b8eb816dc1bebdb27362c3533216459d5e3d

chunk 0 · 447 tokens

Review O ptimal methods of documenting analgesic efficacy in neonatal piglets undergoing castration Meredith Sheil 1 * , Adam Polkinghorne 2 ,3 1 Animal Ethics Pty. Ltd., Yarra Glen, 3775, Victoria, Australia; mlksheil@me.com 2 Department of Microbiology and Infectious Diseases, NSW Health Pathology, Nepean Hospital, Penrith, New South Wales, Australia; adam.polkingho rne@health.nsw.gov.au 3 The University of Sydney Medical School, Nepean Clinical School, Faculty of Medicine and Health, University of Sydney, Penrith, 2750, New South Wales, Australia * Correspondence: mlksheil@me.com.au Simple Summary: Surgical castration in piglets is widely used in commercial pig production systems, however, may cause pain and stress to the animal. There is an urgent need to develop effective pain - relieving medications to use for this procedure. Such products must meet high standards of proof confirming that they are effective. This requires undertaking trials to determine the duration and severity of pain that piglets experience during and after castration, and the extent of pain reduction in anaesthetic/analgesic trea ted piglets. Unfortunately, responses to pain may be transient, subtle or variably expressed. Furthermore, there is no simple “gold standard” method to measure “pain” in neonatal piglets . Instead, researchers must rely on using a range of indirect measures of pain of varying reliability . Without understanding the nature of expression of piglet pain, and the reliability of test measures to detect it, there is the potential of misinterpreting trial outcomes. Although there is a high degree of variability in t he literature of test methods employed and outcomes obtained, there is nevertheless a growing body of evidence to suggest that some piglet responses to

chunk 1 · 447 tokens

e literature of test methods employed and outcomes obtained, there is nevertheless a growing body of evidence to suggest that some piglet responses to pain induced by castration, are more consistently reproduced and specific to the pain experienced during castration than others . In this narrative review, we examine the potential indicators of pain in neonatal piglets undergoing castration to determine the optimal methods , currently available to most accurately detect pain, and assess pain mitigation . Abs tract: Analgesic products for piglet castration are critically needed. This requires extensive animal experimentation such as to meet regulatory - require d proof of efficacy. At present, there are no validated methods of assessing pain in neonatal piglets. T h is poses challenges for investigators to optimize trial design and to meet ethical obligations to minimize the number of animals needed. P ain in neonatal piglets may be subtle , transient and / or variably expressed and, in the absence of validated methods , i nvestigators must rely on using a range of biochemical, physiological and behavioural variables, many of which appear to have very low (or unknown) sensitivity or specificity for documenting pain, or pain - relieving effects. A previous systematic review of this subject was hampered by the high degree of variability in the literature base both in terms of methods used to assess pain and pain mitigation, as well as in outcomes reported. In this setting we provide a narrative review, to assist in determining the optimal methods currently available to detect piglet pain during castration and methods to mitigate castration - induced pain. In overview , the opti mal outcome variables identified are nociceptive motor and vocal response scores during

chunk 2 · 150 tokens

to mitigate castration - induced pain. In overview , the opti mal outcome variables identified are nociceptive motor and vocal response scores during castration , and quantitative sensory - threshold response testing and pain - associated behaviour scores following castration . Keywords: P ig let ; castration; pain; behaviou r; p eri - operative; vocal isation ; nocicepti on ; neonate; anaesthesia; analgesia. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license.

chunk 3 · 448 tokens

1. Introduction A variety of animal husbandry procedures that cause pain to the animal are routinely employed in livestock species as a part of effective animal management syst ems. A primary example of such a procedure is castration, a technique that involves the removal of the testicles or the removal of testicular function [1] . In pigs, castration is employed in commercial swine facilities for several purposes, including improv ing meat flavour, preventing unwanted breeding and modifying animal behaviour. It is generally performed in the first week of life, in male piglets intended to be kept past sexual maturity. Meat quality is improved by reducing the potential for ‘boar taint ’, an unpleasant odour and flavour associated with the presence of androstenone (5 ɑ - androst - 16 - ene - 3 - one), produced in the testes of intact male pigs following sexual maturity [2] . Castration also reduces the risk of unwanted breeding that can interfere with the maintenance of genetic lines [3] , and assists with management of boars by reducing the presence of aggressive behaviours that pose a welfare risk to other animals but also t o the safety of humans interacting with them [3] . The castration procedure itself is rapid, taking under a minute, and is most commonly performed by farmers, in piglets between 2 - 7 days of age. The procedure involves restraining the piglet, incising the skin of the scrotum, extracting the testes and severing the spermatic cords. Antiseptic is commonly sprayed onto the wound, and, less commonly, antibiotics are administered with the piglet finally returned to its sow. The wound is left to heal by secondary intention [1,4 - 6] . Surgical procedures induce pain via a number of mechanisms. The acute phase is primarily neurally mediated . Tissue

chunk 4 · 448 tokens

by secondary intention [1,4 - 6] . Surgical procedures induce pain via a number of mechanisms. The acute phase is primarily neurally mediated . Tissue incision causes trauma to nerve fibres at the incision site, resulting in a barrage of nociceptive neural transmission from the damaged tissue to the central nervous system (nociception) inducing spinal reflexes such as the nociceptive withdrawal reflex, and, on reaching the cerebrum, the perc eption of acute pain and induction of the neuroendocrine response. A second, “sub - acute” or prolonged inflammatory phase arises , primarily due to local release of various mediators in response to tissue damage , that promote ongoing pain or pain hypersensit ivity against thermal , mechanical, and chemical stimuli . P ro - nociceptive mediators such as; ATP, glutamate, kinins, cytokines , tropic factors, and prostaglandins, activate primary afferent neurons directly or indirectly to enhance nociceptive signal transm ission to the central nervous system . Prostaglandins derived from the arachidonic acid cascade are implicated in the production of inflammatory pain, and in sensiti z ing nociceptors to the actions of other mediators . Bleeding and coagulation due to tissue i njury are closely associated with the initiation of inflammation resulting in reflex erythema and acute pain responses. Kallikrein released during coagulation produces bradykinin, a strong al l ogenic factor. Degranulation of activated mast cells results in the release of proteases, cytokines, serotonin and histamine into the extracellular space. These substances sensitize primary afferent neurons to produce hyperalgesia. Sensitization of peripheral and central neuronal structures amplifies and sustains posto perative pain [7] . Consistent with this, piglet

