Zebrafish (Danio rerio) play a major role in scientific aquaculture as the second most used model organism in biomedical research. However, animal welfare surrounding the end of life demonstrates mixed efficacy and aversion behaviors across all life history stages. Both adult and embryonic zebrafish display high resistance and aversion to widely used chemical agents for the euthanasia of laboratory fishes. Electrical euthanasia has been shown to produce a 100% mortality rate and inhibit consciousness within 1 second of current application. With the proper voltage gradient and length of exposure, electricity provides an alternative and improved method of euthanasia over chemical and thermal applications. Since parameters for electro-euthanasia of zebrafish have not been established, the objectives of this study are to determine effective settings and operational procedures for pre and post-hatch embryos and adults.
Two novel electrical systems, the EFS-WASP1 and WASP3 (Electro Fishing Services Ltd. Ireland) were utilized for the euthanasia of pre and post-hatch embryos (12hrs-5 days post fertilization [dpf]) and adult zebrafish (21-40mm), respectively. Embryos (n=5 replicates per treatment, 10 embryos per replicate) were transferred into a square plate electrode exposure chamber (width 3.3cm) and exposed to continuous and pulsed direct current with a rectangular DC waveform at 50Hz with a 50% duty cycle in a water conductivity of 175 μs/cm. The optimal exposure period was initially assessed utilizing the manufacturer-recommended voltage gradient (30V/cm for pre and post-hatch embryos) during pilot testing. Experimental trials sought to validate the minimum voltage gradient and time to achieve 100% mortality. Embryos were randomly exposed to a voltage gradient of 30V/cm for 30-120sec and assessed for survival immediately following and at 5min, 30min, and 12 hours post-exposure.
Adult zebrafish (n=5 replicates per treatment, 3-5 fish per replicate) were initially exposed to manufacturer-recommended settings, 2.8V/cm in a circular electrode with voltage gradients reduced by 0.4V/cm every five consecutive trials achieving 100% mortality. All fish were assessed for indicators of recovery (operculation, coordinated movement) at times 0, 5, and 30min post-exposure. Handling controls were placed in the euthanasia tank but not exposed to current and monitored, and non-handling was left undisturbed for all tested stages.
Post-hatch embryos at 4dpf had 100% mortality utilizing a 90sec exposure to 30V/cm, while 5dpf post-hatch embryos required 120sec exposures of 25V/cm. Adults between 30-40mm experienced 100% mortality following a 90sec exposure of 2.0V/cm, while adults between 21-25mm and 25-35mm had 100% mortality following an exposure of 2.0V/cm for 150 sec. Results show electricity is an immediate acting and viable alternative to traditional euthanasia methods, which require long exposure periods, up to 20 min. Electro-euthanasia provides a more humane ending for zebrafish at all tested life stages by reducing the necessary exposure time during the euthanasia process.