Repeatability analysis improves the reliability of behavioral data
Reliability of data has become a major concern in the course of the reproducibility crisis. Especially when studying animal behavior, confounding factors such as novel test apparatus can lead to a wide variability of data. At worst, effects of novelty or stress related behavior can mask treatment effects and the behavioral data may be misinterpreted. Habituation to the test situation is a common practice to circumvent novelty induced increases in variance and to improve the reliability of the respective measurements. However, there is a lack of published empirical knowledge regarding reasonable habituation procedures and a method validation seems to be overdue. This study aimed at setting up a simple strategy to increase reliability of behavioral data. Therefore, exemplary data from mice tested in an Open Field (OF) arena were used to elucidate the potential of habituation and how reliability of measures can be confirmed by means of a repeatability analysis using the software R. On seven consecutive days, male C57BL/6J, BALB/cJ and 129S1/SvImJ mice were tested in an OF arena once daily and individual mouse behavior (distance travelled, average activity) was recorded. A repeatability analysis was conducted in order to estimate the reliability of measured animal behavior with regard to repeated trials of habituation. Our data analysis revealed that monitoring animal behavior during habituation is important to determine when individual differences of the measurements are stable. Here, the mixed effect model framework proved to be a powerful tool for estimating repeatability values. Repeatability values from distance travelled and average activity increased over the habituation period, revealing that around 60 % of the variance of the data can be explained by individual differences between mice. The first day of habituation was statistically significantly different from the following 6 days in terms of distance travelled and average activity. A habituation period of three days appeared to be sufficient in this study. Overall these results emphasize the importance of habituation and in depth analysis of habituation data to define the correct starting point of the experiment for improving the reliability and reproducibility of experimental data.