Millions of zebrafish (Danio rerio) are used in laboratories around the world, often as part of developmental studies. Zebrafish embryos are transparent and develop externally, facilitating observation and manipulation of the developmental process and leading to greater understanding of developmental defects and diseases. As research subjects, fish are often anesthetized for handling, sample collection and surgical procedures (e.g., fin clipping) and as the first step in euthanasia. Various anesthetic agents may be used for this purpose, but despite routine use, no studies have formally assessed how well the agents are tolerated by fish or whether they trigger aversive behavioral responses. The lack of information impedes the establishment of best practices for zebrafish anesthesia. Joanna C. Murrell, a lecturer in veterinary anesthesia at University of Bristol (Langford, UK), explained, “With tens of millions of fish used in science around the world, it is very important that the anaesthetics used... are the most humane available and do not themselves cause a stress response.”

Murrell worked with Gareth Readman (Brixham Environmental Laboratory, AstraZeneca, Devon, UK) to evaluate nine of the most commonly used fish anesthetics. They carried out preference testing, exposing adult zebrafish of both sexes to water containing each agent at 50% of its standard recommended dose in a flow-through chemotaxic choice chamber and using video tracking software to quantify aversive swimming behavior related to each anesthetic compared with untreated water. They found that seven of the nine anesthetics were aversive, including MS222 (ethyl 3-aminobenzoate methanesulphate) and benzocaine, two of the most often used (PLoS ONE, 8, e73773; 2013). The authors recommend that for ethical best practice, aversive compounds should not be used routinely for anesthesia or for the first step of euthanasia of adult zebrafish.

Two other agents, etomidate and 2,2,2 tribromoethanol (TBE), did not induce aversive behavioral responses. In previous investigations, etomidate has not caused negative allergenic, tissue irritation or blood chemistry responses in fish, although metomidate hydrochloride, an etomidate derivative, has been reported to have negative effects in human and veterinary medicine. Although TBE did not elicit an aversive reaction, the authors caution that improper storage can lead to the formation of potentially harmful degradation products. The authors conclude that etomidate is best suited for routine anesthesia of zebrafish.

The results could improve the welfare standards for zebrafish used in research. In contrast to the many advances in anesthesia for laboratory mammals, this is one of the first studies to assess the welfare implications of anesthesia in fish.