Abstract
Owing to their naturally aggressive behaviour, male mice are often housed individually in toxicity studies. However, several publications advocate group-housing of mice to enable normal social behaviour and interactions between the animals. This refinement project aimed at facilitate group-housing in toxicity studies. A handling procedure, including key factors such as allocation into groups before sexual maturation, transfer of used nesting material into clean cages and avoidance of external changes, that makes group-housing of male CD-1 mice possible in long-term toxicity studies has been developed at Safety Assessment within AstraZeneca, Sweden. Observations on the effect on aggression/fighting in group-housed male mice following different procedures performed in toxicity studies have shown that temporary removal of animals from the group for blood or urine sampling does not affect the group dynamics. However, temporary removal of animals for mating leads to fighting if the animals are taken back to the original group. Treatment with test compound might affect the general condition of the animals and the social hierarchy could be changed. In such cases aggression/fighting might occur and the animals have to be separated. Our experience clearly indicates that group housing of male mice in long-term studies leads to more easily handled animals, as compared with individually housed mice.
General toxicity studies in rodents are required during the developments of new drugs. These studies are most often performed in rats or mice, but also hamsters could sometimes be used. The duration of the studies varies from single dose studies to dosing for six months. 1 For animal welfare reasons it is preferable to keep the animals in groups as far as possible; i.e. single housing should be avoided. Group-housing is no problem for rats (males or females) or for female mice. When it comes to male mice, however, their naturally aggressive nature has usually been accepted as reason to keep them individually housed in toxicity studies. In addition, group-housing of male mice has often been described as a stressful condition.2–4 However, several studies advocate group-housing of mice to enable normal social behaviour and interactions between the animals, which also is proclaimed by the new European Legislation (2010/63/UE). To reduce aggression and also stress, in group-housed male mice, factors such as group size, provision of nesting material, kinship and age have been discussed.3,5–8
Traditionally, toxicity studies are performed in CD-1 mice, for which lot of toxicological reference data are available. CD-1 mice are referred to as highly aggressive compared with other strains, such as BALB/c and C57/BL6N mice.4,9 On the other hand, studies have indicated that CD-1 mice are less susceptible to social stress than BALB/c mice. 4
The published investigations on stressfactors and aggressiveness in group-housed versus single-housed male mice are all performed in naïve mice, i.e. no test compounds or ‘study-like’ handling procedures have been included. In toxicity studies the animals are handled every day and a number of dosing and sampling procedures (blood, urine) are included; in addition test compounds might introduce effects on the general condition of the animal which theoretically could make a dominant male weak. These factors require close observations of the animals throughout the study and immediate actions on signs of changes that can lead to fights.
Safety Assessment, AstraZeneca, Södertälje, Sweden, has as a 3R project developed a process that makes group-housing of male CD-1 mice in long-term toxicity studies possible. The aim of the project was to improve the research situation for male mice in toxicity studies. The male mice were grouped 2–5 per cage and the studies were performed as previous according to regulatory toxicity test guidelines, 1 e.g. administration of test compound once or twice daily, blood and urine sampling approximately 2–3 occasions during the test period. Approximately 1000 mice are yearly included in toxicity studies and half of them are males. The length of the studies varies from one week up to six months. The group-housing procedure was also tested for male mice included in reproductive toxicity studies. As stated in the literature,3,10 and further confirmed by our own experiences, one key factor for group-housing is to allocate the animals into groups before sexual maturation. To ensure sexual maturity during a dosing period, mice are normally included in toxicity studies at an age of 8–10 weeks. Due to animal welfare an acclimation period of approximately two weeks is required, meaning that mice traditionally have been allocated into groups at 6–8 weeks of age. According to our new routines male mice (non-siblings) are allocated into groups before sexual maturation, preferably at 4–5 weeks of age, the younger the better and left for acclimatization for one week before any handling or providing of clean cages. Although the longer acclimatization period requires more personnel and facility resources, big winnings are made when it comes to animal welfare and personnel savings during the study period (more easily handled animals, fewer cages to lift and clean. etc).
In addition to allocation into groups before sexual maturation, our experience has shown that there are a number of general factors that are important for successful group-housing of male mice in toxicity studies. These include:
Change of cage bedding once weekly, i.e. more seldom than in ordinary toxicity studies in which new cages are provided at least twice weekly; At change to clean cages, clean parts on the nesting material/shelters should be transferred from the used cage to the new one.4–6 It is suggested that the nesting material contains aggression-inhibiting pheromones that reduce stress and enhances the animal welfare, hence parts containing urine and/or faeces should not be transferred;4,5 External changes should be avoided as far as possible; e.g. the animals should be kept in the same position in the room throughout the study and the routines regarding background noise and light should be the same throughout the study; Males and females could be kept in the same room – but males should always be handled before females. If the same person handles the female mice before the males, the males most likely will start fighting. Separate sets of gloves and protection cloths should be used at handling of males and females.
We have noted that the first sign of aggressive animals/fight within a group is disorder in the nest (more or less spread out) in the morning. When this is noted, an extra eye should be kept on the specific cage. Typically for such a cage is that the nest is in perfect order during the day when the animals have their sleeping period. Most often it is in the cages with disorder in the nest we first notice animals with wounds and the animals can be separated before the situation has become a problem for the animals or the study.
We have also made the following observations with regard to effect on aggression/fight in group-housed male mice following different procedures performed in toxicity studies:
A daily dosing procedure (oral, intravenous, or subcutaneous dosing) will not affect the group dynamics; Temporary taking away animals from the group for blood sampling procedure will not affect the group dynamics; Temporary taking away animals from the group for urine sampling procedure up to six hours will not affect the group dynamics; In toxicity studies, treatment with test compound might affect the general condition of the animals (e.g. a dominant male shows clinical symptom) and the social hierarchy between the animals might thereby be changed. In such cases aggression/fight might occur and the animals have to be separated; Temporary taking away animals from the group for mating procedure will lead to fight if the animals are taken back to the group; When the animals were anaesthetized for tattoo marking before study start, fighting could occur after the sedation. However our experience is that allocating the animals to groups at four weeks of age gives fewer problems with fighting after anaesthesia as compared with allocating into groups at five weeks of age.
In conclusion, according to our experience group-housing of non-sibling CD-1 male mice is possible in toxicity studies. The key factors are to allocate the animals into groups before sexual maturation and to avoid external changes as far as possible. Used nesting material should be transferred between cages when new bedding material is provided. Clinical signs due to dosing with test compound might affect the group dynamics/the social hierarchy. Most normal study procedures, e.g. urine and blood sampling, will not affect the group dynamics. In addition, our experience clearly indicates that group-housing of male mice in long-term studies leads to more easily handled animals, as compared with individual-housed mice.
In the literature there are several publications discussing group-housing of male mice; all experiments performed on non-treated animals. To our knowledge this is the first time experiences from group-housed male mice used in toxicity studies are described.
Our project is one of many Refinement-projects performed at Safety Assessment, AstraZeneca, including environmental enrichments, handling procedures and method developments. All project ideas from technicians, researchers and veterinarians are carefully evaluated and validated before any changes of research/handling procedures are used as standard operating procedures (SOPs). This refinement project resulted in a change from traditional single-housed male mice to group-housing as a default routine/SOP for all male mice used in long-term toxicity studies at our laboratory.