«October 2009 SCIENTIFIC COORDINATOR Pierre Le Neindre, Senior research scientist, INRA (French National Institute for Agricultural Research) ...»
Other biological markers reflecting the activation of structures of the nervous system involved either in the detection and perception of pain or in the control of pain can be added to those described above. They include the expression of early gene activation such as for the c-fos gene in the dorsal horn of the spinal cord after castration in pigs. In response to pain the body releases endogenous opioids such as endorphins and enkephalins. Hence these can also be used as indicators of pain, as they have been for horses. Lastly, noxious stimuli modify the electrical activity of the brain. Graphical recordings (EEG - Electroencephalograms) obtained via electrodes placed on the skull can be used to analyse the changes in electrical potential that take place at the level of the cortex. The brain electrical activity is classified into four categories of wave frequencies: delta (4 Hz), theta (4-7 Hz), alpha (8-13 Hz) and beta ( 13 Hz). In adult humans, alpha and beta waves are characteristic of a state of wakefulness and delta waves are characteristic of sleep. Beta waves become more abundant under anaesthesia induced by pharmacological agents.
In the same way that the approach based on tissue damage can reveal the origins of pain, detection of proteins during the acute inflammatory stage is an indirect indicator of pain since it reveals tissue inflammation and it is known that inflammation usually causes pain. The measurement of serum concentrations of certain proteins (haptoglobin, fibrinogen, ceruloplasmin, amyloid A serum) may be very useful for detecting subclinical inflammation.
In birds The main physiological variables used as indirect criteria for the assessment of pain are cardiovascular changes, plasma corticosterone concentrations and EEG activity.
Expertise scientifique collective "Douleurs animales" 45 In most cases, exposure to an acute nociceptive stimulus triggers heart rate acceleration in birds. However, no work on heart rate variability during a painful episode has ever been published for poultry. Blood pressure increases after activation of the sympathetic system. This parameter, however, has very rarely been studied in birds because of technical difficulties in recording it without restraining the animals. Changes in the EEG does not seem relevant at the moment because the data available so far have shown similar changes in birds that were subjected to either a fearful situation (tonic immobility during a frightening situation) or to a noxious stimulus (pulling out feathers).
In fish Most studies published so far deal with physiological responses of farmed fish exposed to stressful situations (endocrinological variables such as corticosteroids, or indirect criteria such as respiratory or cardio-vascular changes) and none of them addresses specifically the consequences of a noxious stimulus. It is known that applying a noxious stimulus alters the movement frequency of the gill plate which is an indirect indicator of gill ventilation and therefore of increased breathing rate. Further studies, including the response of the HPA axis to a noxious stimulus, would be very useful.
At slaughter Most studies on the effectiveness of stunning and/or bleeding rely on measures reflecting the level of consciousness or the brain's ability to perceive stimuli from the environment. EEG analyses are also used to measure the brain activity, to characterize its responses to sensory stimuli, or understand the way the brain maintains reflex responses or vital functions in animals.
EEG analysis focuses on the type and intensity of the rhythmic electrical activity of the brain. The presence of delta waves, characteristic of sleep patterns in humans, suggests a reduced level of consciousness. A flat or nearly flat EEG indicates a state of deep anaesthesia, and ultimately brain death. Evoked potentials (EP) correspond to transient changes in the EEG when the animal is subjected to auditory, visual or somato-sensory stimuli. To identify the level of consciousness of an animal at slaughter, some authors refer to the presence of delta waves, others to a significant and sustained reduced brain activity, or to the impairment of EP. Some combine several criteria. In some cases, the different criteria may lead to divergent conclusions. This is partly due to the circumstances in slaughterhouses that make measurement very challenging: EEG recordings may show artefacts because of the difficulty in maintaining the electrodes in place and/or the existence of electrical interference. Furthermore, it should be kept in mind that although impairment of EP clearly indicates a loss in the brain's ability to integrate sensory information, the presence of EP only means that the integrity of the sensory pathways involved has been preserved but not necessarily the perception of stimuli and awareness.
The effectiveness of stunning can be assessed by other methods based on the measurement of blood pressure, the observation of postures (animal collapsing or not) and various reflexes (palpebral-ocular or respiratory reflexes, physical reactions to noxious stimulation, and righting reflex of the head or of the body). However, vestibulo-ocular and respiratory reflexes depend on the activity of the brainstem that may persist despite a state of unconsciousness. Therefore, a lack of reflexes indicates that the activity of the brain stem is profoundly disturbed and that the animal is unconscious while their presence does not necessarily mean that the animal is conscious.
Conclusion Examining physiological criteria renders the identification of pain possible in many animals. The means are often invasive and are generally based on complex methodology. The results may be difficult to interpret since stressful situations void of any nociceptive component often lead to similar physiological changes. Hence, the experimental conditions required to ensure accurate identification of the presence of pain using physiological criteria render this tool impractical for use in situ on farms or in slaughterhouses. Physiological criteria remain nevertheless very useful, especially in mammals, for the identification of sources of pain and in the development and validation of protocols for pain management and of objective scoring systems for pain assessment.
