Implications
This review evaluates the different ways in which unconsciousness after stunning is assessed and weighs the pros and cons of these methods. Assessing unconsciousness is performed in a variety of ways, depending on species as well as the method of stunning. Assessing brain activity by way of electroencephalogram (EEG) analysis is suggested to be the most objective method to evaluate unconsciousness, but this is only applied in experimental set-ups. Studies in which correlations between the EEG and other indicators are looked at in more detail could provide additional information on the exact time points at which animals lose consciousness after stunning.
Introduction
European legislation provides laws, rules and procedures regarding the slaughter of livestock (GWvD, 1992; Council Directive 93/119/EC, 1993; Council Regulation (EC) No 1099/2009, 2009). Article 4 of Council Regulation (EC) No 1099/2009 describes the mandatory pre-slaughter stunning, with exception of particular methods of slaughter prescribed by religious rites, to ensure unconsciousness and insensibility to prevent unnecessary suffering of animals. There is no consensus about the extent to which slaughter of conscious, meaning sensible and/or aware, animals causes them pain and distress. It is claimed that when a clean incision is made with an exquisitely sharp knife, significant pain and distress are avoided (e.g. Grandin, Reference Grandin1994; Rosen, Reference Rosen2004). Johnson et al. (Reference Johnson, Gibson, Stafford and Mellor2012) suggest that massive stimulation of all sensory nerves after the neck cut may lead to shock and distress that would be experienced as pain for the duration of consciousness. Until now, neurophysiological methodology has not provided the ultimate answer to this issue. Because animals are considered not to experience pain when unconscious, it is important to validly determine unconsciousness after stunning. Stunning methods most frequently applied include mechanical stunning (captive bolt), applying an electrical current through the head of the animal or by immersion in a mixture of gasses consisting of (low level) oxygen (O2), carbon dioxide (CO2), argon (Ar) and/or nitrogen (N2). For all stunning methods, it is critical to determine the onset and duration of unconsciousness. Available data from different livestock species to examine the different methods used to assess unconsciousness, include reflexes and behavioural indicators. Less used in practice, but considered the most objective method for the assessment of unconsciousness, involves the evaluation of brain activity as presented in an electroencephalogram (EEG). The possibilities and limitations of the use of EEG for this purpose are further elaborated upon in this manuscript.
Consciousness and unconsciousness
Consciousness is defined in many different ways, but in general is associated with the awake state and the ability to perceive, interact and communicate with the environment and others (Zeman, Reference Zeman2001). The opposite state, that is, unconsciousness, is defined as: ‘a state of unawareness (loss of consciousness) in which there is temporary or permanent disruption to brain function. As a consequence of this disruption, the unconscious animal is unable to respond to normal stimuli, including pain’ (EFSA, 2006). Disruption of brain function can occur as a result of brain concussion, administration of anaesthetics, anoxia or an electroconvulsive shock (Lopes da Silva, Reference Lopes da Silva1982). Some authors prefer the term insensibility over unconsciousness, as they find it less anthropomorphic (Blackmore and Delany, Reference Blackmore and Delany1988). Insensibility refers to the complete inability to experience any sensations, including unpleasant sensations such as pain (Hemsworth et al., Reference Hemsworth, Fisher, Mellor and Johnson2009). Pain is defined as ‘an unpleasant sensory and/or emotional experience associated with actual or potential tissue damage, or described in terms of such damage’ (Merskey, Reference Merskey1986). Pain is considered a conscious experience and needs to be avoided during the slaughter process. The term unconsciousness, as used in this review, also includes insensibility. Stunning of animals aims at inducing unconsciousness and thus insensibility, which lasts until the animal is dead. An animal is considered dead when: ‘respiration and blood circulation have ceased as the respiratory and circulatory centres in the medulla oblongata are irreversibly inactive. Because of the permanent absence of nutrients and O2 in the brain, consciousness is irreversibly lost’ (EFSA, 2004). During the slaughter process, regular checks should be carried out to ensure that the animal does not present any signs of consciousness or sensibility in the period between the end of the stunning process and death (Council Regulation (EC) No 1099/2009, 2009).
