Introduction
Slaughter is a major welfare concern in aquaculture, with an estimated 78–171 billion fish killed annually . Humane slaughter methods must ensure fish are rendered insensible—unable to perceive or respond to stimuli—prior to death and remain so without experiencing fear, pain, or distress. While electroencephalography (EEG) based neurophysiological indicators are the gold standard for assessing sensibility, their use requires specialist equipment and expertise, limiting feasibility in commercial settings.
As a result, operational indicators—such as the loss or recovery of equilibrium, ventilation, and the ’eye-roll’ reflex—are commonly used on farms to assess sensibility. However, concerns remain regarding the time lag between these indicators and the actual cessation or recovery of brain activity. Additionally, these indicators may signify immobilization rather than true insensibility. Misjudging sensibility can compromise animal welfare during procedures such as exsanguination or evisceration.
To ensure their reliability, operational indicators must be validated against neurophysiological benchmarks on a species- and method-specific basis. Yet such validation is lacking for European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata). This study addresses that gap by comparing operational and neurophysiological indicators during gradual (anaesthesia immersion) and rapid (‘in-water’ electrical stunning ) transitions into and out of insensibility in both species, assessing the extent to which operational indicators reliably reflect true insensibility.
Materials and methods
Loss of indicators during gradual induction into insensibility
To evaluate the gradual induction into insensibility in seabass (n = 16) and seabream (n = 14), fish were transferred to a container containing saltwater with an anaesthetic solution (salinity = 9.2 ‰, temperature = 24.0ºC, anaesthetic = 600 ppm 2-phenoxyethanol). Operational indicators (e.g., loss of equilibrium, ventilation, and ’eye-roll’ reflexes) and the loss of VERs were then monitored during the gradual induction into insensibility.
Recovery of indicators following acute induction into insensibility
To evaluate the recovery of seabass (n = 21) and seabream (n = 27) following acute induction into insensibility, fish were transferred to a darkened, in-water electrical stunning chamber containing saltwater (salinity = 9.2 ‰, temperature = 23.8ºC). Fish were then individually subjected to a 30 s electrical stun (i.e., 50 Hz AC, current density of 3.04 A dm-2, electrical field strength of 1.68 V cm-1) and the recovery of operational indicators and VERs were then monitored
Results and discussion
Until neurophysiological monitoring becomes feasible in commercial practice, detailed, species- and method-specific validations of operational indicators remain essential. This study highlights the limitations of operational indicators and underscores the critical need for robust, context-dependent assessments to safeguard fish welfare during slaughter in aquaculture.
Our findings reveal significant temporal mismatches between operational and neurophysiological indicators of sensibility. During anaesthetic immersion, all operational indicators were consistently lost before VERs, with equilibrium lost first and ventilation last (see upper panel of Fig. 1). This mismatch ranged from 1.3 to 8.1 times faster than VER loss, depending on the indicator and species. Similarly, following electrical stunning, operational indicators reappeared substantially later than VERs, by factors ranging from 1.4 to 4.7 (see lower panel of Fig. 1). These findings confirm that an exclusive focus on the absence of operational indicators may lead to incorrect assumptions about the neural state of the individual. Clear interspecies differences also emerged. Seabass lost and regained sensibility faster than seabream across nearly all indicators. Interestingly, the sequence of indicator loss differed between species during anaesthesia, further supporting the need for species-specific welfare assessments.
Despite the rising use of electrical stunning, the majority of fish regained VERs within seconds to minutes following a 30 s ’in-water’ electrical stun. In fact, more than half of individuals already displayed VERs by the time EEG recordings began, underlining how quickly sensibility can return. This rapid recovery poses a serious welfare hazard if not followed by prompt and effective killing. Consequently, there is an urgent need to rigorously evaluate, improve, and validate electrical stunning methods in seabass and seabream before promoting and implementing them as ’humane’ stunning methods.