46 Expertise scientifique collective "Douleurs animales"3.3. Behavioural responses
Behaviour in animals and in humans denied of verbal or written communication may constitute valuable criteria for identifying and locating pain (Table 2). However, as is the case for other criteria, using behaviour as a tool has its limitations. The first one is the variability in behavioural expressions both between animal species and within each species, according to the context. Another limiting factor is that the interpretation of behaviours by the observers, whether breeders or veterinary surgeons, differs according to their knowledge of the behaviour of the species, of the individuals and their personal perception of pain. Some behaviours such as apathy, self isolation or anorexia may also be seen in situations of stress or discomfort without nociceptive components. In addition, the behavioural response to a noxious stimulation may vary over time, or be expressed differently by individuals of the same species or breed. Behavioural responses may be modulated by endogenous analgesic mechanisms that are triggered in response to pain. They may also be influenced by specific physiological states such as pregnancy and parturition in mammals or egg laying in birds. One must therefore remain cautious in the interpretation of behaviour even when it may be very evocative of pain.
Despite the limits mentioned above, observation of behavioural responses (vocalizations, activities and postures, facial expressions) is one of the methods most frequently used by scientists and veterinary practitioners to characterize animal pain. This method has significant advantages because it is generally non-invasive and is fairly sensitive. Methodological precautions must be taken in order to avoid problems of interpretation and to validate the criteria used. Firstly an ethogram should be established to characterize the behavioural repertoire of the species and to define the conditions of expression of each relevant activity, its function, and the ontogenetic and phylogenetic changes.
Several behaviours can be distinguished: automatic behaviours to escape from the noxious stimulus (reflex withdrawal of a limb); behaviours to avoid the stimulation of the painful area (resting, analgesic postures such as limping); behaviours intended to signal the existence of pain to conspecifics and to encourage them to either avoid stimulating the painful area, or to lick, rub or scratch the area to relieve pain (this behaviour probably masks nociception through other sensory signals); behaviours that facilitate learning and thereby help the animal avoid subsequent noxious stimulation. It is important to conduct observations on animals subjected to a painful procedure, in association with an anaesthetic or analgesic treatment for some and without for others, in order to ensure that the criteria used do indeed reflect pain, and not just a stress response resulting from interactions with the operator.
In cattle and sheep, pain induced by numerous husbandry procedures has been analyzed using this method. Most of the behavioural criteria chosen as indices have been validated by cross-comparison between responses and by comparing the responses of animals that were subjected to an intervention to those that did not, either in addition to receiving or not receiving an analgesic treatment for each group. This method has been partly validated for pigs and for birds for some painful situations, but hardly at all for fish exposed to nociceptive stimuli.
In ruminants and pigs Behaviours to be taken into account are well described for a number of painful procedures (tail docking, castration and dehorning) according to the stages of the procedure and the techniques used (see Chapter 4 for details of these procedures). Some studies have compared different behavioural criteria for sensitivity (ability to identify a painful situation) and reproducibility of measurements. Comparisons with physiological criteria have also been conducted to estimate their sensitivity.
Vocalizations are frequently used as indicators of pain in mammals and several types of analyses can be undertaken. The number of vocalizations can simply be counted or the duration or intensity can be measured. The vocalizations can also be analysed by spectral analysis. Experiments during castration in pigs have shown an increase in the number and intensity of vocalizations, as well as changes in their spectral characteristics. All of these changes may be reduced or eliminated by local anaesthesia.
Reflex withdrawal responses are frequently observed in animals subjected to a noxious stimulation. These Expertise scientifique collective "Douleurs animales" 47 behaviours are used to measure the response to a controlled noxious stimulus in cattle or sheep. The measures include, for example, the latency to leg withdrawal or kicking when a limited area of the leg, shoulder or rump is subjected to a painful stimulus. Laser beams heating specific parts of the leg have thus been used to characterize the reaction of cattle to pain in certain contexts.
Defensive behaviours during painful procedures are also very common. Movements of legs and of the body during castration or teeth clipping in young piglets, jumping or kicking during hot-iron or liquid nitrogen branding in cattle are typical examples. During branding, the animals also push much more strongly onto the sides of their restraining cage than in simulated situations. Defensive behaviours (kicking for example) can also be observed when touching a painful area.
Certain other behaviours that are directly linked to a painful area are relatively easy to interpret. This is the case, for example, for licking, rubbing or scratching which may reduce the intensity of the nociceptive signals (see above). Avoidance behaviours and analgesic postures are observed in complement to behaviours stimulating the painful area. One typical example is limping. Lesions of the leg often lead to a reluctance or inability to bear weight on one or more limbs and to a high score for lameness in cattle. Objective scoring systems are available to quantify the degree of lameness (Table 3). That the scores are directly linked to the level of pain is evidenced by the fact that they are lowered when the animal receives analgesic treatment. Instead of conducting a visual observation of the animal, it is possible to determine the degree of lameness by measuring the weight the animal puts on each leg by using sensors, as it has been demonstrated in cattle for example.
General behavioural disturbance such as reduced food intake, reduced mobility, a high level of agitation or, conversely, prostration, as well as changes in behaviour towards humans are often described after a painful procedure or during chronic pain, such as that associated with lameness.
Uneven gait and short strides may be difficult to identify a In birds As for mammals, flight or withdrawal reactions are observed when a painful area is stimulated. Defensive behaviours during painful procedures are also very common.
Vocalizations may be used to reveal the existence of pain in birds as in mammals. The calls emitted when an individual is being pecked by other birds are however described as being only moderately loud, softer than the distress calls emitted during capture. However, methods as elaborate as those used to characterize pig vocalizations during castration are not available for use with birds.
Thomsen P.T., Munksgaard L., Togersen F.A. (2008). Evaluation of a lameness scoring system for dairy cows. Journal of Dairy Science 3 91(1): 119-126.