title: Reduction of variation in behavioural experiments
reg no: ETF6586
project type: Estonian Science Foundation research grant
subject: 3.1. Basic Medicine
status: accepted
institution: TU Faculty of Medicine
head of project: Paavo Pokk
duration: 01.01.2006 - 31.12.2009
description: Since the publication of the classic book by Russell and Burch “The Principles of Humane Experimental Technique” (1959) in animal experiments a growing attention has been paid to the 3 Rs – replacement, reduction and refinement. In this context, “replacement” means using alternative methods instead of laboratory animals. “Reduction” means using a fewer number of animals and “refinement” means causing less stress to laboratory animals. These 3 Rs are not only crucial from the ethical point of view but they can also significantly increase the quality of scientific research, Thus, by using adequate methods of reduction, it is possible to obtain the same amount of information from fewer animals (for review see ECVAM Workshop Report, 1998). One of the main factors, determining the number of experimental animals needed to obtain trustworthy information, is variation within the group. Reduction of variation within the group is possible by using genetically similar inbred animals (Festing, 1999). However, because of lower cost, many scientists use mainly outbred animals. Therefore it is important to study how to reduce variation also when using outbred animals. It has been shown that litter has effect on the investigatory and aggressive behaviour in mice (Barnard et al, 1998, Tanaka, 1998) and on the development of stereotypy (Powell et al, 1999). It has also been proposed in the literature that, in toxicological and teratological studies, instead of individual animals, the litter should be used as the unit of analysis (Spear and File, 1996). Therefore, an approach to reduction is to use litter as a fixed effect in statistical analysis in order to decrease variation. If litter has significant effect on the behaviour of animals, litter coding would decrease noise and therefore reduce the number of animals needed. We also plan to study factors that might affect the expression of litter effect – e.g. adaptation period.
Environmental enrichment can be defined as using objects to improve the quality of life of animals by distracting them from an otherwise monotonous environment (Belz et al, 2003). The European Convention N 123 and Directive of European Council 86/609/EEC state that any restriction on the extent to which the animal can satisfy its physiological and ethological needs shall be limited to absolute minimum. The corrections of the convention are ready but are not officially effective at the moment, the new version of the directive is being revised, and it will probably make environmental enrichment obligatory. Therefore environmental enrichment will be used more frequently in the future. At the same time it has been demonstrated that environmental enrichment has numerous effects on the behaviour of laboratory animals (for review see Key & Hewett, 2002; Key, 2004; Olsson & Dahlborn, 2002) and has influence on the effects of various drugs (Green et al, 2003; Smith et al, 2003). There are contradictory data in the literature concerning the effects of environmental enrichment on variability. It has been proposed that environmental enrichment can increases variability (Tsai et al, 2003), that environmental enrichment has no significant effect (Wolfer et al, 2004) or that the effects of enrichment on variability in results are parameter dependent (Van de Weerd et al, 2002). Furhtermore, it has been proposed in the literature that environmental enrichment could hinder the standardization of experiments (Van de Weerd et al, 2002).
Since, firstly, it has been demonstrated that environmental enrichment significantly affects the behaviour of animals and, secondly, because environmental enrichment is used more and more frequently, it is important to study the effect of environmental enrichment on the behaviour of animals.
In conclusion, the main aim of our work is to study the effects of litter and environmental enrichment on the behaviour of outbred mice in the exploratory models of behaviour. Part of the experiments will be carried out without drug administration and in part of the experiments the effects of selective eNOS and nNOS inhibitors after acute and chronic ethanol administration and withdrawal are observed.The choice of these drugs to study the effect of litter is based on the interesting role of NOergic system in the effects of ethanol. It has been assumed for at least ten years that the effects of ethanol are, at least partially, mediated through L-arginine – NO synthase – NO pathways (Adams et al, 1994). However, data concerning the influence of NOS inhibitors on the effects of ethanol, e.g. ethanol withdrawal are still extremely contradictory. Thus, depending on the NOS inhibitor used and its dose, different authors have reported alleviation, worsening on no changes in the signs of ethanol withdrawal (Uzbay & Oglesby, 2001). It is possible that this is caused by the different selevtivity of used NOS inhibitors for different NOS subtypes – neuronal (nNOS), endothelial (eNOS) and immunological (iNOS). Therefore the use of selective eNOS and nNOS inhibitors might help to clarify the role of NOergic system in the effects of ethanol.
If litter is a significant determinant on the behaviour of outbred mice, then use of litter info could serve for purposes of reduction. Also the determination of an optimal adaptation period and environmental enrichment could help to reduce variation and thereby the necessary number of laboratory animals. If we are able to demonstrate the role of different NOS subtypes in the development of ethanol withdrawal this might help to explain ethanol’s mechanism of action and aid in the search of new drugs for ethanol withdrawal syndrome.

project group
no name institution position  
1.Aavo Lang 
2.Paavo PokkTartu Ülikool 
3.Kai Õkva