Assessing unconsciousness is important to safeguard animal welfare shortly after stunning at the slaughter plant. Indicators that can be visually evaluated are most often used when assessing unconsciousness, as they can be easily applied in slaughter plants. These indicators include reflexes originating from the brain stem (e.g. eye reflexes) or from the spinal cord (e.g. pedal reflex) and behavioural indicators such as loss of posture, vocalisations and rhythmic breathing. When physically stunning an animal, for example, captive bolt, most important indicators looked at are posture, righting reflex, rhythmic breathing and the corneal or palpebral reflex that should all be absent if the animal is unconscious. Spinal reflexes are difficult as a measure of unconsciousness with this type of stunning, as they may occur more vigorous. For stunning methods that do not physically destroy the brain, for example, electrical and gas stunning, most important indicators looked at are posture, righting reflex, natural blinking response, rhythmic breathing, vocalisations and focused eye movement that should all be absent if the animal is unconscious. Brain stem reflexes such as the cornea reflex are difficult as measures of unconsciousness in electrically stunned animals, as they may reflect residual brain stem activity and not necessarily consciousness. Under commercial conditions, none of the indicators mentioned above should be used as a single indicator to determine unconsciousness after stunning. Multiple indicators should be used to determine unconsciousness and sufficient time should be left for the animal to die following exsanguination before starting invasive dressing procedures such as scalding or skinning. The recording and subsequent assessment of brain activity, as presented in an electroencephalogram (EEG), is considered the most objective way to assess unconsciousness compared with reflexes and behavioural indicators, but is only applied in experimental set-ups. Studies performed in an experimental set-up have often looked at either the EEG or reflexes and behavioural indicators and there is a scarcity of studies that correlate these different readout parameters. It is recommended to study these correlations in more detail to investigate the validity of reflexes and behavioural indicators and to accurately determine the point in time at which the animal loses consciousness.

European legislation states that after stunning regular checks should be performed to guarantee animals are unconscious between the end of the stunning process and death. When animals are killed without prior stunning these checks should be performed before the animal is released from restraint. The validity of certain indicators used to assess unconsciousness under different stunning and slaughter conditions is under debate. The aim of this study was to validate the absence of threat-, withdrawal-, corneal-and eyelid reflex as indicators to assess unconsciousness in calves subjected to different stunning and slaughter methods. Calves (201 ± 22 kg) were randomly assigned to one of the following four treatments: (1) Captive bolt stunning followed by neck cut in an inverted position (n = 25); (2) Non-stunned slaughter in an upright position (n = 7); (3) Non-stunned slaughter in an inverted position (180°rotation) (n = 25); (4) Non-stunned slaughter in an upright position followed by captive bolt stunning 40 s after the neck cut (n = 25). Each calf was equipped with non-invasive electroencephalogram (EEG) electrodes before the slaughter procedure. All reflexes were verified once before the slaughter procedure. At the beginning of the procedure (T = 0 s) calves were stunned (treatment 1) or neck cut in an upright position (treatment 2, 4) or inverted position (treatment 3). Calves of treatment 4 were captive bolt stunned 34 ± 8 s after the neck cut. Reflexes were assessed every 20 s from T = 15 s for all treatments until all reflex tests resulted in a negative response three times in a row and a flat line EEG was observed. In addition, reflexes were assessed 5 s after captive bolt stunning in calves of treatments 1 and 4. Visual assessment of changes in the amplitude and frequency of EEG traces was used to determine loss of consciousness. Timing of loss of consciousness was related to timing of loss of reflexes. After captive bolt stunning, absence of threat-, withdrawal-, corneal-and eyelid reflex indicated unconsciousness as determined by EEG recordings. After non-stunned slaughter, both threat-and withdrawal reflex were on average lost before calves were unconscious based on EEG recordings. The eyelid-and corneal reflex were on average lost after calves had lost consciousness based on EEG recordings and appeared to be distinctly conservative indicators of unconsciousness in non-stunned slaughtered calves since they were observed until 76 ± 50 and 85 ± 45 s (mean ± SD), respectively, after EEG-based loss of consciousness.Keywords: electroencephalogram, calves, reflexes, slaughter, (un)consciousness ImplicationsMonitoring unconsciousness at slaughter is mandatory by European legislation and is often determined by the absence of behavioural indicators (i.e. loss of posture), physical signs (i.e. rhythmic breathing) and reflexes. Previous research in sheep has indicated that the absence of certain reflexes at slaughter is not a reliable indicator of unconsciousness. The present study showed that absence of the withdr...

