Background
Nile tilapia (Oreochromis niloticus) is an important aquaculture species and was the third most produced species globally in 2020 (FAO, 2022). T he most common killing procedures for this species include asphyxiation, exsanguination through gill cutting or decapitation and/or direct evisceration (Lines and Spence, 2011; Robb and Kestin, 2002). To meet requirements of humane slaughter and provide strong animal protection, an efficient stunning prior to killing is crucial (WOAH, 2019). Yet all of the above-mentioned methods are merely killing pro cedures that do not include any form of stunning and therefore will compromise the welfare of the fish (Robb and Kestin, 2002). For a stunning method to be efficient, it should induce immediate loss of consciousness and the unconscious state should be irreversible, or at least persist long enough for death to ensue before consciousness is regained (WOAH, 2019; EFSA, 2004). Efficiency of stunning methods for Nile tilapia is largely unknown and there is an urgent need for validation and/or development of humane slaughter methods for this important aquaculture species.
Material and methods
Unconsciousness was determined by absence of visually evoked responses (VERs) in the electroencephalogram (EEG) of the Nile tilapia (Figure 1 ). EEG was obtained by inserting two 21G needles, soldered to 1.5 mm shielded wire electrodes acting as positive and negative electrodes , approximately 1 cm posterior to the eyes and 0.5 cm lateral to each side of the sagittal suture . A 29G needle electrode that served as the ground was placed subcutaneously at the tail. To study VERs, or lack thereof, EEG was measured in a dark room where a strobe light delivered repeated 20:480 ms light:dark episodes. The EEG signal was analyzed using the 13-32 Hz frequency range (Figure 1) as this is associated to conscious, waking states in many animals including fish ( Bowman et al., 2019; Verhoeven et al., 2015 ). The different stunning methods assessed were i) percussive stunning, ii) live chilling, iii) electrical stunning and iv) a combination of electrical stunning followed by throat cut and placement in ice slurry. EEG was recorded before, during (when applicable) and after the stunning procedure. Percussive stunning was achieved using a handheld pneumatic bolt gun and live chilling was carried out by replacing the warm water with ice slurry. For electrical stunning , 1 second stun durations was initially performed to verify that the electrical exposure managed to induce an immediate loss of consciousness (Cook et al. 1995). When this was verified, 30 second stun durations using similar settings were applied to evaluate if the unconscious state could be prolonged by prolonging the stun duration . For the combinational procedure (i.e., combining electrical stunning with throat cutting and placement in ice slurry) , a 30 s electrical stun duration was applied before the fish was throat cut and placed in ice slurry.
Results
Percussive stunning rendered all fish immediately and irreversibly unconscious. However, this required handling, air exposure and restraining the fish, which, each on its own is stressful. Also live chilling rendered all fish unconscious but had a long induction time (4.92±2.26 min ). Electrical stunning for 1 s managed to induce immediate loss of consciousness when an electric field strength of 8.2 VRMS cm-1 , a current density of 0.68 ARMS dm-2, a frequency of 50 Hz sinusoidal alternating current (AC) and a water conductivity of 753 µS cm-1 was used. When prolonging the stu n duration to 30 s using similar electrical parameters (i.e., 8.2 VRMS cm-1 , 0.68 ARMS dm-2 , 50 Hz sinusoidal AC and 753 µS cm-1), fish lost VERs for at least 30 s. When electrical stunning using similar settings (i.e., for 30 s using 8.2 VRMS cm-1 , 0.6 2 ARMS dm-2 , 50 Hz sinusoidal AC and 753 µS cm-1 ) was followed by throat cutting and ice slurry immersion in two fish, VERs came back after 80 and 96 seconds, respectively . However, when the strength of the exposure was increased (electric field strength and current density of 14.2 VRMS cm-1 and 1.07 ARMS dm-2, respectively) , VERs did not come back within 30 minutes after the electrical exposure when it was followed by throat cutting and ice slurry immersion.
Conclusion
Here, we show that when combining electrical stunning with throat cut ting and ice slurry immersion, an immediate and irreversible loss of VERs can be accomplished. Also percussive stunning using a handheld bolt gun is efficient in rendering the fish immediately and irreversibly unconscious. However, the latter method require handling and could benefit from a pre-stunning procedure, such as electrical stunning. T hese results show that electrical stunning followed by both percussive stunning or throat cut and immersion in ice slurry have the potential to ensure humane slaughter of Nile tilapia. However, the electrical exposures needed to guarantee that the effect persists long enough for death to ensue is largely depending on the stun-to-kill time of the selected killing procedure. Collectively, our results shows that h umane slaughter of Nile tilapia is possible. If the outcome of our study is i mplemented this may aid in safeguarding the welfare of billions of tilapia at time of slaughter.
References
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