Evaluating Clicker Training as a Novel Refinement Method for Reducing Stress in Laboratory Mice During and After Subcutaneous Injection

Abstract

The main aim of this study was to investigate if clicker training laboratory mice to stand still during a subcutaneous (SC) injection with minimal restraint could effectively reduce stress both during and after the injection, potentially serving as a novel refinement method. In order to investigate this, three treatment groups were created: a trained group, an exposed group and an unexposed group. The trained group (n=8) underwent 19 sessions of three minutes following a training protocol teaching them to stand still during a SC injection. The exposed group (n=8) underwent the same amount of sessions, exposing the mice to the reward, researcher and training environment, but were not trained. The unexposed mice (n=8) underwent no sessions at all, meaning they were not exposed to the reward or training environment. Ultimately, all 24 mice were injected with 0,1 ml NaCl 0,9% to assess the effect of training on the behavioral response of the animals during and after the injection, specifically regarding stress-related behaviors and behaviors directed to the researcher. The analysis showed a difference between treatment groups regarding standing still during injection, with trained mice standing still longer than unexposed mice, indicating that trained mice potentially experience less stress during injection than unexposed mice. A trend was found between the exposed and unexposed mice, with the exposed mice standing still longer, indicating that exposure alone may affect the stress response of mice during a SC injection. Another significant correlation was found between treatment groups and reward acceptance, with the exposed mice eating longer than unexposed mice, again highlighting the potential impact of exposure on reducing stress during injection. No other significant differences between the treatment groups were found. Several limitations in the study, like small sample size, lack of naivety in the unexposed mice, and their advanced age may have influenced the results of the remaining behaviors. Clicker training appears to have been effective, as evident by trained mice standing still longer during injection. However, no effects of training on any stress-related behaviors were observed yet, suggesting exposure alone might yield stronger effects. Further research on clicker training mice is warranted to understand its comprehensive impact on stress levels and whether exposure is as effective or even more so.