Fish are now widely recognised to be sentient animals . Good welfare practices are therefore not only ethical, but they also are demonstrated to improve fish quality and may even drive consumer choice. A critical point in the life of a captive fish is the final stages of the ir production. Not only this moment has the risk of dramatically affecting the welfare of the individuals, but it can also cause serious economic impacts to the farm, since the slaughter process can affect meat quality and carcass appearance. The most common method to slaughter fish (including trout) is by asphyxia either in ice-water or in the open air. However, this method induces prolonged and intense suffering, which is a poor practice in ethical, commercial, and legal terms. To achieve a humane slaughter practice, a stunning method needs to be implemented before slaughter, and it must render the fish immediately unconscious until death. In this regard, electrical stunning prior to slaughter has been proposed as a humane stunning method for trout , although there are anecdotal reports of trout carcass and fillet damage, with consequent decrease in value. In this experiment we tested a newly devised temperature stunner, in which the fish were immersed in water that achieves water temperatures of -8ºC still in liquid state. The objective 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 (FC) as a novel method, asphyxia (ASP) as the current method, electrical stunning (ES) as a humane method, and anaesthesia with MS-222 (AN) as a positive control. We used a multi-level approach to address the welfare of 176 juvenile trout, combining behaviour ( individual swimming activity, equilibrium, opercular movement and eye-roll), physiology (heart rate and amplitude of electrocardiogram signal ) and stress biomarkers (plasma cortisol and osmolality) , and proteomics. We then proceeded to analyse the effects on shelf-life fillet and quality of fish subjected to each of these methods, using a wide range of indicators (namely rigor mortis , water content, fillet colour, pH and ATP degradation) . This is, to our knowledge, the most detailed assessment of rainbow trout welfare at slaughter.
Behavioural indicators showed that ES presented quick and effective induction of unconsciousness (i.e., absence of all consciousness indicators in 20s ), while AN was slower yet 100% effective. Fish subjected to FC showed signs of extremely poor welfare (e.g., gill haemorrhage , brain damage, eye freezing and thawing, heavy mucus release) and this method was not only ineffective (only 13% lost consciousness) but also inconsistent (they recovered consciousness quickly). ASP also induced extremely poor welfare , with fish dying slowly (up to 20 min or more) and maintaining consciousness until death in 44% of cases.
Physiology indicators showed that ES had higher osmolality than AN , while ASP higher osmolality than AN and FC . Fish subjected to ASP had higher cortisol levels than AN , while ES and FC showed large variation in this hormone level . Regarding electrocardiogram signal, ES presented stronger heartbeat than ASP , while AN showed stronger heartbeat than ASP and FC. No differences were found in heart rate.
Regarding proteomics, FC and ASP had the highest impact on the brain proteome with bimodal effects. On one hand, FC registered a significant number of reactions, biological and metabolic processes. On the other hand, ASP results suggest suppression of the coping mechanisms and therefore an allostatic cost that may infer higher suffering. Interestingly, AN also induced a significant impact on the brain proteome as indicated by significant involvement of programmed cell death processes, which is probably linked to the opioid nature of MS-222.
Regarding quality indicators, ASP and FC showed faster rigor onset than AN and ES until 6h post-mortem . Energy depletion was lower in AN and ES than in ASP and FC , while AN showed higher pH than all the other treatments, and ASP presented the lowest pH. Fish subjected to ASP showed the highest fillet weight loss after rigor , ES and AN the lowest.
Our results demonstrate that, other than anaesthesia (which is generally not permitted in fish farmed for food), electrical stunning consistently showed good results as a humane stunning method. This was presented at all levels of welfare analysis, as well as in fillet quality terms. The fast-chilling method, on the other hand, presented very poor results both in welfare and in quality and did not seem to be a viable humane alternative to asphyxia.
Additionally, the proteome analysis opens new windows into the selection of fine-scale biomarkers of welfare and, more specifically, provides very interesting insights into the brain mechanisms of rainbow trout at slaughter.