chunk 5 · 418 tokens

yperalgesia. Sensitization of peripheral and central neuronal structures amplifies and sustains posto perative pain [7] . Consistent with this, piglet castration is reported to cause pain and stress to the animal involving (i) discomfort and stress prior to the procedure due to handling and restraint; (ii) acute pain and stress during the procedure itself a ssociated with incision of the scrotum, separation of the tissue to release each testicle, followed by severing of the spermatic cord; and (iii) post - operative pain and / or discomfort in the hours and days following the procedure [1,6] . Despite this, castr ation is typically performed without any pain relief, including in North America [8] and the EU [5] . In a detailed survey of 26 European countries, undertaken as part of the PIGCAS project in the European Union [5] , it was estimated that 79.3% of the about 98 million male pigs were castrated and analgesic use was reported as “very rare” or “never” in all EU member countries surveyed, excluding Norway and Latvia. One of the major obstacles has been the absence of practical and effective pain relief products lic ensed for farmers to use in piglets. Over the past decade, welfare concerns and ethical objectives have led to a drive to develop effective pain relief strategies for piglet castration, along with strategies to support the phasing out of the procedure wher e possible. In 2010, for example, the ‘European Declaration on alternatives to surgical castration of pigs’ was agreed, stipulating the intention that from January 1, Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 6 · 447 tokens

2012, surgical castration of pigs should only be performed with prolonged analgesia and/o r anaesthesia. From 2018, surgical castration of pigs should be phased out altogether. Whilst there has been progress towards these objectives, investigators in these fields face a number of challenges. There is a growing body of literature exploring the use of pain - mitigation strategies for piglet castration including; general anaesthesia, local anaesthesia, and general analgesia, such as with Non Steroidal Anti - Inflammatory (NSAID) medication [6] . While many of these methods are known to address pain in other surgical settings, the challenge faced by stakeholders involved in studies in neonatal piglets is to identify options that are effective in mitigating pain but are also safe, practical and economically sustainable for use in commercial swine facilit ies. Our group has been investigating the use of a topical anaesthetic formulation , administered via intra - operative wound instillation, as a method to mitigate subsequent acute peri - operative pain in piglets. This has required reviewing methods of assessi ng analgesic efficacy in neonatal piglets to identify those most valid, sensitive and specific for the assessment of surgical and post - surgical pain. Proof of anaesthetic / analgesic efficacy is challenging in neonatal piglets. There is no one “gold stand ard” or validated measure of “pain” in piglets. S igns of pain in neonatal piglets can be subtle and variably expressed, and readily confounded by extraneous variables, particularly when required to be examined in “the field” setting (as opposed to in a lab oratory) as is a standard requirement for regulatory approvals . Nevertheless, it is generally accepted that piglets react to stimuli in a number of

chunk 7 · 448 tokens

b oratory) as is a standard requirement for regulatory approvals . Nevertheless, it is generally accepted that piglets react to stimuli in a number of ways including: physiologically, behaviorally and through resistance movements and vocalisation [1,6] . On this basis, a range of outcome variables have been used to assess piglet pain during and following castration, and to assess amelioration of pain due to use of local anaesthetics or analgesia. These include; (a) physiological responses during the procedure [9 - 27] , (b) nociceptive motor responses during the procedure [19,2 6,28 - 30] ; (c) vocal responses during the procedure [18,19,24,25,29 - 34] ; (d) mechanical sensory testing in the minutes and hours following the procedure [23,30,35] and; (e) post - operative pain - related behaviours in the minutes and hours following the procedur e [10,11,18,19,23 - 25,33,34,36 - 39] . More recently, newer technologies have been explored including (e) facial expression [ 23,34, 37 ] , and (f) infra red thermography (IRT) [ 16,19 ,20 ] . Unfortunately, the methods used to examine analgesic efficacy in the reported literature have varied considerably between investigators, and the detail and quality of reporting has been highly variable, precluding the ability to make standardized assessments of the validity of each measure. As highlighted in previous reviews on this topic [40 - 42] , this variation in the methods has impeded efforts to develop science - based guidelines for pain management protocols for castration . To be valuable as indicators of pain mitigation , measures must be capable of consistently detecting a sign ificant difference in pain - associated responses during and / or following castration as compared with pre - operative values, and / or as compared

chunk 8 · 327 tokens

n ificant difference in pain - associated responses during and / or following castration as compared with pre - operative values, and / or as compared between castrated and non - castrated piglets. Secondly, variables must optimally be physiologically and/or clini cally relevant to the evaluation of the type of pain being measured e.g. intraoperative pain or post - operative pain. Ideally, these measures (i) must be practically measured within the study without being confounded by the assessment of other endpoints); a nd; (ii) have the ability to be measured using an analytical method or measurement device/subjective assessment tool that has sufficient validation. In the current review, we summarise literature on the currently available methods for assessing peri - ope rative 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 developme nt of more standardized methods and optimise (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

chunk 9 · 447 tokens

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 a n increase in the level of glucose and lactate in the blood . A ctivation 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 - neuro peptides) 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 - endorphi n release are th us often used as indirect measure s of pain. Th ese physiological response s 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 p ain. 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 concent ration, as well as tissue content of pro - inflammatory cytokines; (including tumour necrosis factor - alpha (TNF - a) and interleukin - 1 alpha (IL - 1 a ) , IL - 6 and IL10), and opiates have a proposed role in regulating the

chunk 10 · 448 tokens

tumour necrosis factor - alpha (TNF - a) and interleukin - 1 alpha (IL - 1 a ) , IL - 6 and IL10), and opiates have a proposed role in regulating the hemodynamic response to blood loss [45] . In a porcine model of abdominal surgery, for example, a standardized laparotomy without visceral involvement was performed on 24 an a esthetized pigs. Surgery gave rise to dramatic increases in plasma ACTH and cortisol (p < 0.01 and p < 0.001, respectively) wit hin 15 min of incision, while animals were still under full general anaesthesia [43] . The activation of the HPA axis, and inflammatory cascade in response to surgical tissue trauma is generally termed the “surgical stress response”, and plays an important role in haemostasis and fluid homeostasis, immune defence , endogenous pain mitigatio n and wound healing [44] . Similar to other surgical procedures, piglet castration results in an acute physiological response with activation of the HPA - axis and SNS , and opiate neuropeptide release . Prunier et al. [4] reported that castration of piglets in duced significant (P < 0.05) increases in ACTH from 5 to 60 min, cortisol (from 15 to 90 min), and lactate (from 5 to 30 min) following the procedure , although no significant changes in blood glucose were observed. These authors hypothesised that glucose m ay not increase in neonatal piglets due to lack of glycogen stores. There is also a very rapid and transient increase in plasma adrenaline , followed by a longer lasting increase in plasma noradrenaline [4] as well as an i ncrease in heart rate , blood pressu re, and other signs of activation of the SNS such as reduced skin temperature have also been reported [4,31] . Elevated β - endorphin levels have been reported in piglets castrated via cutting, but not via tearing the