Brain regions that are involved in consciousness are the cerebral cortex and thalamus, together forming the thalamocortical complex, which is regulated by the brainstem. A well-functioning brainstem and thalamus are essential for the maintenance of consciousness and damage to (one of) these regions can cause rapid loss of consciousness (Gregory and Shaw, Reference Gregory and Shaw2000). However, localised lesions in the cortex, for instance in the sensory cortex, do not necessarily cause unconsciousness, but may only change specific features such as colour vision or the way visual objects and faces are interpreted (Seth et al., Reference Seth, Baars and Edelman2005). The central core of the brainstem is formed by the reticular formation, a large network of neural tissue located in the central region of the brain stem. The reticular formation receives sensory information from the cortex and several subcortical regions and its axons project to the cerebral cortex, thalamus and spinal cord. The reticular formation plays not only a role in sleep and arousal, but also in attention, muscle tone, movement and various vital reflexes (Carlson, Reference Carlson2007). When the reticular formation fails, the cerebral cortex will be switched off or cannot be switched on. When the cortex is (functionally) damaged, neuronal integration of signals from the central nervous system necessary for conscious perception and subjective experience cannot occur. The disruption of normal electrical brain activity is considered to be incompatible with consciousness (Savenije et al., Reference Savenije, Lambooij, Gerritzen and Korf2002; Lambooij, Reference Lambooij2004; Adams and Sheridan, Reference Adams and Sheridan2008). To maintain consciousness, a constant supply of O2 and energy to the brain and continuous removal of metabolic waste, such as CO2, is needed. If one of the mechanisms fails, for instance due to stunning, an animal will become unconscious (Adams and Sheridan, Reference Adams and Sheridan2008).
Time to and duration of unconsciousness
In a large-scale study by von Wenzlawowicz et al. (Reference von Wenzlawowicz, von Holleben and Eser2012), stunning effectiveness was assessed in over 37 000 pigs and cattle, stunned by different methods. The mean percentages for animals showing signs compatible with insufficient stunning ranged from 3% to 14%, depending on the stunning method and with a high variability between slaughter plants. Gregory (Reference Gregory2008) found that 8% of electrically stunned cattle (n=67) were not deeply stunned and showed signs of consciousness at 20 and 90 s post stunning. If stunning is reversible, the chance for recovery should be minimised and the stun-to-stick interval should be kept to a minimum to prevent recovery during exsanguination. With electrical stunning in pigs, an interval under 15 s was recommended, where after exposure to gas a stun-to-stick interval of 25 to 45 s was advised, depending on the gas mixture and concentration used (Anil, Reference Anil1991; Raj, Reference Raj1999). Recommendations on the duration of stun-to-stick interval depend on different factors including the amount of current or concentration of gas used and the exposure time. When the stun is found not to be effective, the animal should be re-stunned as soon as possible. Animals that are conscious at time of the neck cut lose consciousness as a consequence of the severe decrease in cerebral blood flow leading to a rapid onset of disorganised brain function and thus unconsciousness (Mellor et al., Reference Mellor, Gibson and Johnson2009). Sheep and poultry lose spontaneous brain activity after on average 14 and 23 s when both carotid arteries are severed (Gregory and Wotton, Reference Gregory and Wotton1984 and Reference Gregory and Wotton1986). In cattle, however, consciousness after the neck cut is prolonged, as the vertebral arteries, which are not severed by the neck cut, supply blood to the circle of Willis and play a direct role in the blood supply to the brain (Baldwin and Bell, Reference Baldwin and Bell1963). Cattle lose spontaneous brain activity 75±48 s post neck cut (range 19 to 113 s), but Newhook and Blackmore (Reference Newhook and Blackmore1982) suggested possible intermittent sensibility for up to 123 to 323 s after slaughter in cattle (Daly et al., Reference Daly, Kallweit and Ellendorf1988). The time to loss of consciousness in non-stunned animals, emphasises the need to verify unconsciousness after stunning and take sufficient time for full bleed out before the start of carcass processing, especially in cattle.
Assessing unconsciousness
Unconsciousness, caused by temporary or permanent disruption to the brain, is generally assessed by the observation of behavioural indicators, which are internally coordinated responses to internal or external stimuli (Levitis et al., Reference Levitis, Lidicker and Freund2009). They include reflexes originating from the brain stem (e.g. eye reflexes) or spinal cord (e.g. pedal reflex) and behavioural indicators such as loss of posture, vocalisation and rhythmic breathing. In an experimental set-up, the assessment of brain activity as presented in an EEG, derivatives of the EEG, and evoked potentials can be used to assess unconsciousness.
