Zebrafish are the most utilized fish in research, and it’s estimated that five million fish are used for research annually. Every year, vast quantities of Zebrafish must be euthanized, usually achieved via immersion in chemical solutions or rapid chilling. Aversive behavior is often seen, and extended exposure periods are needed for these methods to be effective. AVMA humane slaughter guidelines state that electrocution is also an acceptable euthanization method, provided that the electric current is sufficient to immediately induce insensibility before or simultaneously with killing the fish. Electro-euthanasia was shown to be effective on zebrafish at 12 hours and 5 days after fertilization with no aversive behavior. This research aims to validate the manufacturer’s recommended settings and develop standard operating procedures for electro-euthanasia for zebrafish across various developmental stages.
Wild-type zebrafish and two electrical systems, WASP3 and EFS-WASP1 (Electro Fishing Services, Ltd. Ireland) were utilized. All developmental stages were exposed to a continuous and pulsed direct current with a rectangular DC waveform at a frequency of 50 pulses per second with a 50% duty cycle. All tests included handling and non-handling controls. All trials included five replicates (n=5 replicates per treatment). The voltage gradient was reduced every five successful trials until 100% mortality was not achieved. If 100% mortality was not achieved, trials were discontinued.
Pre- and post-hatch embryos (<5 days post fertilization [dpf]) were transferred to a 33mm3 electrode chamber. Pilot testing began with manufacturer-recommended settings (Voltage gradient of 30V/cm and a 60 sec exposure period). Trials (n=5 replicates per treatment, 10 fish per replicate) began with a voltage gradient of 30V/cm and a 120 sec exposure. Trials were assessed immediately after for signs of survival and at 5 min, 30 min, and 12 hours post-exposure. 100% mortality was achieved for embryos 4 dpf following a 90 sec exposure to 30 V/cm. Embryos 5 dpf required a 120 sec exposure period to a voltage gradient of 25V/cm. Trials for adults (n=5 replicates, 3-5 fish per replicate) began with the manufacturer’s recommended voltage gradient of 2.8V/cm. 100% mortality was observed in Adult zebrafish with lengths 21-25mm and 26-35mm, following a 150 sec exposure to 2.0V/cm. Adults 36-40mm required only a 90 sec exposure to 2.0V/cm to achieve 100% mortality. Results of these experiments show that electricity is a viable alternative to currently utilized methods of euthanasia.