chunk 11 · 446 tokens

emperature have also been reported [4,31] . Elevated β - endorphin levels have been reported in piglets castrated via cutting, but not via tearing the spermatic cord, despite equivalent rises in cortisol, as well as motor and vocal responses during the procedure. This was hypothesised as due to the increased risk of blood loss when cutting as opposed to tearing the cordal tissues [46] . Highlighting concerns over interpreting such physiologica l markers as being indicative of pain rather than in response to surgical tissue trauma, comparison s of anaesthetised and non - anaesthetised castrated piglets have found no significant difference in stress hormone responses [26] . Plasma cortisol, ACTH and β - endorphins did not differ significantly between the anaestheti s ed and non - anestheti s ed castration groups indicating that tissue trauma (with inflammatory mediator release) and / or blood - loss , rather than pain , is primarily responsible for the physiological HPA - axis activation and opiate neuropeptide response. Cortisol was reported as “not a sensitive tool to judge castration stress” in piglets castrated under gener al anaesthesia [27] . This indicates that v ariability in wound size, blood loss and a piglet’s neuroendocrine and immune response to wounding may all have a greater impact on cortisol levels than pain in piglets undergoing castration. Furthermore , activat ion of the HPA axis and SNS may occur simply through handling and restraining piglets. Marchant - Forde et al. [46] reported that cortisol and β - endorphin levels were increased 45 min following the procedure in castrated piglets versus sham handled controls (p < 0.1) , Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 12 · 449 tokens

however this was associated with a significant difference in the duration of handling and restraint, and was no longer evident when these factors were taken into account. Hay et al. [47] did not find differences in urinary levels of corticoster oids and catecholamines over the 4 days following surgical castration of piglets, as compared with sham - handled controls. This was considered most likely due to the short - lived activity of the adrenal and sympathetic axes [4] . Lonardi et al. [48] reported a short - lived increase in cortisol levels in castrated versus sham handled animals at 20 min but not at 3 – 24 hours following the procedure. Lactate and glucose levels were not significantly different between the two groups. Sutherland et al. [ 2 4] reported increased cortisol levels in castrated versus sham handled piglets 30 - 120 minutes, but not 180 minutes or 24 hours following procedure, however the study involved prolonged handling of piglets for blood collection and / or administration of anesthetic trea tments prior to castration, and the actual duration of restraint and handling was not documented for each piglet to allow group comparisons. S ubstance P (SP ), however, was not significantly different between groups. (SP is a neurotransmitter released direc tly from damaged nerve fibres at the site of tissue damage, that is associated with increase pain perception and, hence, used as a biomarker of pain[50] ) . Other stud ies have reported that castrated piglets tended to have higher cortisol levels than sham handled pigs, however this did not reach statistical significance at the p<0.05 level [25,49] . Interestingly, where duration of restraint was controlled to be equivalent betwee n groups, there were also not significant differences between castrated and

chunk 13 · 448 tokens

tingly, where duration of restraint was controlled to be equivalent betwee n groups, there were also not significant differences between castrated and sham handled piglets in plasma levels of pro - inflammatory cytokines; TNF - a and interleukin - 1beta (IL - 1b), or on acute phase proteins C - reactive protein (CRP), serum amyloid A (SAA ) and haptoglobin (Hp) and Moya et al . [49] concluded that pro - inflammatory cytokines and acute phase proteins did not provide relevant information on the physiological consequences of castration in neonatal piglets. Together, th ese data suggest that handl ing stress alone induce s a physiological response similar to that of castration in neonatal piglets. Despite the significant impact that the duration of restraint and handling may have on results , this variable is not always detailed in study reports or included as a variable in analyses . Local anaesthetics and NSAIDS act to block pain via different mechanisms . This has important implications regarding interpreting the validity of biomarkers of HPA axis, neuroendocrine and / or inflammatory cascade activation as indicators of pain in this setting. NSAIDs mitigate pain via blockade of the conversion of arachidon ic acid to prostaglandins by cyclooxygenase enzymes ( COX ), preventing activation of the inflammatory cascade and release of pain - inducing inflammatory mediators. Prostaglandins also directly stimulate ACTH and cortisol release. Separate to mitigating pain, NSAIDs thus also may directly mitigate the humoral aspect of the surgical stress response to tissue trauma [51,52] . A reduction in cortisol following NSAID administration, may be anticipated to indicate a collateral reduction in production of prostaglandins and other associated pain - inducing inflammatory

chunk 14 · 412 tokens

dministration, may be anticipated to indicate a collateral reduction in production of prostaglandins and other associated pain - inducing inflammatory mediators in piglets post castration , and hence also an associated decrease in pain . Hence , cortisol or ACTH level s may provide an indirect biomarker of pain in pigl ets following NSAID administration . T his is not the case for local or general anaesthetics , however. Local anaesthetics act by blocking nerve fibre conduction of pain signals . These prevent pain sensation via local or central nervous system effects, without primary effect on the humoral / inflammatory response to tissue trauma or associated HPA - axis activation . Biomarkers associated with the surgical stress response may thus be eleva ted, even al though pain induced by them is blocked . Such variables are thus unlikely to be reliable indicators of pain in animals administered local or general anaesthesia. An additional confounding factor in the case of local anaesthetics is that, in many cases, these are administered in combination with adrenalin. This is to enhance local anaesthetic effects and minimize risks of systemic absorption. Adrenalin and n or - adrenalin, produced by the adrenal medulla, positively feedback to the pituitary and inc rease the breakdown of proopiomelanocortins into ACTH and β - endorphins . Exogenously administered adrenalin may thus clearly impact and confound markers of endogenous HPA - axis and SNS activation and opiate - peptide production in castrated piglets. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 15 · 447 tokens

