The main purpose of this study was to measure the short-and long-term effects of two lengths of tail resection on post-amputation tail stump sensitivity in pigs subjected to surgical tail amputation at two different ages (9 and 17 weeks). The rationale for the study was to investigate the likely effects of such injury in the context of the issue of tail mutilations in pig production, where a proportion of the tail can be fully bitten off, but using an experimental and ethically-approved approach to assess the effects of tail-amputation injury. The ages at which the tail resections were performed also links to the age at which the on-set of tail biting typically occurs in weaner and grower pigs26. However, the study also has more general scientific relevance for the fundamental understanding of pain response mechanisms evoked by damages to the peripheral pain pathways.

Data from the present study clearly show that tail amputation at both ages induced short-term changes in mechanical nociceptive thresholds (MNT) representative of an increased sensitivity to noxious mechanical stimulation that appeared to be sustained for a period of up to 16 weeks (4 months) and possibly beyond. MNT data were obtained using a handheld PAM device in conjunction with previously validated assessment and data analysis protocols27, 28, through which the consistency of individual levels of MNT, and the variability associated with different animal ages, and anatomical tail locations were determined.

This is the first study to report evidence of immediate and sustained mechanical hyperalgesia following tail amputation injury caused in later life. In addition, the findings of the present study are the first to show that the age or stage of development at which tail amputation injury occurs can affect threshold response profiles, suggesting that 9 week-old animals exposed to the injury experience mechanical sensitisation for a longer period of time than 17 week-old animals, regardless of the extent of tail removed. It is also evident from the results of this study that the amount of tail that is amputated can have a significant effect on the thresholds of tail sensitivity, notably the removal of 1/3 (long tail resection group) of the tail resulting in lower MNT compared with pigs with 2/3 of the tail amputated (short tail resection group), depending on the age of exposure to the surgery.

Similar to our previous observations on pigs with intact tails (97.2%)27, a high level of responsiveness to individual stimuli was recorded in this study (98.9%). In addition, the average individual variability of 28.9% observed in the present study corresponds to that reported in our previous experiment (30–32%)27, and confirms the efficacy of the habituation procedure on obtaining reliable MNT data. The presence of mechanical hyperalgesia one week after tail resection demonstrated in the present study is consistent with reports of measures of increased sensitisation reported in previous experiments specifically targeting tail amputations or more general tissue injuries. Increased sensitivity to mechanical stimulation following surgical incision of the tail or the plantar surface of the paw can be sustained for a period of up to seven days post-surgery in rodent inflammatory pain models29, 30. Similar findings have also been observed in pigs subjected to full skin and muscle incision performed in the lower back5. The impact of tail tip removal on mice for DNA profiling has also been previously described in two related studies. Zhuo22 and Kim and Zhuo23 characterised the development of mechanical and thermal hyperalgesia measured on the dorsal surface of the adult mouse tail, in proximity to the site of injury. Once again, tail sensitisation was sustained for a period of seven days following surgery. None of the previous investigations provided information on MNT profiles extending beyond 7 days post-injury.

It is well recognised that the amputation of limbs and appendages may cause altered sensitivity and possible long-term pain in the affected body part31. These effects can be observed in addition to the activation of nociceptive responses at the time of injury and the heightened pain sensitivity that occurs in response to tissue injury and inflammation (i.e. inflammatory pain). Neuropathic pain can arise from injury that occurs to primary sensory neurons responsible for transmitting pain signals, especially when these nerves are resected and subsequently form into neuromas32. In the present study, prolonged increases in tail sensitivity were observed up to 4 months after tail amputation in pigs undergoing tail resection at 9 weeks of age. Similarly, enduring sensitivity to noxious mechanical stimulation up to 2 months after injury was also seen in pigs subjected to tail resection at 17 weeks of age, suggesting in both treatment groups that sustained peripheral and spinal somatosensory changes linked to amputation injury are still evident sometime after post-injury tissue inflammation appears to have resolved.