Reflexes
Reflexes are automatic, stereotyped movements that are produced as the direct result of a stimulus and are mediated by the central nervous system (Carlson, Reference Carlson2007). The presence of central reflexes are indicators of consciousness that are linked to functioning of the brain stem or spinal cord. Brain stem reflexes are regulated by 12 pairs of cranial nerves that enter and exit the brain and are not under cortical control. Two cranial nerves (I and II) enter from the forebrain and the other nerves (III to XII) enter and exit from the brain stem (Carlson, Reference Carlson2007; Rubin and Safdieh, Reference Rubin and Safdieh2007). Brain stem reflexes that are used to assess unconsciousness after stunning in livestock are cornea or blinking, palpebral, pupillary light and threat reflex. The cornea reflex causes involuntary blinking of the eyelids in response to stimulation of the cornea and is in general the last reflex to be lost in anaesthetised animals (Dugdale, Reference Dugdale2010). The palpebral reflex also results in blinking as a response to touching the medial canthus of the eye and disappears earlier than the cornea reflex in anaesthetised animals. Both the cornea and palpebral reflex require a functional afferent cranial nerve V (trigeminal) and efferent cranial nerve VII (facial) and the relevant eye muscles to function adequately (Adams and Sheridan, Reference Adams and Sheridan2008). The pupillary light reflex is tested by letting light fall on the eye and observing whether the pupil adapts to it. The reflex is controlled by cranial nerves II (optic) and III (oculomotor) and is not considered a reliable reflex during exsanguination, as exsanguination interferes with the blood supply to the retina (Blackman et al., Reference Blackman, Cheetham and Blackmore1986). When testing the threat reflex, an object (finger or pencil) suddenly approaches the eye and a conscious animal will close its eye or withdraw the head. This reflex requires a functional efferent cranial nerve VII (facial) and integration of the motor cortex, but is not often applied, as it requires the eye to be open. Focused eye movement, not a reflex, is considered a definite sign of consciousness, as it needs cortical activity for perception and controlled motor activity from the eyeball muscles (Grillner et al., Reference Grillner, Wallén, Saitoh, Kozlov and Robertson2008; Vogel et al., Reference Vogel, Badtram, Claus, Grandin, Turpin, Weyker and Voogd2011). It is pointed out that positive eye reflexes alone do not necessarily indicate consciousness, as positive brain stem reflexes might occur on the basis of residual brain stem activity and do not distinguish clearly between consciousness and unconsciousness (Anil, Reference Anil1991). This especially holds true for animals that are electrically stunned, which was documented as early as 80 years ago (Roos and Koopmans, Reference Roos and Koopmans1936; Blackmore and Delany, Reference Blackmore and Delany1988; von Holleben et al., Reference von Holleben, von Wenzlawowicz, Gregory, Anil, Velarde, Rodríguez, Cenci Goga, Catanese and Lambooij2010). In both sheep and calves, brain stem reflexes were present long after electrical stunning, even though the EEG was suppressed or iso-electric (Anil, Reference Anil1991; Anil and McKinstry, Reference Anil and McKinstry1991). On the other hand, eye reflexes may be inhibited after electrical stunning, whereas the cerebral cortex still functions and the animal may be conscious (Blackmore and Delany, Reference Blackmore and Delany1988). There is no literature available on the frequency of such incidences and its risk for animal welfare is therefore difficult to estimate. After effective captive bolt stunning, however, no eye reflexes should be present, because of the brain trauma produced (Finnie, Reference Finnie1995; Gregory and Shaw, Reference Gregory and Shaw2000). Thus, cranial nerve reflexes can be good indicators for impaired midbrain or brain stem activity, but only work reliably in one way: when absent, it is very likely that the animal is unconscious, but when they are present, the animal is not necessarily conscious. Spinal reflexes include stretch and flexor reflexes. The stretch reflex, a monosynaptic reflex, is the most basic reflex and plays an important role in control of posture. It does not involve the brain and is therefore not used to assess