
Where severe cases of aggression are occurring and the actions outlined in recommendations for reducing aggression have been carefully reviewed and implemented, then there may be no choice but to house singly. Single housing of animals that are accustomed to group living should be a last resort but grouping should be reviewed on a case-by-case basis . If mice are single housed they will require additional nesting material to maintain their body temperature and additional environmental enrichment should also be provided. Visual and olfactory contact with other mice may be beneficial to the welfare of singly-housed individuals  but this should be evaluated on an individual basis as it can also be a stressor .
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