The formation of traumatic neuromas in the stump of tail-docked piglets has been recently described20, 21. Both investigations confirmed the presence of traumatic neuromas and neuromatous tissue development in tail stumps collected from pigs of up to 22 weeks of age and suggested that neuroma formation is still incomplete four months after tail docking injury, with possible implications for sensitivity of the tail stump. MNT were not measured in these studies and therefore it was not possible to confirm if the presence or stage of neuroma development had an effect upon post-docking tail stump sensitivity, although it has been previously suggested that the presence of traumatic neuromas may lead to increased sensitivity to pain in the amputation stump19.

Amputation-induced injury to the peripheral nerve system, producing neuroanatomical changes such as the development of traumatic neuromas, can be associated with changes in somatosensory nerve function and with consequent alterations in nociceptive afferent fibre activity following nerve transection. These are important factors leading to the experience of abnormal sensations in humans, such as paraesthesia and dysesthesia, and in extreme cases lead to the phenomenon of stump or phantom limb pain8. Neuromas have been described as predominantly susceptible to mechanical pressure33,34,35. In addition to showing increased sensitivity to mechanical force, it has been reported in electrophysiological studies on rodents that peripheral nerve traumatic neuromas can exhibit ectopic or spontaneous activity that is linked to mechanical hyperalgesia mediated by damaged and non-damaged A-delta and C-fibre primary afferent neurons12. Evidence of sustained reductions in MNT reported in the current study suggests that tail amputation (irrespective of the amount removed) can cause prolonged increases in tail stump sensitivity reflecting mechanical hyperalgesia. This would indicate that tail amputation causes alterations in peripheral and spinal nociceptive processing that lead to central sensitisation36 that are still present 2–4 months later and, although the measured responses to mechanical pressure reflect a polysynaptic withdrawal reflex to noxious stimulation, it is reasonable to suggest that the pigs with amputated tails would perceive such mechanical stimulation as more painful (pain hypersensitivity) than their intact counterparts during this time. It is also plausible that the observed mechanical hyperalgesia in pigs subjected to tail amputation in later life may be functionally-linked to the presence of traumatic neuromas in the resected tail stumps, although no histological examination was carried out as part of this study to confirm it. Increased mechanical sensitivity associated with neuroma development has been reported in humans undergoing both traumatic and elective digital amputations37.

While the effects of tail amputation on the central nervous system have not been directly examined in this study, reports from human and rodent studies suggests that peripheral nerve injury induces hyperexcitability changes that extend from the periphery, spinal cord and brainstem to several cortical structures16. Long-term synaptic depression in the insular cortex has been previously characterised in mice as a consequence of tail amputation38, supporting the hypothesis of central sensitisation following a peripheral injury. In order to confirm whether peripheral mechanical sensitisation is a valid proxy measure for the experience of pain in pigs, more comprehensive experimental approaches would be required, coupling quantitative sensory testing with more complex behavioural non-reflexive assays (e.g. motivational tasks) that provide more information on the involvement of the central nervous system in the affective component of pain experienced by pigs following tail amputation.

Age at the time of tail amputation appears to have influenced the temporal changes in MNTs. Significantly lower values were recorded 16 weeks post-tail resection in pigs exposed to surgery at 9 weeks of age when compared to MNTs of intact tails. In pigs undergoing tail resection at 17 weeks of age, MNTs were significantly lower 8 but not 16 weeks post-surgery.

Previous studies on surgical neuropathic pain models in both young and aged rats have demonstrated the occurrence of tactile allodynia up to 35 days following surgery39, 40. In both studies the effect of age at the time of injury was evaluated and a lower magnitude of change in allodynia, with less robust (i.e. lower withdrawal response thresholds) neuropathic pain behaviours, was observed in older rats. Age-specific differences in the duration of sensitisation may reflect different stages of maturation of the peripheral and central components of the nervous system at the time of the insult. In humans and rodents, the level of maturity of the nervous system has been suggested as a determining factor in the responsiveness to tactile and noxious stimulation41. Younger subjects appear to exhibit more pronounced and prolonged behavioural and electrophysiological responses to noxious stimulation, which decreased in duration over time42, 43. It is possible that tail resection had a different impact on long-lasting changes in nociceptive neural pathways according to the neuroanatomical maturity of the animals at the time of surgery although, based on the small sample size of pigs (8 animals per treatment) tested at 16 weeks following surgery at 17 weeks of age, interpretation of the temporal pattern of sensitisation should be made with some degree of caution. Nine and seventeen week-old pigs are at distinct stages of growth (i.e. early juvenile and pubertal late juvenile) and have different degrees of brain and spinal cord development44,45,46, which may influence somatosensory and affective responses to amputation injury. In addition, it should be recognised that another factor that can determine the intensity and duration of post-amputation sensitivity is the different degree of hyper-innervation by A- and C-fibres around the injured area during the healing process41. Sprouting of sensory nerves after traumatic injury is a known phenomenon in humans and rodents and is particularly marked in young subjects (neonates) and linked to peripheral hypersensitivity and central sensitisation47. It is not known if such hyper-innervation of healed tissues after amputation injury is associated with sustained peripheral sensitivity in juvenile pigs.