unconsciousness (Carlson, Reference Carlson2007). The flexor reflex, a polysynaptic reflex, involves activation of nociceptors and is used to assess unconsciousness (Anil, Reference Anil1991; Erasmus et al., Reference Erasmus, Turner and Widowski2010). An example of a flexor reflex is the pain withdrawal reflex, which is elicited by applying a painful stimulus to the animal, such as a nose or ear prick. In a survey on expert opinion, the pain withdrawal reflex was ranked high, and thus valued highly, as an indicator to assess unconsciousness after all types of stunning (Gerritzen and Hindle, Reference Gerritzen and Hindle2009). The pedal reflex is elicited by, for instance, pinching the skin between the toes of an animal. This reflex is often used for assessment of depth of anaesthesia in laboratory animals, such as rodents and rabbits, but is only occasionally applied in livestock after stunning, as all spinal reflexes are difficult to assess when animals exhibit convulsions or body movements (Tidswell et al., Reference Tidswell, Blackmore and Newhook1987). This especially holds true for animals that are physically stunned, for example, captive bolt stunning, when there is lack of inhibition from the brain and spinal reflexes may occur more vigorously (Blackmore and Delany, Reference Blackmore and Delany1988). Again, electrically stunned animals may exhibit this reflex long after losing consciousness and the reflex may occur more vigorously when the animal is handled (Blackmore and Newhook, Reference Blackmore and Newhook1982). The righting reflex refers to any reflex that tends to bring the body into its normal upright position. It is often assessed when animals are removed from the stunning box or are hung to the bleeding rail and is also referred to the head righting reflex. This reflex is also difficult to assess when animals exhibit convulsions or involuntary body movements (Blackmore and Newhook, Reference Blackmore and Newhook1982; Anil, Reference Anil1991). Table 1 shows an overview of the different brain stem and spinal reflexes used to assess unconsciousness after stunning.
Reflexes used to assess unconsciousness in livestock after stunning
1 Presence and absence of reflexes are presented as follows:+=present, −=absent, (+)=may be present, (−)=may be absent.
Behavioural indicators
Loss of posture, the inability of the animal to remain in an initial standing or sitting position, is considered a valuable indicator as it is often the first sign to be lost after successful stunning and indicates that the cerebral cortex is no longer able to control posture (Raj et al., Reference Raj, Wotton and Gregory1992; Raj and Gregory, Reference Raj and Gregory1996; Llonch et al., Reference Llonch, Rodríguez, Jospin, Dalmau, Manteca and Velarde2013). Both mechanical and electrical stunning should lead to immediate collapse (AVMA, 2013). Nystagmus, involuntary rapid horizontal eye flickering, is caused by damage to the vestibular, labyrinthine or central nervous system and was more present in cattle that had a shallow depth of concussion following captive bolt. It was observed in only 3% of 1608 cattle, but was associated with a greater chance of rhythmic breathing. Its presence could add strength to the conclusion that the depth of concussion has been shallow (Gregory et al., Reference Gregory, Lee and Widdicombe2007). In a study by Bourquet et al. (Reference Bourguet, Deiss, Tannugi and Terlouw2011), nystagmus was observed in one out of 95 captive bolt shot cattle. This animal was reshot, and this supported the study by Gregory et al. (Reference Gregory, Lee and Widdicombe2007), which indicated that when nystagmus was observed, there was a one in three chance that the quality of the stun was insufficient. Nystagmus may occur as a result of electrical stunning (Grandin, Reference Grandin2002), but in CO2-stunned pigs, nystagmus was not observed once (Atkinson et al., Reference Atkinson, Velarde, Llonch and Algers2012). It is stated that under no circumstances should a stunned animal vocalise, as vocalisation after stunning indicates consciousness and probably distress and pain (Grandin and Smith, Reference Grandin and Smith2004; Gouveia et al., Reference Gouveia, Ferreira, da Costa, Vaz-Pires and da Costa2009). A large network of brain regions is involved in the production of vocalisations, including the frontal lobe and primary motor cortex and