Refining a ‘toolkit’ for objective assessment of pain and stress in ruminants

Abstract

Pain is a complex phenomenon, and to address pain management in animals, the presence of pain needs to be determined. Most studies assess the presence of pain based on the appraisal of either a single biomarker or a combination of a few parameters, which were often not examined simultaneously. In ruminants, measurements of cortisol and haptoglobin (Hp), and the assessment of behavioural changes have been the standard practice for this purpose. Nevertheless, there are still limitations in using these parameters, as they are also indicators of psychological stress, and can be misinterpreted as to indicate the presence of pain. Assessment of behavioural changes in ruminants can be difficult as prey animals conceal their experience of pain to avoid predators’ attention. Therefore it is recommended that a reliable assessment tool be devised that includes a combination of various parameters to determine the presence of pain in ruminants in order to address pain management effectively. The primary aims of this study were to evaluate the conventional and contemporary biomarkers to determine the response of an animal to a noxious experience, and to refine a multi-parameter ‘toolkit’ that can be applied in assessing pain in ruminants. For these purposes, castration was used as a model of a common painful husbandry procedure that presumably evokes pain associated with the tissue injury. Merino cross lambs were assigned to two groups that were either surgically castrated or subjected to restraint stress (control). Castration was an open surgical technique without the provision of anaesthesia or analgesia. The conventional biomarkers estimated were plasma cortisol, Hp and beta-endorphin (β-EP). The contemporary biomarkers measured were interleukin-6 (IL-6), substance P (SP), and prostaglandin E2 (PGE2). The peripheral leukocytes expression of IL-6 and proopiomelanocortin (POMC), the precursor of β-EP were also estimated. Behavioural changes were recorded to assess pain-related behaviour caused by the surgical procedure. Following treatment, the cortisol concentration in the castrated lambs was higher than control animals from ten minutes to two hours post-castration, indicative of the acute noxious experience associated with the surgical procedure. Surgical castration also caused a response in SP indicative of nociception and neurogenic inflammation, which was increased from 30 minutes and became significant eight hours following castration. The behavioural assessment showed that the castrated lambs demonstrated statue standing, a pain-like behaviour, from three to five hours post-castration, which were associated with the increase in SP, indicative of the pain sensitisation caused by the tissue injury. Increase in the systemic inflammatory mediators, the Hp and IL-6 were observed from day two, in which Hp remained significant until day four and resolved by day five. The IL-6 continued to increase over time, suggesting of an ongoing inflammatory response and inflammatory pain possibly until wound healing had occurred. No significant changes of β-EP and PGE2 were found, suggesting a local response. The quantification of IL-6 and POMC gene expressions by the circulating leukocytes showed a depletion of response compared with the plasma levels, which indicated that the cells might have migrated to the injury area. This pattern of changes supports the assumption of a local tissue response and the lack of detectable plasma IL-6 in the initial phase of a post-tissue insult. The response of plasma IL-6 and (interleukin-1) IL-1 were also measured in samples of a study in cattle that were castrated surgically and using tension banding in two age groups (weaner and mature bulls). Interleukin-1 was below detection level and IL-6 did not change in the surgically castrated bulls. Nonetheless, the IL-6 increased significantly in the bulls that were castrated using banding from as early as three days following castration, more evidently in the mature bulls indicative of a chronic inflammatory pain. Some variability was detected, which was also observed in the surgically castrated lambs suggesting that breed background could influence the variation in the immune response in both species. In conclusion, the studies conducted in this thesis were aimed at understanding the underlying relationships of the various biomarkers in the assessment of pain in ruminants. It was found that these results support the hypothesis that more than one parameter is required to assess pain in ruminants due to tissue injury caused by a common husbandry procedure. Substance P and IL-6 are recommended as potential good markers in the assessment of nociception and inflammatory pain. The trends and magnitude of response of these markers until point of tissue healing will be more informative of the pain induced and the implications of the procedure on the animal. A local insult such as castration may have evoked a local response in β-EP, PGE2, and IL-1 that could not be detected in plasma. Cortisol and Hp should still be measured together with these parameters until assessment of pain can be refined further on other forms of pain models in ruminants.