Abstract
Reduction of animal suffering during in vivo experiments is usually ensured by continuously monitoring the health status using a score sheet and by applying humane endpoints. However, most studies do not evaluate the plausibility of score sheets and do not attempt to reduce the suffering of animals by determining earlier and, therefore, more humane endpoints. The present study uses data from BALB/cANCrl mice after bile duct ligation to retrospectively analyze which score sheet criteria are informative to determine humane endpoints. The performance of each single as well as combinations of multiple animal welfare parameters was analyzed by a Cox proportional-hazards model followed by Harrell’s concordance index. The addition of behavioral parameters, such as burrowing activity, helped to define a more humane early endpoint for euthanizing these animals. Using this approach, we determined that a body weight loss of 10-20% combined with a reduction of burrowing activity by more than 79.4% was able to predict that these animals would die within two days. Thus, this approach successfully determined an earlier humane endpoint and will reduce the suffering of animals in future experiments. Application of such an approach or similar methods can contribute to the refinement of various animal experiments.
Highlights
According to animal protection laws enacted by most nations (EU, 2010; Germany, 2013), high animal welfare standards are a prerequisite to obtain permission to perform animal-based research
3.1 Retrospective analysis of distress parameters After removing distress parameters that were never observed during the experiment (II-a, II-d, II-g, II-h, III-c, III-d, V-a, V-b, V-c) and parameters that demanded immediate euthanasia (I-b, II-i, IV-b), 11 variables were evaluated by Kaplan-Meier estimator (Fig. 1)
All mice with a positive status of I-a had to be euthanized within 14 days, and mice with pronounced apathy (III-b) or who were passive or overactive after being touched by the researcher (IV-a) had to be euthanized within 1-2 days. This suggests that I-a, III-b and IV-a might be powerful criteria to define early humane endpoints for the bile duct ligation (BDL) model
Summary
According to animal protection laws enacted by most nations (EU, 2010; Germany, 2013), high animal welfare standards are a prerequisite to obtain permission to perform animal-based research. Mice with a positive status of I-a (body weight loss of 10% to 20%), II-c (eyes unclear or squinted), II-f (dehydration), III-a (spontaneous behavior: passive or overactive), III-b (pronounced apathy, hyperkinetic, or isolation), IV-a (passive or overactive after being touched by the observer), VI (reduction of burrowing activity more than 79.4%), or VII (decrease of nesting score more than 2.5) had a significantly (P ≤ 0.005) shorter survival time than mice with negative status of these criteria (Fig. 3).
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