Hypothalamic-Pituitary-Adrenal-responses

e (reduce and refine) future analgesic efficacy trials in this field. 2. Physiological measurements of pain in piglets Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1 Physiological responses occur in response to pain and stress, including activation of the hypothalamus-pituitary-adrenal axis (HPA-axis) and sympathetic nervous system (SNS), and release of opiate neuropeptides. This acts to increase the metabolic rate in preparation for “flight or fight” as well as mediate the inflammatory response and mitigate pain. Adrenalcorticotrophic hormone (ACTH) is released by the pituitary and acts on the adrenal gland. Cortisol and adrenalin are released and, in turn, result in an increase in the level of glucose and lactate in the blood. Activation of the SNS may result in an increased heart rate and blood pressure and reduced skin temperature as blood is diverted to muscles and vital organs. β-endorphins (endogenous opioid-neuropeptides) are released from the anterior pituitary and act on opiate receptors in the peripheral and central nervous system to induce analgesia principally through effects on mu-opioid receptors. Indicators of the HPA axis and SNS activation, or β-endorphin release are thus often used as indirect measures of pain. These physiological responses however, are not specific to pain. They may be triggered by stress alone, and / or by tissue trauma (such as induced by surgical incision), even in the absence of pain. Surgical studies reveal that animals under a general anaesthetic increased cortisol and ACTH production, irrespective of the animal’s sensation of pain [43,44]. Haemorrhage alone is known to result in an increase in ACTH, cortisol, β-endorphin concentration, as well as tissue

e (reduce and refine) future analgesic efficacy trials in this field. 2. Physiological measurements of pain in piglets Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1 Physiological responses occur in response to pain and stress, including activation of the hypothalamus-pituitary-adrenal axis (HPA-axis) and sympathetic nervous system (SNS), and release of opiate neuropeptides. This acts to increase the metabolic rate in preparation for “flight or fight” as well as mediate the inflammatory response and mitigate pain. Adrenalcorticotrophic hormone (ACTH) is released by the pituitary and acts on the adrenal gland. Cortisol and adrenalin are released and, in turn, result in an increase in the level of glucose and lactate in the blood. Activation of the SNS may result in an increased heart rate and blood pressure and reduced skin temperature as blood is diverted to muscles and vital organs. β-endorphins (endogenous opioid-neuropeptides) are released from the anterior pituitary and act on opiate receptors in the peripheral and central nervous system to induce analgesia principally through effects on mu-opioid receptors. Indicators of the HPA axis and SNS activation, or β-endorphin release are thus often used as indirect measures of pain. These physiological responses however, are not specific to pain. They may be triggered by stress alone, and / or by tissue trauma (such as induced by surgical incision), even in the absence of pain. Surgical studies reveal that animals under a general anaesthetic increased cortisol and ACTH production, irrespective of the animal’s sensation of pain [43,44]. Haemorrhage alone is known to result in an increase in ACTH, cortisol, β-endorphin concentration, as well as tissue

fficient validation. In the current review, we summarise literature on the currently available methods for assessing peri-operative pain in surgically castrated neonatal piglets and provide a critical analysis of the outcome variables identified to ascertain those that most closely meet these criteria. It is anticipated that this critical analysis may assist the future development of more standardized methods and optimise (reduce and refine) future analgesic efficacy trials in this field. 2. Physiological Measurements of Pain in Piglets Physiological responses occur in response to pain and stress, including activation of the hypothalamus-pituitary-adrenal axis (HPA-axis) and sympathetic nervous system (SNS), and release of opiate neuropeptides. This acts to increase the metabolic rate in preparation for flight or fight as well as mediate the inflammatory response and mitigate pain. Adrenalcorticotrophic hormone (ACTH) is released by the pituitary and acts on the adrenal gland. Cortisol and adrenalin are released and, in turn, result in an increase in the level of glucose and lactate in the blood. Activation of the SNS may result in an increased heart rate and blood pressure and reduced skin temperature as blood is diverted to muscles and vital organs. β-endorphins (endogenous opioid-neuropeptides) are released from the anterior pituitary and act on opiate receptors in the peripheral and central nervous system to induce analgesia principally through effects on mu-opioid receptors. Indicators of the HPA axis and SNS activation, or β-endorphin release are thus often used as indirect measures of pain. These physiological responses; however, are not specific to pain. They may be triggered by stress alone, and/or by tissue trauma (such as induced by surgical incision),

…acute physiological responses (increased the method employed to prevent taint in meat from male Hypothalamic-Pituitary-Adrenal-responses and protein c- pigs, as well as to obtain more docile pigs. However, since…

