Effects of litter and cage furniture on mouse anxiety-like behaviour

Discussions on laboratory animal welfare issues often refer to the Three Rs – replacement, reduction and refinement. Replacement means substituting living animals with nonsentient systems; reduction refers to using fewer animals and refinement causing less pain, suffering and distress to the animals or improving their welfare. This work is focusing on two R-s: reduction and refinement in mice. If one considers reduction as meaning obtaining trustworthy information from using fewer animals, then this can be achieved by improved research strategies, better experimental design and more sophisticated statistical analyses. One can reduce variation within the group by using isogenic inbred animals or by finding ways to reduce variation in outbred animals. One approach to achieve reduction in outbred animals is to include litter and individual features of the animals, e.g weight dynamics, in the statistical analyses. Since the elevated plus-maze (EPM) test is one of the most common tests to evaluate anxiety-like behaviour, it was used to assess the possible effects of litter and weight on the behaviour of outbred mice and the effects of environmental enrichment (EE) on the behaviour of inbred mice. As a research tool, the effects of acute or chronic administration of ethanol or acute therapy with the nitric oxide synthase (NOS) inhibitor L-NOARG were examined in outbred NIH/S mice. The administration of L-NOARG had no effect on the behaviour of mice after acute or chronic ethanol administration but attenuated the anxiogenic effect of ethanol withdrawal. The litter from which the mice had originated had a significant effect on their behaviour in the EPM test. The behavioural indices of mice, originating from different litters, tended to be above or below the mean of the corresponding drug-treatment group, irrespective of the drug treatment. Litter had a significant effect on the initial weight and also on the weight changes occurring during the adaptation period and ethanol inhalation. An approach to refinement, EE, has been introduced to create more natural speciesspecific living conditions for laboratory animals. At the same time, it has been claimed that EE can affect the results of behavioural studies and also increase variation. The effects of different types of EE and different time periods were studied in inbred C57BL/6J and BALB/c mice. The exposure of male C57BL/6J mice to the different types of EE objects in the form of cage furniture (CF) -nest box, corner and stairsinduced an anxiolytic-like effect in the EPM test and tended to increase the locomotor activity of mice. This apparent anxiolyticlike effect was most pronounced in the third week. The CF, in the form of modified Tapvei OY mouse stairs, produced an anxiolytic-like effect and increased the locomotor activity in female C57BL/6J mice, but not in BALB/c female mice. In conclusion, the NOS inhibitors may have effects on the behavioural changes caused by ethanol withdrawal. Information about the litter of outbred mice could and should be used in statistical analysis in order to reduce variation and the number of mice needed. In the EPM test, different CF items induced an anxiolytic-like effect in male and female C57BL/6J mice, but not in BALB/c female mice. This effect depended on the type of objects and was influenced by time. This anxiolytic–like effect can be interpreted as refinement of the housing by improving animal welfare. The effects of CF should be considered in planning enrichment programs for housing institutions, in designing behavioural experiments and in analyzing the results obtained. Therefore the evaluation of CF could provide the valuable information and it is recommended that CF manufacturers collate and distribute the refinement results on the specific CF items they produce.