In view of these factors, it is not surprising that studies investigating the impact of local anaesthesia or analgesia on physiological parameters in piglet castration have shown highly variable and , at times , apparently conflicting results (Table 1). The mor e consistent results are seen with the use of NSAIDs. Compared with piglets castrated without analgesic treatment , significantly reduced plasma cortisol and / or ACTH levels have been documented in NSAID - treated piglets at 30 mins [ 10, 14,17,53] 60 min [ 13, 23 ,24 ] , or up to 4 h rs post - procedure [ 11, 13, 23] . Others however, have reported no significant (p<0.05) effect of NSAIDs administered prior to [12,15, 18 ] or at the time of the procedure [20 , 25] , on cortisol and/or ACTH , nor acute phase reactants , Hp, SAA and / or CRP [ 10, 19,2 4 ] . Bates et al. [16] reported significantly greater amount of prostaglandin E 2 (PGE 2 ) inhibition at 10hrs, and from 30 - 100hrs post castration in piglets of meloxicam - as opposed to placebo treated sows . Cortisol and SP concentrations , however , were not significantly different (p<0.05) between the two groups. O’Connor [42] and associates concluded a weak recommendation for use of NSAIDS for pain alleviation in piglets 1 - 24 h rs post - castration following a systematic review of available trial data, based principally on impact on cortisol. In the same review, NSAID s were not fo und to have any impact of vocalisation to suggest an effect to mitigate procedural pain, which is discussed further below. Together , these data support the conclusion that some NSAID s may have activity to reduce the inflammatory response and HPA - axis activ ation resulting from tissue trauma in piglets in the hours following castration, consistent with their known

chunk 16 · 448 tokens

nflammatory response and HPA - axis activ ation resulting from tissue trauma in piglets in the hours following castration, consistent with their known mechanism of action . Where cortisol and ACTH levels are reduced post castration, (despite equivalent handling duration between treatment and contro l groups), this may be indicative of efficacy of NSAIDs to mitigate post - operative pain. By contrast, as expected, the majority of studies have found little or no impact of either local or general anaesthesia on markers of the tissue trauma / inflammatory response to piglet castration and resulting activation of the HPA axis. Pre - emptive use of local anaesthesia via in tra - testicular (i.t.) or infundibular injection, or via topical wound instillation , has been associated with reduced cortisol levels as compared with untreated animals in some trials [ 1 8 ,23 ] , while not in others [ 9, 13, 17 , 25 ] , or only where local anaestheti cs and NSAIDs have been used in combination [20] . As detailed above, the lack of efficacy of local or general anaesthesia to reduce cortisol or ACTH does not , however , represent lack of efficacy to mitigate pain . These agents act via a different mechanism, and mitigate pain via block ade of neural transmission. Neural markers of pain mitigation, such as t he expression of the c - fos gene and its protein product , Fos , in neurons of the spinal cord [54] , are significan tly reduced when piglets are castrated under effective local or general anaesthesia, as compared with piglets castrated without anaesthesia , and furthermore, this is associated with a dramatic reduction in the nociceptive motor and vocal response to castration [21,22] . Additionally , reduced post - operative hyperalgesia has been documented in local anesthetic -

chunk 17 · 294 tokens

eptive motor and vocal response to castration [21,22] . Additionally , reduced post - operative hyperalgesia has been documented in local anesthetic - treated piglets [30,35] . Together, these factors are considered to indicate that biomarkers of activation of the HPA axis, and inflammatory response lack specificity for pain mitigating effects of local and general anaesthetics, and are poor indicators of pain in piglets castrated under general or local anaesthesia [1] . They are similarly not suited to comparative efficacy trials with NSAIDs . Based o n this review, it is concluded that biomarkers of activation of the HPA axis, SNS, opiate neuro - peptides and immune response, lack specificity as indicators of pain associated with neonatal piglet castration, and are confounded by the physiological respons e to restraint and to tissue trauma . They may provide some indication regarding the efficacy of NSAIDs to reduce post - operative inflammatory pain, however are very poor markers of potential pain mitigating effects of local or general anaesthetics. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 18 · 445 tokens

Table 1. Summary of studies investigating physiological responses during piglet castration 1 Authors Piglets N, age Castration experimental groups Significant findings Prunier et al. [55] 18, 7 - 9 days Castrated without analgesia /anaeshesia (CAST), Sham - handled (SHAM) or No handling  ACTH; (5 to 60 min), cortisol (15 to 90 min), and lactate (5 to 30 min) in CAST animals. No effect on g lucose. Marchant - Forde et al. [46] 328, 2 - 3 days CAST (cut or tear) , SHAM Blood sampling immediately bef ore and at 45 min, 4 h, 48 h, 1 and 2 wk s post procedure . 45 min post castration -  cortisol (trend) in CAST vs SHAM piglets. and  β - endorphin (trend) in cut vs tear and SHAM piglets. S ignificantly longer duration of procedure noted in CAST piglets vs SHAM piglets , however. Moya et al. [49] 40, 5 days CAST, SHAM *controlled for time of restraint B lood sampl ing before (0 h) and 1, 2, 3 and 4 h after procedures (cortisol, TNF - a and IL - 1b ) and before (0 h) and 12, 24, 48 and 72 h after procedures (CRP, SAA and Hp).  cortisol trend only (P < 0.1) in CAST vs SHAM and no statistically significant difference between groups (NS D ) for TNF - a , IL - 1b, CRP, SAA or Hp . Lonardi et al. [48] 32, 4 days CAST, SHAM B lood sampl ing 1 hr before and at 20 mins, 3, 5 and 24 hrs after procedures .  c ortisol in CAST vs SHAM animals 20 min but not 3 - 24 hrs post castration ;  l actate and g lucose (SHAM and CAST) 3 - 24 h rs post - castration. Car r oll et al. [56] 90, 3 , 6, 9 and 12 days CAST, SHAM B lood sampl ing before and at 0.5, 1, 1.5, 2, 24, and 48 h after castration .  cortisol for 0.5 - 2 h r after procedure CAST > SHAM and  cortisol in older versus 3 day old piglets . Hay et al. [47] 84; 5 days CAST; SHAM; ( Animals previously tail - docked )

chunk 19 · 154 tokens

r procedure CAST > SHAM and  cortisol in older versus 3 day old piglets . Hay et al. [47] 84; 5 days CAST; SHAM; ( Animals previously tail - docked ) NS D between CAST vs SHAM animals during 4 days of urinary measurements Keita et al. [10] 90 ; mean 5 days CAST; SHAM ; NSAID (NSAID = Meloxicam (M) i.m. 10 - 30mins prior to castration) . 30 minutes post castration -  cortisol in CAST and M versus SHAM.  cortisol and ACTH in M vs CAST g roup, (ACTH in M group similar to SHAM). NS D for Hp at 24 hrs. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 20 · 443 tokens

