The zebrafish (Danio rerio ) accounts for approximately 50% of fishes u sed in research and its adoption has grown rapidly since the 1990’s (Kinth et al. 2013). The high number of individuals used in research warrants for an accessible, effective, and humane manner to stun and kill zebrafish in the laboratory. Common methods adopted in research today include overdose of anaesthetics, rapid cooling followed by brain destruction or exsanguination. However, there is evidence that these methods are aversive (e.g., many anaesthetics) and require capture, handling and transferring to another tank, thus causing stress (Wong et al. 2014). Electrical stunning could be a more effective and less stressful euthanasia method but although it is extensively used in aquaculture, very little is known about the use of electrical stunning for zebrafish (Mocho & von Krogh 2022). One study has demonstrated that euthanizing zebrafish embryos and larvae with electricity is rapid and reliable (Mocho et al. 2022 ). Here, we have developed a suitable electrical stunning method for euthanizing zebrafish in laboratory settings. Our aim is to investigate i) whether adult zebrafish can be euthanized humanely and effectively using electricity, and ii) what is the minimal, electrical field required to achieve a humane and effective euthanasia without using unnecessarily high voltage. Furthermore, we have developed portable electrode panels that can be placed in a common zebrafish housing tank to reduce the amount of handling and therefore, minimize the unnecessary stress that zebrafish may experience both before and during the procedure of euthanasia.
Material and methods
In all of the trials, water temperature, conductivity, and frequency were kept constant at 28±0.3 °C, 800±5 µS/cm and 50 Hz, respectively, whereas the voltage was manipulated to achieve different electrical field strengths. The first trials focused on finding a field strength that is sufficient to stun the zebrafish immediately without the fish showing any adverse reaction. This was done to verify that the fish experienced an immediate loss of consciousness, and therefore would not endure extreme stress and pain during the stunning. In these trials, the electrical stun duration was 1 s. A behavioural assessment was done by visually inspecting the fish during and after the stun. Video footage was used to revise the reaction and to confirm all the trials. After the appropriate minimal field strength was verified, single zebrafish where stunned for 30 s to demonstrate the efficacy of the field strength to eliminate the subject. The fish were inspected for 30 min after the stun to verify that the stun was irreversible. Zebrafish where then stunned in groups of 5 fish per tank and later in groups of 10 fish per tank for 30 s. After a sufficient field strength was concluded, portable and adjustable electrode panels were constructed. The final design was made to be suitable for an 8 L standardized zebrafish tank (Tecniplast S.p.A, Italy), which is commonly used for zebrafish housing. The electrode panels were designed to have adjustable handles and supporting staffs to be able to adjust the electrode separation to be fitted also into the smaller, 3.5 L standard tank (Tecniplast S.p.A, Italy). The portable electrodes where then tested using both 8- and 3.5 L tanks by stunning groups of 10 fish for 30 s as well as stunning the number of fish equal to the maximum capacity for each tank, i.e., 40 fish in the large tank and 15 fish in the small tank for 30 s. Only trials where all fish were stunned irreversibly were determined successful. If the trial was deemed unsuccessful, the voltage was adjusted so that the electric field strength was 1.0 V/cm higher.
Zebrafish that that were stunned irreversibly showed no aversive nor aggravated behaviour during the stun . It was conducted that 5±0.3 V/cm was sufficient to euthanize 1, 5 or 10 fish per trial in a 3.5 L tank , but stunning the maximum capacity of fish in the same tank required a 6.7±0.1 V/cm. For the larger, 8 L tank, 6.0±4 V/cm was sufficient to euthanize 10 fish at once. The trials conducted with the standard housing tanks are presented in Table 1.
Electrical stunning was highly effective in causing immediate loss of conscious and death without recovery in zebrafish. Many large facilities are killing hundreds of zebrafish every week thus this technique represents a more refined method without the use of chemicals, cooling stress or handling of the animals. Depending on the number of zebrafish and tank size, the sufficient field strength does need to be adjusted . Ou r results comply with the previous assumptions regarding electrical stunning of young forms of zebrafish (Mocho et al. 2022 ), and therefore electrical stunning could contribute to a more humane euthanization and better welfare of adult zebrafish in the future. This approach could be extrapolated to other species of fish used in research including aquaculture species where killing large numbers of fish humanely remains a challenge.
Kinth, P., Mahesh, G. & Panwar, Y. (2013). Mapping of zebrafish research: a global outlook. Zebrafish, 10(4), 510-7.
Lines, J. & Kestin, S. (2004). Electrical stunning of fish: the relationship between the electric field strength and water conductivity. Aquaculture, 241(1-4), 219-234.
Wong, D., von Keyserlingk, M.A., Richards, J.G. & Weary, D.M. (2014). Conditioned place avoidance of zebrafish (Danio rerio) to three chemicals used for euthanasia and anaesthesia. PLoS One, 9(2), e88030.