vocalisations are considered a conscious response (Carlson, Reference Carlson2007). The involuntary passage of air along the vocal cords, however, may cause sounds that can be mistaken for vocalisations. Absence of vocalisations on the other hand, is certainly no guarantee for absence of pain or distress, as the occurrence of vocalisations also depends on the species. A sheep often does not vocalise when injured, where a pig will scream loudly (Broom, Reference Broom2001; EFSA, 2004). Grandin (Reference Grandin2002) believes an animal to be unconscious when it shows a limp head and protruding tongue. The tongue is controlled by nerve XII (hypoglossal) and when relaxed this may indicate loss of cranial nerve function. A study by Gregory et al. (Reference Gregory, Lee and Widdicombe2007) showed that a protruding tongue was not associated with depth of concussion after captive bolt stunning, but was proposed as indicator following exsanguination, when 40% of the cattle had a protruding tongue while hanging on the bleeding rail. Similarly, relaxation of the jaw may be taken into account, but can be observed in conscious animals (Gregory et al., Reference Gregory, Spence, Mason, Tinarwo and Heasman2009). Both jaw relaxation and tongue protruding are not used as single indicators to assess unconsciousness, but can support other indicators of unconsciousness (Grandin, Reference Grandin2002; von Holleben et al., Reference von Holleben, von Wenzlawowicz, Gregory, Anil, Velarde, Rodríguez, Cenci Goga, Catanese and Lambooij2010). Beside the important role regarding consciousness, the brain stem also houses the regulatory centres for respiratory and circulatory systems. Rhythmic breathing movements after stunning indicate that the corticospinal, ventral and lateral columns of the spinal cord are still intact and may thus indicate consciousness (Mitchell and Berger, Reference Mitchell and Berger1975). The presence of rhythmic breathing after stunning is generally accepted to indicate that an animal may not be fully unconscious and is thought to be one of the first signs of recovery after CO2 and electrical stunning (Gerritzen and Hindle, Reference Gerritzen and Hindle2009; Anastasov and Wotton, Reference Anastasov and Wotton2012). In captive bolt stunned cattle, rhythmic breathing immediately disappears after an effective shot because of axonal injuries to the brainstem (Finnie et al., Reference Finnie, Blumbergs, Manavis, Summersides and Davies2000). The occurrence of convulsions, observed as uncontrolled movements of the body, indicates effective stunning in electrical or mechanical stunned animals, but also occur in unconscious animals that are gas stunned (Adams and Sheridan, Reference Adams and Sheridan2008; Marzin et al., Reference Marzin, Collobert, Jaunet and Marrec2008; von Holleben et al., Reference von Holleben, von Wenzlawowicz, Gregory, Anil, Velarde, Rodríguez, Cenci Goga, Catanese and Lambooij2010). These convulsions are thought to be incompatible with consciousness due to the absence of higher motor control (Lambooij, Reference Lambooij2004). They can, however, sometimes be mistaken for rhythmic breathing, as they can occur as almost rhythmic body movements (Wotton and Sparrey, Reference Wotton and Sparrey2002). Gagging refers to low-frequency inhalations with the neck positioned towards the front legs and occasional emission of sounds similar to snoring and is considered an indicator of deep unconsciousness (Rodríguez et al., Reference Rodríguez, Dalmau, Ruiz-de-la-Torre, Manteca, Jensen, Rodríguez, Litvan and Velarde2008). Gasping is seen when an animal takes deep breaths through an open mouth and is considered an indicator of onset of breathlessness during CO2 stunning, which continues long after loss of consciousness even when brain activity is no longer recorded, but may also occur after electrical stunning (Blackmore and Petersen, Reference Blackmore and Petersen1981; Newhook and Blackmore, Reference Newhook and Blackmore1982; Grandin, Reference Grandin2013). Interpretation of all individual indicators mentioned above can be doubtful unless supported by other information (Blackmore, Reference Blackmore1984; Gerritzen and Hindle, Reference Gerritzen and Hindle2009; Anastasov and Wotton, Reference Anastasov and Wotton2012). Table 2 shows an overview of the different behavioural indicators used to assess unconsciousness after stunning.
