The choice of euthanasia techniques can affect experimental results in aquatic behavioural studies

Abstract

With the use of animal models in research comes the responsibility of treating them ethically. While general anaesthetics are used to reduce pain, they have the potential to interfere with research results. This is a concern in behavioural ecology, particularly in aquatic systems where organisms use chemicals as a form of communication. One well-studied source of chemical information are alarm cues – compounds released from injured skin of conspecifics that mediate antipredator responses in nearby individuals and facilitate learned predator recognition when paired with novel predator cues. To determine whether chemical euthanasia interferes with the ecological functioning of alarm cues, we first exposed woodfrog tadpoles, Lithobates sylvaticus , to alarm cues prepared using different euthanasia, preparation and use methods (physical versus chemical euthanasia; mortar and pestle homogenization versus tissue homogenizer; cues used fresh versus frozen versus aged) and compared the intensity of their antipredator responses. Second, we exposed tadpoles to a novel predator odour paired with alarm cues obtained using either physical or chemical euthanasia techniques to elicit learning, and subsequently compared their learned responses. We found that tadpoles exposed to alarm cues prepared with a chemical euthanizing agent (MS222) displayed a weakened antipredator response that was not significantly different from our water (negative) control. Similarly, tadpoles were unable to learn novel predator odours as a threat when alarm cues were prepared with MS222. In contrast, alarm cues obtained using a physical euthanasia method yielded the highest responses regardless of preparation and use methods, with the exception of cues aged 24 h, which became nonfunctional. Our results confirmed the potential for chemical euthanasia methods to interfere with the functioning of ecologically relevant chemicals. Therefore, a single euthanasia method cannot be applied to all experimental situations – options are needed so researchers can select a method that provides adequate animal welfare without interfering with results. • Tadpoles exposed to MS222 prepared alarm cues had a weakened antipredator response. • Tadpoles were unable to learn novel odours as threats with MS222 prepared alarm cues. • Alarm cues remained effective after freezing but become ineffective if aged for 24 h. • Chemical euthanasia can interfere with ecologically relevant chemicals. • A single euthanasia method cannot be applied to all experimental situations.