Another explanation for the apparent lack of a sustained effect on MNT observed between intact and tail-resected pigs 4 month after surgery at 17 weeks of age may be an effect of age on the rate of peripheral axonal regeneration after amputation injury. Age-related changes in the time and extent of post-operative tissue re-innervation (density and number of regenerating axons) is slower and decreased in older animals48.

The amount of tail removed following amputation (1/3rd “long” vs. 2/3rds “short”) had different effects on MNTs depending on the age at which tail amputation was performed and the sampling time after tail resection. No tail length treatment effect was observed one week following tail amputation in either age group. Similarly, short and long tails exhibited similar MNTs in later testing of pigs undergoing surgery at 9 weeks of age. In contrast, a later tail length effect was observed in pigs exposed to surgery at 17 weeks of age, with significantly lower MNTs in the long tail group 2 months after tail amputation compared to the short tail pigs, although it was not possible to evaluate the effect of tail amputation length 4 months after surgery owing to a mechanical breakdown of the PAM device which prevented the assessment and collection of data from the long tail resected pigs at this time point. The finding that pigs exposed to a greater extent of tail amputation appeared less sensitive to mechanical stimuli may contradict previous human literature. It is recognised that the greater the level of amputation injury in limbs and appendages in humans the greater the potential for the presence and severity of pain over a larger affected area due to proportionally greater damage occurring to somatosensory peripheral nerves49. Nonetheless, the emergence of phantom pain appears independent of the level of amputation50. The observed reduction in nociceptive thresholds measured in the current study may be explained by gradients in somatosensory innervation in different regions of the tail27. Proximal-distal variations in intra-epidermal nerve fibre innervation densities have been reported in human studies with greater densities recorded in the most distal parts of the leg51. Confirmation of a similar pattern of innervation density has yet to be corroborated in the pig tail. In addition to possible regional changes in tail somatosensory nerve distribution, it is possible that reductions in MNT in amputated tail stumps of differing lengths may be affected by the relative thickness of the soft-tissues at the site of mechanical stimulation. In humans, distinct values of nociceptive thresholds are linked to the varying thickness of cutaneous, adipose and muscle tissues52, 53. Lower MNT observed in the long tails may therefore reflect greater and more rapid focal noxious mechanical force generation linked to the reduced thickness of the overlying soft-tissues in the most distal part of the amputated tail. The converse may be true where the tissues at the site of stimulation are thicker, which may dissipate some of the mechanical force before response thresholds are achieved.

In conclusion, tail amputation injury in pigs appears to evoke acute as well as sustained changes in peripheral mechanical sensitivity with hyperalgesia observed one week and up to several weeks following the surgical procedure. The age at which the animals are exposed to the injury seemingly influences the temporal profile of sensitisation, with younger pigs being affected for a longer period of time. Further studies on age and body size-specific morphological characteristics are needed to fully comprehend the relationship with post-injury pain sensitivity in pigs. In addition, a smaller extent of tail amputated appears to be associated with higher sensitisation; however the partial loss of data collected in older animals warrants particular caution in interpreting our findings. In the near future, it will be of pivotal importance to correlate these mechanical threshold measures of pain sensitivity with changes in molecular mediators of pain signalling to provide novel information on the mechanisms and pathways involved in the development of hyperalgesia in pigs following tail amputation.