…acute physiological responses (increased the method employed to prevent taint in meat from male Hypothalamic-Pituitary-Adrenal-responses and protein c- pigs, as well as to obtain more docile pigs. However, since…

response than 2.5 or stress response compared to handling on the lap, however, we 5 min intervals. It is possible that a longer interval between stays would expect that the placement of piglets in a calm, heated area in the bench resulted in a different shape of the curve of cortisol with littermates between injection and castration contributed response, the peak of which is not known. We also hypothesize to limiting the stress response compared to the typical on- that the lack of sensitivity of cortisol may have resulted in farm practice of placement in a cartwheel in the farrowing ceiling effects following handling alone, as already suggested by room. Similarly, application of the procedures in a calm previous authors (4, 11), especially in neonatal piglets, whose environment may limit the emotional social contagion of stress HPA-axis may be highly responsive (33). This suggestion is responses (among littermates, and with the sow) potentially supported, in our study, by comparable cortisol levels recorded happening when several litters are processed simultaneously in sham handled and castrated piglets. As a consequence of these in the farrowing room (39). Yet, further studies are required known concerns, the present study examined acute responses to to investigate these suggestions. Throughout the study, the injection of anesthetic and castration across several indicators same trained herdsman, hired for this specific role, performed including cortisol, vocalizations and resistance movements, the procedures on all experimental piglets. This was chosen thereby taking a multi-modal approach as suggested by Sheil and to strengthen accuracy, lower variation and thus increase the Polkinghorne, and Baysinger et al. (36, 37). power to detect differences when comparing

response than 2.5 or stress response compared to handling on the lap, however, we 5 min intervals. It is possible that a longer interval between stays would expect that the placement of piglets in a calm, heated area in the bench resulted in a different shape of the curve of cortisol with littermates between injection and castration contributed response, the peak of which is not known. We also hypothesize to limiting the stress response compared to the typical on- that the lack of sensitivity of cortisol may have resulted in farm practice of placement in a cartwheel in the farrowing ceiling effects following handling alone, as already suggested by room. Similarly, application of the procedures in a calm previous authors (4, 11), especially in neonatal piglets, whose environment may limit the emotional social contagion of stress HPA-axis may be highly responsive (33). This suggestion is responses (among littermates, and with the sow) potentially supported, in our study, by comparable cortisol levels recorded happening when several litters are processed simultaneously in sham handled and castrated piglets. As a consequence of these in the farrowing room (39). Yet, further studies are required known concerns, the present study examined acute responses to to investigate these suggestions. Throughout the study, the injection of anesthetic and castration across several indicators same trained herdsman, hired for this specific role, performed including cortisol, vocalizations and resistance movements, the procedures on all experimental piglets. This was chosen thereby taking a multi-modal approach as suggested by Sheil and to strengthen accuracy, lower variation and thus increase the Polkinghorne, and Baysinger et al. (36, 37). power to detect differences when comparing

ntinuous excised area from the tip of the tail to the caudal surface of the hind leg. The mulesing operator was blinded to the experimental treatments of lambs that were mulesed, The lambs in the sham mules treatment were placed in the lamb marking cradles for approximately 20 to 30 seconds, which was the typical time taken to complete the mulesing and tail docking procedure, but were not tail docked or mulesed and then were released back with their dams. Cortisol determination The plasma cortisol concentrations were determined using a commercial radioimmunoassay (Spectria Cortisol RIA, Orion Diagnostica, Espoo, Finland), adapted and validated for ovine plasma. Briefly, human serum standards were used as provided by the supplier or diluted in phosphate-buffered saline (PBS) and 20 pl of standard, control or unknown sample were pipetted into tubes coated with antibody to cortisol. Five hundred microlitres of the '"I-labelled cortisol was then added and tubes incubated for 2 h at 37°C. Tubes were decanted, washed once with 1 ml of distilled water and counted for 1 min in a gamma counter. Parallelism between the standard and unknown samples was demonstrated by serial dilutions of two ovine plasma samples: the calculated slopes of the binding vs log cortisol concentration or dilution curves for the cortisol standards and the two samples were —0.172, -0.165 and ~0.131, respectively. The calculated recovery of cortisol added to spiked ovine plasma samples was 102% and the sensitivity of the assay was 10 nmol/L. The stated Figure 1. Diagram of the areas of skin removed in the mulesing cross-reactivities of the anti-cortisol antibody with corticoster- operation. Hatched areas represent portions of skin removed. one, cortisone, dexamethasone, prednisolone and prednisone were

on occurring 40 min post procedural intervention. There was a clear effect of handling and procedural pain on cortisol response, with no difference in plasma cortisol concentrations (PCC) between the two cohorts of surgically dehorned calves. We identified statistical trends for lower PCC and AUC in DTA compared to D calves. In addition, the PCC of dehorned calves exceeded that of sham dehorned calves. This suggests that timing of anaesthetic delivery may partly account for the observed cortisol response. Blockage of local nociception after the administration of a noxious stimuli is unlikely to greatly influence the HPA response, particularly as the elevated cortisol response presumably reflects both the stress of prolonged restraint plus haemorrhage from the wound site [36–38]. Animals 2020, 10, 312 8 of 11 There was no significant difference in AUC0–40 min between sham dehorned calves and dehorned calves regardless of treatment with TA, indicating the stress of handling and restraint on cortisol response [36]. The stress of sham dehorning has been well documented and causes a small yet significant rise in cortisol concentrations from base level [9,10,13,25,39,40]. This is due to the activation of the HPA system by a number of external stressors including restraint, handling and the presence of humans [36,41]. In the current study, there was no significant difference in peak cortisol concentration between sham dehorned and dehorned calves, which contrasts previous dehorning studies [9,10,13,25]. This difference may be attributed to the type and duration of restraint used in this study. In other experiments, calves were manually restrained for a relatively short period of time in a standing position (<15–45 s) [9,10,13,25,39,40], whereas in the present study,