Welfare of rainbow trout at slaughter: Integrating behavioural, physiological, proteomic and quality indicators and testing a novel fast-chill stunning method

A critical point in the life of a captive fish is the final stages of production, not only in welfare terms but also due to effects on meat quality, carcass appearance and derived economic impacts. The most common method to slaughter fish is by asphyxia either in ice-water or in the open air. In humane slaughter procedures, however, a stunning method needs to be implemented to render the fish immediately unconscious (within one second) until death. The objective of this research was to evaluate and compare the effectiveness and welfare effects of four types of stunning methods in rainbow trout (O. mykiss): cold shock by fast-chilling as a novel method, where the fish were immersed in liquid water at − 8 ◦C, asphyxia (as the currently used method), electrical stunning, and anaesthesia with MS-222. We used a total of 176 trout (mean weight 524 ± 138 g), combining behavioural (individual swimming activity, equilibrium, opercular movement and eye-roll), physiological (heart rate and electrocardiogram amplitude) and circulating (plasma cortisol and osmolality) indicators with brain proteomic signatures. We also analysed the effects on fillet shelf-life and quality in each method (rigor mortis, water content, fillet colour, pH and ATP degradation). Anaesthesia effectively induced unconsciousness, with regular and strong heartbeat and low cortisol. Quality indicators were the best among all the methods assessed. Electric shock was found to be an effective and irreversible method for inducing unconsciousness, with strong heartbeat and large variation in cortisol response and quality indicators similar to anaesthesia. On the contrary, asphyxia presented indicators of poor welfare (e.g., long-lasting consciousness throughout the slaughter process, high cortisol levels), with very low flesh quality parameters. Fast-chilling also resulted in extreme signs of stress (intense mucus release, haemorrhage and no loss of consciousness), low ATP content and the worst proteomic signatures, along with an early onset and resolution of rigor mortis (6 and 48 h, respectively). Our results reinforce the idea that electric stunning is a promising humane method to stun farmed trout. In contrast, the fast-chilling method showed very poor results both in welfare and in quality, indicating that it is not a viable humane alternative to asphyxia. Moreover, the proteome analysis provided valuable insights into the brain mechanisms of rainbow trout at slaughter, offering potential fine-scale biomarkers of welfare