Langhoff et al. [11] 245; 4 – 6 days CAST, SHAM, NSAID (NSAID = M, flunixin(F), metamizole(MET), carprofen(C)), or saline i.m . 15 - 30 min prior ) Blood sampling before a nd at 30mins, 1,4 and 24 hrs following procedures .  cortisol in CAST piglets 1 & 4 hrs post castration;  cortisol in all NSAID vs CAST piglets; (  cortisol in M and F vs CAST at 30 min, 1 hr and 4 hrs; NS D vs S HAM treated animals at 1 hr ) . Reiner et al. [12] N/A SHAM, NSAID (M or F)  cortisol in NSAID vs SHAM piglets 30 min post - castration Zöls et al. [13] 78 ; 4 - 6 days CAST, SHAM, NSAID (M) i.m. prior  cortisol in CAST vs NSAID and SHAM piglets 1, 4 (but not 2 8 ) hrs post castration. Schwab et al. [14] 130; < 7 days CAST , SHAM , NSAID (K etoprofen, ( K ) i.m. 10 - 30 mins prior ) 30 min post - castration -  cortisol and ACTH CAST > NSAID > SHAM piglets . Wavreille et al. [15] 66; 5 - 6 days CAST, SHAM, NSAID (T olfenamic acid (T) or M ) NS D CAST vs SHAM or M;  cortisol 30 min post - castration in T - pigs. Bates et al. [16] 10 sows; 60 piglets; 5 days CAST (M) - (piglets from M treated sows), CAST(p) - (piglets from placebo treated sows)  PGE 2 inhibition, 10 hrs and 30 - 100hrs post (castration + tail docking + iron injection) in CAST - M vs CAST - p piglets . NSD between groups for plasma cortisol and SP. (Peak cortisol occurred 1hr post procedures). Mars á lek et al. [9] 36, 4 days CAST, SHAM, Local anaesthesia (LA) (LA = Lignocaine (L) + Noradrenalin (N - adr ) , administered i.t. 3 mins prior)  c ortisol CAST and LA vs SHAM at 1 h r after castration. (L+N - adr did not modify cortisol concentrations ) . Zöls et al. [ 13 ] 124; 4 - 6 days CAST, SHAM, LA ( LA = Procaine Hydrochloride (P) i. t . 15 mins prior)  cortisol in CAST and LA vs SHAM piglets

chunk 21 · 202 tokens

. Zöls et al. [ 13 ] 124; 4 - 6 days CAST, SHAM, LA ( LA = Procaine Hydrochloride (P) i. t . 15 mins prior)  cortisol in CAST and LA vs SHAM piglets 1, 4 (but not 2 8 ) hrs post castration. (P did not modify cortisol concentrations) Courboulay et al. [17] 96 CAST, SHAM, NSAID (K) , LA (L).  cortisol at 30 mins in L and CAST vs K and SHAM. Kluivers - Poodt et al. [ 18 ] 160 , 3 - 5 days CAST, SHAM, NSAID (M) , LA ( L ) , L +M (L - i.t.+s.c. M - i.m. administered 15mins prior) Cortisol, lactate glucose and creatinine kinase(CK) measued before and 20 mins following procedures.  cortisol all grp vs SHAM .  cortisol L vs CAST and M. NS D any treatment group s, for lactate, glucose or CK . Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 22 · 444 tokens

Hansson et al. [19] 564; 1 – 7 days CAST; NSAID ( M ); LA (L+adr) , LA + NSAID ( Administration L+adr - i.t. 3 - 30 mins prior, M - i.m. post castration). Trend to reduced SAA in NSAID - treated piglets. Bonastre et al. [20] 120; 4 – 7 days CAST, SHAM, SHAM+NSAID (M) , CAST+ M , CAST+L A(L) , CAST+L+M, CAST+L+ bupivacaine (B), CAST+L+B+M (Administration ; L and B i.t. 20 mins prior, M i.m. immediately post castration).  Cortisol (20 min) in all groups except SHAM and CAST+L+M;  Glucose (20 min) in all groups except SHAM and CAST+L . Nyborg et al. [21] NA CAST, LA. (LA= L+B administered intrafunicularly (bilateral) and subcutaneously prior to castration)  cFos protein (spinal cord) in CAST vs L A piglets Svendsen [22] 20 CAST, CAST+ L A , CAST+ CO 2 / O 2 general anaesthesia (GA)  cFos protein (spinal cord) in CAST vs L A and G A piglets Gottardo et al. [23] 196; 4 days CAST; SHAM ; NSAID ( M , K or T); CAST+topical anaesthesia( TA ) ( TA= 2% or 6% topical tetracaine hydrochloride prior and applied to wound immediately post procedure );  cortisol and ACTH at 30 and 60 mins in CAST vs NSAID, T A and SHAM groups . Sutherland et al. [25] 36; 3 days CAST; SHAM ; TA (tetracaine) ; TA (L + B+ adrenalin ) . (TA administered post incision, to spermatic cords and skin edge immediately prior to castration). Trend (P=0.06)  cortisol in CAST and TA piglets 0.5 - 1 hr post castration but not at 90 – 180 mins ;  cortisol (P<0.05) in TA+adrenalin piglets between 30 - 180 minutes post - castrat ion. Sutherland et al. [24] 70; 3 days CAST; SHAM ; SHAM+ NSAID, SHAM+ GA(CO 2 ), CAST+ NSAID , CAST+GA (CO 2 ) , CAST+both (NSAID = F, i.m. immediately prior to procedure) Bld sampled before, and 30, 60, 120, and 180 min, 24 h, and 3 d after castration for cortisol,

chunk 23 · 140 tokens

T+both (NSAID = F, i.m. immediately prior to procedure) Bld sampled before, and 30, 60, 120, and 180 min, 24 h, and 3 d after castration for cortisol, Substance P (0 - 180 min) and CRP (24hr - 3 days).  cortisol (30min ) in all CAST vs SHAM grps .  cortisol (60 - 120min) in CAST and CAST+NSAID versus SHAM grp.  CRP in CAST (trend) and CAST+GA( CO 2 ) piglets. (  CRP CAST+GA(CO2) vs CAST piglets).  SP in all piglets grps receiving GA( CO 2 ). Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 24 · 141 tokens