Behavioural indicators used to assess unconsciousness in livestock after stunning
1 Presence and absence of reflexes are presented as follows:+=present; −=absent; (+)=may be present; (−)=may be absent.
Brain activity (EEG)
When monitoring brain functioning, activity can be presented in an EEG, which displays electrical activity derived from electrodes attached to various locations on the surface of the head. The EEG is considered the most objective method for assessing unconsciousness and reflects the sum of underlying electrical activity of populations of neurones supported by glia cells (Murrell and Johnson, Reference Murrell and Johnson2006). There are four different types of wave patterns in the EEG that can be distinguished based on their respective frequencies and that are related to the state of consciousness: δ (0 to 4 Hz), θ (4 to 8 Hz), α (8 to 12 Hz) and β (>12 Hz) waves. Both δ and θ (slow wave) activity is related to sleep or reduced consciousness. α activity is prominent in subjects that are conscious, but mentally inactive (closing eyes and relaxation) and β waves are associated with active movements and increased alertness (Kooi et al., Reference Kooi, Tucker and Marshall1978; Niedermeyer et al., Reference Niedermeyer, Schomer and Da Silva2011). Depending on the method of stunning, the EEG shows a characteristic pattern of change when animals lose consciousness. Generally, an increase in low frequency activity is accompanied by an increase in amplitude. When neurons depolarise at the same time or frequency, they fire in a synchronised fashion creating slow high amplitude waves as seen in unconscious states suggesting a depression of the reticular formation (Lopes da Silva, Reference Lopes da Silva1982). Consciousness on the other hand is characterised by high frequency (α and β), low amplitude waves (Seth et al., Reference Seth, Baars and Edelman2005). When looking more specifically at EEG wave patterns, the EEG can be broken down in different time segments, better known as epochs. These epochs can be analysed for frequency (Hz), amplitude (µV) and power (µV2), together representing the amount of activity in the brain. Four stages of EEG can be distinguished during the process of stunning and slaughter and are related to the level of consciousness, namely: active, transitional, unconscious and iso-electric (flat) EEG. In the first (active) stage, normal awake activity is recorded with high frequency, low amplitude waves, indicating the animal is conscious. In the second (transitional) stage, the amplitude of the EEG increases together with a decrease in frequency. When these changes become more profound, the animal is considered unconscious. When loss of consciousness progresses, the EEG turns iso-electric and brain activity is no longer recorded (Gibson et al., Reference Gibson, Johnson, Stafford, Mitchinsont and Mellor2007; McKeegan et al., Reference McKeegan, McIntryre, Demmers, Lowe, Wathes, van den Broek, Coenen and Gentle2007). The exact moment when unconsciousness sets in, based on the EEG, is difficult to determine as changes are often gradual. The iso-electric EEG, however, is never compatible with consciousness. There is no consistency in the literature regarding the number of stages used in the assessment of unconsciousness. Other research may only differentiate between the stages conscious and unconscious or contrary, use additional stages besides the four mentioned above.
Derivatives of the EEG
Another way of analysing raw EEG data, next to visual appraisal of the EEG, would be to compute a Fast Fourier Transformation (FFT). The output thereof represents the frequency composition of the signal, or alternatively formulated, how much power is presented in the different frequency bands. The principle is similar to defining the EEG in different EEG types that consist of slow or fast waves with high or low amplitudes (Davidson, Reference Davidson2006). Further (automatic) calculations of the FFT can lead to EEG derivatives presenting a single value or percentage that is easier to standardise.
