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In rodents, the T-maze is commonly used to investigate spontaneous alternating behaviour, but it can also be used to investigate preference between goods. However, for T-maze preference tests with mice there is no recommended protocol and researchers frequently report reproduction difficulties. Here, we tried to develop an efficient protocol with female C57BL/6J CrL mice for preference tests. We used two different designs, adapting habituation, cues and trial timing. However, in both experiments mice did not show any preference, although we used goods which we knew mice find rewarding. Instead, they alternated choices indicating that exploratory behaviour overruled preference. We argue that this behavioural strategy has evolved as an adaptive trait in saturated conditions where there is no need to take the reward immediately. Therefore, we deem the T-maze unsuitable for preference testing with the procedures we used here.

In rodents, the T-maze test is commonly used to investigate spontaneous alternating behaviour but it can also be used to investigate memory, stimuli discrimination or preference between goods. However, especially regarding T-maze preference tests there is no recommended protocol and researchers frequently report reproduction difficulties of this test using mice.Here, we aimed to develop an efficient protocol with female C57BL/6J mice, conducting two preference tests with different design: In a first test, on two consecutive days with five trials, thirteen mice had to choose between two fluids. In a second preference test, on five consecutive days with two (week 1) or three (week 2) trials, twelve mice had to choose between one arm containing bedding mixed with millet and one containing only bedding. This test design resembled a simple learning test (learn where to find the rewarded and the unrewarded arm on the basis of spatial, olfactory and visual cues).In both experiments, mice took only a few seconds per trial to run the maze and make their choice. However, in both experiments mice failed to show any preference for one of the arms. Instead, they alternated choices. We therefore believe the T-maze test to be rather unsuitable to test preference or learning behaviour with C57BL/6J mice.

Simple Summary In 2017, 9.4 million animals were used for research and testing in the European Union. Animal testing always entails the potential for harm caused to the animals. In order to minimize animal suffering, it is of ethical and scientific interest to have a research-based severity assessment of animal experiments. In the past, many methods have been developed to investigate animal suffering. Initially, the focus was on physiological parameters, such as body weight or glucocorticoids as an indicator of stress. In addition, the animals’ behavior has come more into focus and has been included as an indicator of severity. However, in order to obtain a comprehensive understanding of animal suffering, an animal’s individual perspective should also be taken into account. Preference tests might be used, for example, to “ask” animals what they prefer, and providing such goods in turn allows, among other things, to improve housing conditions. In this review, different methods are introduced, which can be used to investigate and evaluate animal suffering and well-being with a special focus on animal-centric strategies. Abstract It has become mandatory for the application for allowance of animal experimentation to rate the severity of the experimental procedures. In order to minimize suffering related to animal experimentation it is therefore crucial to develop appropriate methods for the assessment of animal suffering. Physiological parameters such as hormones or body weight are used to assess stress in laboratory animals. However, such physiological parameters alone are often difficult to interpret and leave a wide scope for interpretation. More recently, behavior, feelings and emotions have come increasingly into the focus of welfare research. Tests like preference tests or cognitive bias tests give insight on how animals evaluate certain situations or objects, how they feel and what their emotional state is. These methods should be combined in order to obtain a comprehensive understanding of the well-being of laboratory animals.