Walker et al. [26] 85; 2 - 12 CAST; CAST + GA (Isofluorane)  cortisol , ACTH and β - endorphins in CAST animals; NSD between anaesthetized and non - anesthetized groups despite obvious behavioural differences . Kohler et al. [27] 21 – 28 days CAST, CAST+GA (CO 2 /O 2 ) , CAST+GA(Halothane)  cortisol, ACTH, β - endorphin ; NSD between groups despite obvious behavioural differences. 2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 25 · 446 tokens

3 3. Nociceptive motor responses during piglet castration 4 5 Piglet castration without anaesthesia induces protracted violent struggling and escape 6 behaviour in piglets during the procedure [26] . This piglet motor response is usually accompanied by 7 a loud vocal response and is attributable to the nociceptive withdrawal response to acute pain 8 induced during the procedure. It is referred to in the literature by a variety of terms including ‘escape 9 attempts’ [46] ; ‘defense behaviour’ [29] or; ‘resistance movements’ [19] . Measurement o f the 10 nociceptive motor response is typically conducted by use of a variety of methodologies including (i) 11 ordinal scales [29,46] (ii) focal assessments [ 10, 24 ] , (ii i ) a visual analogue scale (VAS) [58] , or; (i v ) the 12 use of a numerical rating scale (NRS) [26,35] . Regardless of the methods used, a nalysis of the 13 nociceptive motor responses of piglets consistently detects a marked and significant increase in 14 castrated versus sham - handled animals, and successful miti gation of this response through use of 15 general or local anaesthesia, indicative of sensitivity to detect pain mitigating effects (Table 2). 16 17 Numerous studies have demonstrated that the piglet nociceptive motor response to castration 18 is significantly increa sed in piglets undergoing castration as compared with sham - handled controls 19 and/or following the application of effective local or general anaesthesia (Table 2 ). Marchant - Forde 20 et al. [46] reported that castration triggered significant escape attempts in pi glets undergoing 21 castration compared to sham - handled controls. Focal sampling observations revealed that the piglet’s 22 nociceptive motor response often involved a sequence of sequential leg kicks

chunk 26 · 449 tokens

ndled controls. Focal sampling observations revealed that the piglet’s 22 nociceptive motor response often involved a sequence of sequential leg kicks in an attempt to escape, 23 followed by a pause. Injectable anae sthesia (i.e. 2% Lignocaine ) applied via intratesticular or 24 infundibular injection with an effective wait time has been shown to reduce the relative proportion 25 of resistance movements from the entire period of fixation, including during the cutting of the 26 spermatic cords, which elicits the greatest response and is considered to be the most painful step of 27 the procedure [28] . A subsequent study investigating lignocaine effectiveness also confirmed less 28 resistance movements during castration in piglets pre - inj ected with 10 mg/ml lignocaine into each 29 testicle as compared to untreated animals [19] . By contrast, pre - emptive i.m. administration of an 30 NSAID did not result in a significant reduction in nociceptive motor response [10 ] . 31 32 To investigate the efficacy of topical anaesthesia to mitigate piglet castration pain when instilled 33 into the wound and allowed a 30 sec wait time, our group recently employed a m ethod in which 34 piglet castration was recorded on video - tape, and the nociceptive motor response was graded off - 35 line by a blinded trained observer using an NRS (0 - 2, based on nil, partial or vigorous full body 36 response) including scoring at four specific time points during the surgical procedure (i.e. during 37 traction of each testicle and severance of each spermatic cord). Piglets were settled at the time of 38 commencing procedures. Nociceptive motor response scores were increased at all four time p oints in 39 untreated piglets, and were also shown to be significantly reduced in animals treated with

chunk 27 · 158 tokens

sponse scores were increased at all four time p oints in 39 untreated piglets, and were also shown to be significantly reduced in animals treated with topical 40 anaesthe t i c via wound instillation with 30 sec dwell time [30] . Together, this literature is considered 41 to indicate that assessment of nociceptive mo tor response can provide a consistent, sensitive and 42 repeatable method for documenting piglet pain responses during the castration procedure, and the 43 efficacy of pain management strategies. 44 45 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 28 · 443 tokens

46 Table 2 . Summary of st udies measuring motor response movements during castration 47 48 Authors Piglets N, age Castration experimental groups Method Significant findings (p<0.05) Marchant - Forde et al. [46] 32 ; 2 – 8 days Castration without anaesthesia (CAST); Cutting or tearing spermatic cord; sham - handled animals (SHAM) No. escape attempts (sequential kicks) during procedure ↑ escape attempts CAST vs sham groups; no significant differen ce (NSD) in response between castration method (cut versus tear) Horn et al. [28] 36; 10 – 14 days CAST, Local Anaesthesia (LA) (LA = L ignocaine (L) administered i.t. +/ - intrafunicularly prior to castration) Relative p roportion of resistance movements ↑ resistance movements in CAST, particularly prominent during spermatic cord cutting . ↓ in L - treated group Leidig et al. [29] 61; 3 – 4 days CAST; SHAM; L A ; (LA = L or Procaine(P) i.t. prior to castration) Ordinal scale measuring duration and intensity . ↑ scores in CAST animals; ↓ scores in SHAM, L and P - treated animal s Sheil et al. [30] 40; 3 – 7 days CAST; Topical wound anesthetic (TA), applied by wound instillation 30s prior to excising testes. Numerical rating scale ↑ scores in CAST piglets with traction on each teste and cutting of each spermatic cord; significantly reduced in TA treated group Walker et al. [26] 85; 2 - 12 CAST; CAST under general anaesthesia (GA) (Isofluorane) Numerical rating scale ↑ scores in CAST piglets with skin incision and testis excision ; significantly reduced in GA group Keita et al. [10] 90 ; mean 5 days CAST; SHAM; NSAID (NSAID = Meloxicam (M) i.m. 10 - 30mins prior to castration). “Global” behaviour score (GBS) calculated from presence or absence of: foreleg ; or hind leg ; or other body movements ; urine

chunk 29 · 180 tokens

s prior to castration). “Global” behaviour score (GBS) calculated from presence or absence of: foreleg ; or hind leg ; or other body movements ; urine or faeces emission ; tremors. GBS was similar in the meloxicam and placebo groups. There was a behavioural response (i.e. global score of 1 or more) in more than 95% of all pigl ets in the study during castration Hansson et al. [19] 564; 1 – 7 days CAST ; LA ( L +adrenalin) ; NSAID( M ) ; LA + M (Administration L+adr - i.t. 3 - 30 mins prior, M - i.m. post castration) Visual analogue scale ↑ scores in CAST animals; ↓ scores in L and LM - treated animals Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 30 · 447 tokens

