Dunford, K., Hakkesteeg, J., Wilson, C. 2019. Stiff as a board: Measuring rigor mortis in zebrafish. Animal Technology and Welfare 18(3), 203-205.

Schedule one killing (S1K) methods require a two-step process: a humane method of death, typically for Zebrafish an anaesthetic overdose and confirmation of death, such as confirmation of rigor mortis. There is widespread variation of anaesthetics used for S1K and there is debate about refining anaesthesia for Zebrafish, as adverse effects are becoming a concern. Anecdotal evidence suggests that different anaesthetics can inhibit or reduce the rate of the onset of rigor mortis, with some taking an hour whilst others take more than three. We conducted a trial in order to determine the rate of rigor mortis for four different agents. A total of 57 hybrid/(AB:TupLF) 10 month old adult fish of the same stock were used. Each anaesthetic [benzocaine, MS-222, 2-PE] was tested into two different post-mortem media: ‘removed’ into fresh water and ‘staying’ in dosed water. The 2-PE dose followed UCL protocol, whilst the other three were based on published recommendations. For each treatment, three fish were added to the predosed water and left for ten minutes to ensure death. After a knock test to ensure death, individual fish were transferred to labelled petri dishes with the assigned post-mortem media. At 30 minute intervals, each fish was held by the caudal peduncle and measured against a protractor to measure the angle. The most notable difference was 2-PE ‘stay’, which reached 0˚ at a faster rate than Benzocaine and MS-222; 2-PE reached 0˚ at approximately the three-hour mark, whereas the other two took more than five hours. The dosages may have played a role: benzocaine and lidocaine dosages are quantitatively small compared to 2-PE.