Derivatives of the EEG include: the total power (P tot), which is the area underneath the frequency spectrum curve, the medium frequency (F 50), which is the frequency below which 50% of the total power is located and the spectral edge frequency (F 95), which is the frequency below which 95% of the power is located. These readout parameters are considered quantitative tools to describe changes in EEG activity (Murrell and Johnson, Reference Murrell and Johnson2006). An initial increase in P tot may represent a loss of functional cerebrocortical activity when amplitudes of EEG waves increase because of synchronised firing of neurons. But as the level of unconsciousness deepens, a decrease in all three derivatives is seen (Bager et al., Reference Bager, Braggins, Devine, Graafhuis, Mellor, Tavener and Upsdell1992; Martoft et al., Reference Martoft, Jensen, Rodriguez, Jorgensen, Forslid and Pedersen2001). In electrically stunned livestock, an increase in power of all frequency bands is first observed in the first 5 to 15 s post-stun because of initial epileptiform activity (Velarde et al., Reference Velarde, Ruiz-de-la-Torre, Roselló, Fàbrega, Diestre and Manteca2002; Beyssen et al., Reference Beyssen, Babile and Fernandez2004). Automatic FFT is applied during human surgeries and on a smaller scale during animal surgeries, where the raw EEG and its FFT are used to assess anaesthetic depth. Established anaesthesia monitors are used to assess depth of anaesthesia, but they differ in the algorithm used to analyse the EEG (Bruhn et al., Reference Bruhn, Myles, Sneyd and Struys2006). To the authors’ knowledge, only one of such monitors, namely the Index of Consciousness or IoC, has been used in a study concerning stunning in animals. During gas stunning of pigs, the raw EEG was recorded and based on that data a dimensionless variable (IoC) was calculated (Llonch et al., Reference Llonch, Rodríguez, Jospin, Dalmau, Manteca and Velarde2013). This variable ranges from 100 (awake) to 0 (iso-electric) and decreases with increasing anaesthetic and sedative depth. Values between 40 and 60 are suggested to represent an adequate hypnotic effect of the subject under general anaesthesia (Grover and Bharti, Reference Grover and Bharti2008). In the study by Llonch et al. (Reference Llonch, Rodríguez, Jospin, Dalmau, Manteca and Velarde2013), time to loss of posture occurred almost 20 s earlier then the accompanying decrease in IoC. A delay in IoC reading, compared with loss of balance, was also seen in pigs anaesthetised with propofol, but with a delay of only 7 s (Llonch et al., Reference Llonch, Andaluz, Rodríguez, Dalmau, Jensen, Manteca and Velarde2011). Muscular excitations that occur during CO2 stunning probably affected the IoC calculation, as movement artefacts are known to influence EEG data and calculations made in anaesthesia monitors (Teplan, Reference Teplan2002). This is one of the reasons offline calculation is used to more adequately compare and correlate brain activity data with behavioural indicators. Though many studies have looked at behavioural indicators or the EEG separately, only a few have studied correlations between these different read-out parameters for assessing unconsciousness. In a study by Benson et al. (Reference Benson, Alphin, Rankin, Caputo, Kinney and Johnson2012), loss of posture was correlated to the α/δ ratio extracted from the EEG, in an effort to find a more objective and alternative method (as opposed to loss of posture) to assess loss of consciousness in broilers. A correlation and no difference was found between time to unconsciousness as observed by the two methods, supporting the use of α/δ ratio as method to assess unconsciousness. The study shows that such correlations can provide additional, more objective data to support the use of behavioural indicators as a measure of unconsciousness and provide details when certain behaviours may be present or absent in an animal that loses consciousness.
Evoked responses
The EEG recording is also used to assess unconsciousness by way of generating evoked responses. Evoked responses are responses in the EEG following external stimuli (visual, somatosensory or auditory), generated in specific areas of the cerebral cortex, mid brain and brainstem (Schneider and Sebel, Reference Schneider and Sebel1997; Grover and Bharti, Reference Grover and Bharti2008). Evoked responses are frequently used as additional indicators to assess unconsciousness next to behavioural indicators, and have been applied in sheep, cattle, poultry and pigs. No correlations, however, have been calculated for the presence or absence of evoked potentials and presence or absence of behavioural indicators. Though, similar to EEG derivatives, evoked potentials may in this way provide additional support for the use of certain behavioural indicators. As for now, evoked responses are only used in experimental set-ups. Rapid changes in consciousness are difficult to observe with evoked potentials, as repeated stimulation and averaging of data (EEG) is needed to see these changes (Beyssen et al., Reference Beyssen, Babile and Fernandez2004). Differences in time to loss of consciousness based on the loss of spontaneous EEG or evoked responses have been observed in multiple studies. In hens stunned with different gas mixtures, evoked responses were observed to disappear ∼15 s after the EEG became suppressed, but almost 30 s before the occurrence of an iso-electric EEG (Raj et al., Reference Raj, Gregory and Wotton1991 and Reference Raj, Wotton and Gregory1992). In poultry slaughtered by nine different methods, all without prior stunning, spontaneous brain activity was lost after 23 to 233 s, where visual evoked potentials were lost after 90 to 349 s (Gregory and Wotton, Reference Gregory and Wotton1986). The loss of somatosensory evoked potentials was also recorded before an iso-electric EEG, but after a suppressed EEG in gas-stunned turkeys (Raj and Gregory, Reference Raj and Gregory1993). The presence of an evoked response implies that the afferent pathways to the higher brain centres are intact, but not necessarily that the animal is aware of the stimulus (Raj et al., Reference Raj, Gregory and Wotton1991). Visual evoked potentials have been observed in, for instance, anaesthetised animals (Gregory and Wotton, Reference Gregory and Wotton1986; Gregory, and Wotton, Reference Gregory and Wotton1989). Conversely, the absence of evoked potentials may not always guarantee unconsciousness (Anil et al., Reference Anil, Raj and McKinstry2000). Gregory and Wotton (Reference Gregory and Wotton1990) looked at the effects of multiple electrical stunning currents on spontaneous physical activity and evoked responses and found that the loss of somatosensory evoked potentials indicated a deeper level of unconsciousness than absence of neck tension. All these studies show that the use of different methods to assess unconsciousness may lead to different findings regarding the time to loss of consciousness. The use of absence of evoked responses or iso-electric EEG, may provide more conservative times to loss of consciousness compared with loss of spontaneous EEG. The indicators based on brain activity that can be used to asses unconsciousness after stunning are presented in Table 3.