Exposure to CO2 at high concentration is a much debated stunning method in pigs. Pigs respond aversively to high concentrations of CO2, and there is uncertainty about what behaviors occur before and after loss of consciousness. The aim was to assess timing of unconsciousness in pigs during exposure to high concentrations of CO2 based on changes in electroencephalogram (EEG) activity and the relation with the behaviors sniffing, retreat and escape attempts, lateral head movements, jumping, muscular contractions, loss of posture, and gasping. Pigs (108 ± 9 kg) were randomly assigned to 80% CO2 (80C, n = 24) or 95% CO2 (95C, n = 24). The time at which the gondola started descending into the well pre-filled with 80C or 95C was marked as T = 0. The CO2 exposure lasted 346 s after which the corneal reflex and breathing were assessed for 1 min. Visual assessment of changes in the amplitude and frequency of EEG traces after T = 0 was used to determine loss of consciousness. Time to loss of consciousness was longer in 80C pigs (47 ± 6 s) than in 95C pigs (33 ± 7 s). Time to an iso-electric EEG was similar in 80C pigs (75 ± 23 s) and 95C pigs (64 ± 32 s). When pigs descended into the well, the earlier entry of 95C pigs into high CO2 atmosphere rather than the concentration of CO2 by itself affected the latency of behavioral responses and decreasing brain activity. During exposure to the gas, 80C and 95C pigs exhibited sniffing, retreat attempts, lateral head movements, jumping, and gasping before loss of consciousness. 95C pigs exhibited all these behaviors on average earlier than 80C pigs after T = 0. But the interval between onset of these behaviors and loss of consciousness and the duration of these behaviors, except gasping, was similar for both treatments. Loss of posture was on average observed in both groups 10 s before EEG-based loss of consciousness. Furthermore, 88% of 80C pigs and 94% of 95C pigs demonstrated muscular contractions before loss of consciousness. The findings provide little reason to conclude on a behavioral basis that these atmospheres are greatly different in their impact on pig welfare.

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The stunning quality of animals for slaughter remains under constant scrutiny. In response to previous research showing low stunning efficiency in poultry, the conventional water bath will be phased out in the Netherlands. Presently, the main practical alternative to water bath stunning of poultry is a 2-phased gas stunning method. Gas stunning methods are recognized by governments and animal welfare organizations across Europe. In this study, 3 sets of experiments were conducted on gas stunning methods using CO(2) in 2 phases. Two methods were examined to identify potential effects on bird behavior and investigate their practical implications: a 5-stage incremental CO(2) scheme lasting 6 min (treatment 1) and a 4-stage incremental CO(2) scheme lasting 4 min (treatment 2). The onset and duration of unconsciousness were specifically tested in experiment 2 by using 25 birds equipped with electrodes monitoring brain and heart activity. Behavioral responses were observed on 15 non-instrument-monitored birds kept in the same cages at that time. Results in all 3 sets of the experiments showed that multistage gas stunning was stable and consistent, and increases in CO(2) concentrations were rapid and reliable. Ambient temperatures and RH of the air remained within acceptable levels at all times. Induction of unconsciousness occurred below 40% CO(2) and did not significantly differ between treatments. Conscious birds were never exposed to high CO(2) concentrations (>40% CO(2)), yet some birds showed signs of distress (e.g., head shaking, wing flapping) before losing consciousness. Discomfort experienced during exposure to low (<40%) CO(2) concentrations compares favorably with the experiences of handling, tilting, and or shackling of conscious birds when using alternative stunning methods, implying that multistage gas stunning has distinct advantages for bird welfare. Compared with the multibird water bath system, this method provides an opportunity to guarantee that all birds are properly stunned. The risk of convulsions, which was higher with treatment 2, leading to possible injuries, indicates a preference for the 5-stage treatment.