4.0 Vocal responses during piglet castration 49 A review of the literature indicates that some changes in piglet vocalisation (i) can be detected 50 during surgical castration , (ii) can be moderated with the use of anaesthesia and ; (iii) are considered 51 to be indicative of pain (Table 3 ). Although piglets commonly vocalise when they are handled, and 52 particularly when restrained, the lit erature shows that during castration piglets may squeal more 53 often, more loudly and/or at a higher frequency than piglets that undergo sham handling [46,60 - 62] . 54 Castration is reported to produce changes in piglet vocalisation sound parameters that are 55 compr ehensively different to those detected from handling alone [32] . A wide range of p arameters 56 have been employed to measure piglet vocal response includ ing measurement of ; duration, energy 57 or loudness (dB) , peak frequency or pitch (Hz) , or highest energy (Hz) , vocalisation rate, and/ or the 58 percent of piglets that vocalise d . Parameters that describe a single event in a call, such as peak level 59 or peak frequency are considered to provide more consistent results than parameters that describe 60 an average, such as w eighted frequency and main frequency [63] . Most recently, s pecifically designed 61 software (Stremodo® ( S tre ss M o nitor and D o cumentation unit ) has been developed to detect stress 62 vocalisations in piglets [64,65] . This uses linear prediction analysis [66] to extr act features of calls and 63 categorise them as stress calls, non - stress calls or background noise. 64 65 S tudies have reported that piglets during castration produced more high - frequency calls (>1000 66 Hz), ( referred to as screams [63] ) , than non - castrated controls. Pulling and severing of the

chunk 31 · 450 tokens

ation produced more high - frequency calls (>1000 66 Hz), ( referred to as screams [63] ) , than non - castrated controls. Pulling and severing of the spermatic 67 cords lead to the greatest vocalisation response, greater than those normally e mitted during handling 68 and restraint as well as during the initial incision [63,67] . Vocalisation responses were also used to 69 compare the castration procedure itself with cutting or tearing of the spermatic cord found to have 70 little difference on the durat ion of responses [46] . Interestingly, intra - muscular injection of analgesics 71 induces vocalisations of similar power (dB), frequency (Hz) and energy as that induced by pulling 72 and tearing the spermatic cords duri ng castration, and of significantly greater p ower (dB), frequency 73 (Hz) and energy than skin incision [34] . 74 75 The majority of studies identify that local and general anaesthesia are effective in mitigating 76 piglet vocal response to castration. Piglets castrated without local anaesthesia produce a higher 77 number of screams with higher frequencies compared to piglets castrated with 78 anaesthesia [19,29,31,32,57] . Hansson et al. [19] used a decibel meter during castration to record the 79 highest vocal int ensity level ( dB ) of piglets castrated with and without a local an a esthetic ( lignocaine ). 80 Piglets castrated with out the local an a esthetic produced calls of a significantly higher intensity than 81 those administered lignocaine . Leidig et al. [29] summed the to tal duration of stress calls relative to 82 the total time of the procedure, finding that duration of vocalisations of piglets receiving intra - 83 testicular anaesthesia with injectable p rocaine was half of that emitted by piglets without anaesthesia. 84 Animals tha t

chunk 32 · 419 tokens

iglets receiving intra - 83 testicular anaesthesia with injectable p rocaine was half of that emitted by piglets without anaesthesia. 84 Animals tha t have received local anaesthetic injection to the testicle on one side vocalise less when 85 the anestheti s ed testicle is removed than the non - anaesthetised testicle, although there was wide 86 variability in the result [68] . Trials examining the impact of NSAID administration prior to castration 87 however, have uniformly reported little to no impact on piglet vocal responses during castration 88 [12, 23, 24,57] compared to piglets castrated without NSAID treatment. 89 90 Despite the overall consistency of reported outcomes, the actual metric s reported by authors are 91 very diverse and reporting of measures of variation i s poor , such that it is difficult to combine these 92 data or quantify the effect of anaesthetic interventions on vocalisation [41] . A confounder to studies 93 that rely on the quality of vocalisation responses to assess pain in piglets is that, in most cases, these 94 findings have been recorded in rooms acoustically isolated from farrowing pens where piglet 95 castration usually takes place. S ince regulatory safety and efficacy trials require demonstration in 96 ‘real - life’ situations, the sensitivity of pig vocalisation measurements and the consistency of results 97 needs to be considered against the normal background noise levels, and confounding f actors of a 98 farrowing pen in a commercial farm setting. The presence of the sow and littermates can have 99 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 33 · 448 tokens

confounding effects on piglet vocal responses. In view of these factors, it may be anticipated that 100 analysis of vocal responses may not be as sensitive an indicator of pain in regulatory field trial settings 101 as in acoustically separated research environments. 102 103 W e recently developed a modified method for quantifying piglet vocal responses in the on - farm 104 setting [30] . Piglet vocal response was recorded usi ng a decibel meter as well as time - stamped video - 105 tape recording. Off - line analysis by a blinded technician allowed generation of standardised 106 decibel/time wavefo r m recordings for each piglet, on which the time of various specific procedural 107 events were abl e to be marked. This allowed comparison of the peak (dB) and total auditory response 108 (area under the dB/time waveform curve (AUC)) of each piglet, during specific procedural event - 109 time periods (e.g. piglet vocal response during traction and severing of eac h cord). This provided 110 consistency and specificity to the measurement period. Using this technique, we identified that both 111 the peak dB and AUC recording were significantly reduced in piglets (n=20) treated with topical 112 anaesthesia instilled to the wound f ollowed by a 30 sec wait time, as compared with untreated piglets 113 (n=20) during traction and severing of the first cord . (A trend effect was evident for traction and 114 severing the second cord however statistical power was affected by increased variability) . This 115 finding was in contrast to a previous report [23] in which vocal responses in castrated piglets treated 116 with topical anaesthetics or an NSAID were compared with untreated controls (n=10 per group) using 117 Stremodo software. No measurable difference ha d been recorded