Indicators based on brain activity as presented in an electroencephalogram (EEG) used to assess unconsciousness in livestock after stunning
Difficulties in the use of EEG
Though the EEG may be considered most objective when assessing unconsciousness, there are some disadvantages to its use. First, there is no golden standard for the way in which the division of stages of consciousness is described and this also limits the use of brain function monitors in differentiating between consciousness and unconsciousness, especially during transitional stages (Alkire et al., Reference Alkire, Hudetz and Tononi2008). Second, it is difficult to compare EEG values between species and individuals, because of animal variation caused by electrode placement, skull thickness and differences between equipment. Third, the EEG can be influenced by artefacts that are animal related (eye or muscle movements) or technical related (cable movements, impedance fluctuation or 50/60 Hz interference) (Teplan, Reference Teplan2002). Experimental controlled situations provide a significantly better environment to limit these artefact sources than slaughter plants. These artefacts, however, limit possibilities for EEG application as an evaluation method in slaughter plants at this stage.
Conclusion
This literature review shows that there is a wide range of indicators available to assess unconsciousness in livestock after stunning. In general, pathophysiology of the consequences of the stunning method should be taken into account when assessing unconsciousness, as applicability and reliability of the different indicators vary per stunning method. When physically stunning an animal, for example, captive bolt, most important indicators are posture, righting reflex, rhythmic breathing and the corneal or palpebral reflex that should all be absent when 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 are posture, righting reflex, natural blinking response, rhythmic breathing, vocalisations and focused eye movement that should all be absent when the animal is unconscious. Brain stem reflexes such as the cornea reflex are difficult as measures of unconsciousness in electrically stunned animals, as when present they may reflect residual brain stem activity and not necessarily consciousness. It is highly recommended to use multiple indicators to definitively assess and determine unconsciousness before starting invasive dressing procedures such as scalding or skinning. The EEG is generally considered to be a most reliable indicator for assessing unconsciousness, but is (the most) difficult to apply during slaughtering because of technical- and animal-related artefacts that can occur. Furthermore, the lack of a golden standard for determining (un)consciousness makes the evaluation of the EEG somewhat subjective. It is recommended to put further effort into resolving these difficulties so that the EEG can be more easily used in the assessment of unconsciousness after stunning. A substantial number of controlled studies have used the EEG to assess unconsciousness, but only one focussed on the correlation between an EEG derivative and a behavioural indicator. More research in this area should provide additional information on the absence of behavioural indicators in relation to the EEG and validate the use of certain behavioural indicators. Overall, better validated and applicable indicators are needed to reliably and reproducibly assess unconsciousness. These indicators could potentially also provide additional information on the onset of unconsciousness during the transitional period, as at present this is highly subjective, as it is often based on visual appraisal. Knowledge derived from studies using EEG in combination with other indicators in experimental set-ups could subsequently lead to improvements regarding stunning methods and subsequently animal welfare at the slaughter plant.
Acknowledgements
This study was funded by the Ministry of Economic Affairs, The Netherlands.
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