Introduction
Current animal welfare guidelines, based on the five-domain framework, state the need to prevent animal suffering, particularly during slaughter. The guidelines recommend that fish must be rendered unconscious through appropriate stunning methods such as live chilling, electronarcosis, or percussive stunning (OIE, 2019) , but the effectiveness of these methods varies by species and requires validation. Consequently, turbot farmers have become interested in alternative methods to conventional live chilling, leading to studies that assessed electrical and percussive stunning using behavioural indicators. However, these indicators were found to be unreliable for determining consciousness due to their poor correlation with neurological activity (Morzel et al., 2003). In this context, e lectroencephalography (EEG) has been recommended as a preferred method to evaluate the effectiveness of stunning techniques (Bowman et al., 2019). The present study aimed to provide new evidence on the effects of electrical and live chilling stunning methods on turbot, using EEG to assess their effectiveness in industry settings.
Materials and methods
Commercial-sized turbot (Scophthalmus maximus) weighing 1–2.5 kg from Stolt Sea Farm S.A. and CETGA (A Coruña, Spain) were used to evaluate the efficacy of industrial electric stunning and live chilling methods . Individually, each fish was anaesthetised with MS-222 (80 mg/L) prior to cranial placement of two stainless steel EEG electrodes over the telencephalon and optic lobe , with a reference ele ctrode in the dorsal muscle. After recovery of the fish from anaesthesia, basal EEG recordings and strobe light-induced visual evoked responses (VERs) were collected for 10 min. F or electrical stunning, the electrodes were removed prior to placing the fish in an industrial stunner (5 0 V, 50 Hz), and repositioned upon exit from the stunner , with brain activity being recorded for the next 15 min. For live chilling, recordings were taken at 10- min intervals before and during exposure to ice slurry , as well as after transfer to normal temperature water . EEG data were filtered to remove artefacts and analysed using Spike2 software, with power spectrum analysis (band pass filter 0.5-32 Hz, 10 Hz resolution) used to calculate total power (Ptot) and αβ power (Pαβ), both normalised to baseline . VERs were assessed by averaging twenty 1-second windows during light transitions.
Results
This study evaluated the effect of two industrial stunning methods, electrical stunning and live chilling, on EEG activity in commercial-sized turbot. Our results showed that electrical stunning slightly increased power parameters values, although this was not significant due to the high variability of the recordings, especially towards the end of the post-stunning period studied. As for VERs, no attenuation or disappearance was evident following electrical stunning ; on the contrary, a twofold increase in VER amplitude was noted during the early post-stunning recording.
Regarding live chilling, a progressive reduction in EEG power parameters was observed after immersion of fish in ice slurry. Significant decreases were observed within two minutes in ice slurry, reaching levels approximately 60% lower than those prior to cooling . VERs also showed a gradual decrease starting after two minutes of immersion in the ice mixture, disappearing complete after five minutes . Upon returning the fish to water of normal temperature, a gradual recovery of power parameters was observed, reaching pre-cooling levels after six min . The VERs also reappeared and gradually recovered to pre-cooling amplitude levels within two minutes of exposure to normal water temperature.
Discussion
This study analysed and compared the EEG effects of electrical stunning and live chilling to assess their effectiveness in rendering turbot unconscious during industrial slaughter. We evaluated the electric stunning using the optimised set-up determined by previous operational indicator tests. The results showed that this method strongly immobilizes the fish by muscular blockage for a few min, but it does not cause redu ction in brain activity and/or loss of VERs . This indicates that fish may remain conscious after stunning and highlights the poor correlation between behavioural signs and EEG-based measures of unconsciousness (Bowman et al., 2019).
On the other hand, live chilling resulted in a progressive decrease in brain activity, reaching a reduction of more than 50% in power parameters . In addition, a progressive disappearance of VERs within five minutes of live chilling was observed. These EEG findings are consistent with standards used in rainbow trout studies, where the absence of VERs is considered a reliable indicator of unconsciousness (Bowman et al., 2019), suggesting that this method could induce loss of consciousness in turbot. However, despite these results, live chilling also does not induce unconsciousness immediately, so with the current protocol it cannot be claimed to be an optimal method for stunning according to welfare guidelines (OIE, 2019).
Overall, our results show that the current method of electrical stunning is ineffective in rendering turbot unconscious, while live chilling appears more effective, although with time constraints that need to be considered . This study also highlights the limitations of operational indicators and emphasises the importance of using EEG techniques when assessing slaughter in turbot.
References
Bowman, J., Hjelmstedt, P., & Gräns, A. (2019). Non‐invasive recording of brain function in rainbow trout: Evaluations of the effects of MS‐222 anaesthesia induction. Aquaculture Research , 50 (11), 3420-3428. https://doi.org/10.1111/are.14300
Morzel , M., Sohier , D., & Van De Vis, H. (2003). Evaluation of slaughtering methods for turbot with respect to animal welfare and flesh quality. Journal of the Science of Food and Agriculture , 83(1), 19-28. https://doi.org/10.1002/jsfa.1253
OIE. (2019). Aquatic Animal Health Code. 133-145.
Acknowledgements
This study received funding from the Industrial Doctorates program of Xunta de Galicia (Consellería de Educación , Ciencia, Universidades e Formación Profesional ) to CRR , and from Marine Science programme (ThinkInAzul ) supported by Ministerio de Ciencia e Innovación and Xunta de Galicia with funding EU NextGeneration (PRTR-C17. I1).