chunk 34 · 449 tokens

aesthetics or an NSAID were compared with untreated controls (n=10 per group) using 117 Stremodo software. No measurable difference ha d been recorded between treatment and non - treated 118 castrated groups in this trial . T his may have been due to lack of sufficient dwell time allowed for 119 efficacy of the topical anaesthetic agents employed , and / or insufficient power . More recently, we 120 commis sioned a further trial examining vocal response to castration following wound instillation of 121 a topical anaesthetic formulation (with 30 second dwell time) (n= 44 per group) using peak dB and 122 area under the dB/time waveform (as above) to compare vocal respo nse to castration between treated 123 and untreated piglets. With increased power, a significant reduction in vocal response (peak dB and 124 AUC) to traction and severing of both the first and second spermatic cords was recorded. (Sheil, M; 125 unpublished observatio ns , manuscript in preparation ). 126 127 In summary, it is considered that with careful application to ensure targeting of the 128 measurement period to coincide with the time points of pain generation, and avoidance of 129 confounding factors (particularly duration of restraint or recordings ) , measures of piglet vocalisation 130 in response to castration including ; the peak dB, total vocal response ( such as area under the dB/time 131 waveform ), the frequency (Hz) of call with the highest intensity (dB (A)), rate of high frequency calls 132 (>1000Hz)) or stress vocalisations using Stremodo, appear to provide a relatively consistent and 133 sensitive method of assessing procedural pain associated with castration, and pain mitigation in 134 neonatal piglets. 135 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020

chunk 35 · 47 tokens

associated with castration, and pain mitigation in 134 neonatal piglets. 135 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 36 · 445 tokens

Table 3 . Summary of studies measuring piglet vocal responses during castration 136 Authors Piglets Age, number Castration experimental groups Measurement method Significant findings (p<0.05) Wemelsfelder and van Putten [60] 4 weeks CAST; Female litter mates Calls highest in amplitude Incising the scrotum did not result in a change in vocalisation, however pulling and cutting spermatic resulted in a marked ↑ in vocalisation. White et al. [31] 172; 1 – 28 days Injectable Lignocaine (L) ; Castration without anaesthesia (CAST) Frequency with highest decibel level (HEF) Ligating cord produced ↑ HEF during castration; Significantly ↓ HEF in pigs treated with L Weary et al. [61] 102; 8 - 12 days CAST; sham - castrated (SHAM) Mean High (>1000 Hz) and low (<1000 Hz) calls Significantly > high frequency calls in castrated vs sham - handled piglets. G reatest differences occurred during the severing of the spermatic cords and lesser differences when t he scrotum was incised and the testicles extruded Taylor and Weary [67] 139; 7 – 10 days CAST; SHAM Mean High (>1000 Hz) and low (<1000 Hz) calls Significantly > high frequency calls in castrated vs sham - castrated piglets; pulling and severing produced hi ghest call rate Taylor et al. [62] 84; 3, 10, 17 days CAST; SHAM Mean High (>1000 Hz) and low (<1000 Hz) calls Significantly > high frequency calls in castrated vs sham - castrated piglets; No signifcant age effect observed on frequency of calls Marx et al. [63] 70; 7, 13, 19 days CAST; L 12 variables Calls classifed into three types (screams, grunts squeals); 2 x number of screams in untreated castrates vs treated Puppe et al. [65] 19; 14 days CAST Rate of stress calls; STREMODO automated call monitoring system ↑ Stress calls (>1000 Hz) during surgical parts of

chunk 37 · 128 tokens

Puppe et al. [65] 19; 14 days CAST Rate of stress calls; STREMODO automated call monitoring system ↑ Stress calls (>1000 Hz) during surgical parts of castration procedure Kluivers - Poodt et al. [ 18 ] 160; 3 – 5 days CAST; L; Meloxicam (M); L + M; SHAM Temporal, waveform & spectral parameters CAST piglets squealed longer and louder than piglets treated with L ± M; M - treated piglets similar to CAST Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 38 · 444 tokens

137 Keita et al. [10] 150 ; mean 5 days CAST; Meloxicam (M) O ccurrence of vocali s ation during castration recorded as ‘cry’, ‘growl’ or ‘silence’. Vocali s ation (crying) during castration occurred in 149 of the 150 piglets in the study . NSE of M treatment . Marchant - Forde et al. [46] 32; 2 – 8 days CAST; ( Cutting or tearing spermatic cord ) ; SHAM Duration, mean frequency, and frequency of peak amplitude Signi ficantly > peak frequency of call in castrated piglets vs sham handled controls Leidig et al. [29] 61; 3 – 4 days CAST ; SHAM ; L; Procaine (P) STREMODO CAST pain vocalisations significantly different from other treatment groups; no significant difference (NSD) between other groups Sutherland et al.[25] 36; 3 days CAST; SHAM; topical anesthetic(TA) , NSAID STREMODO Significant difference between SHAM pigle ts and castrated piglets (with or without treatment) Sutherland et al. (2012) 70; 3 days CAST ; SHAM ; NSAID, GA (C O 2), NSAID+GA (CO2) STREDMODO frequency of stress vocalisations % of stress vocalisation s was greater (P < 0.05) in CAS T and CAS T +NSAID pigs than all other treatments. Hansson et al. [19] 564; 1 – 7 days CAST ; L; M; L + M Calls highest in amplitude L and L+M piglets produced calls with significantly lower intensity than CAST and M - treated piglets Sheil et al. [30] 40; 3 – 7 days TA(+30s wait) ; CAST Peak dB and Area Under the dB / time (waveform) Curve (AUC) Significant reduction in vocal responses in TA(+30s wait) vs CAST piglets during traction and severance of first spermatic cord. Viscardi and Turner [34] 60; 5 days CAST; SHAM Buprenorphine (BUP); SHAM + BUP S pectrograms from v ideo - recording s . M aximum ; frequency (Hz), amplitude (μ) , power (dB) ; and energy (dB) of each call was determined comparing

chunk 39 · 146 tokens

pectrograms from v ideo - recording s . M aximum ; frequency (Hz), amplitude (μ) , power (dB) ; and energy (dB) of each call was determined comparing skin marking, i.m. injection, skin incision and castration i.m. injection an d castration (pulling and severing the spermatic cord) induced vocalisation s of ↑ frequency (Hz), power (dB) and amplitude (u) and / or energy , than skin incision, and/or spray marking / sham handling - all groups. NSE of Buprenorphine treatment. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 40 · 445 tokens

5.0 Post - operative pain - related behaviours In general, measures of behaviour have proven to be more reliable indicators of pain than physiological measures in animals following castration [1,46] . In other animal species , behaviours such as decreased or abnormal locomotion, turning the head towards t he rump, abnormal postures including prostration (standing or sitting with head below the shoulders), “hunching” (standing with kyphosis), “stiffness” (lying with legs tense and extended or walking with a stiff gait), and reduced movements of the tail are considered indicators of pain resulting from castration [69 - 72] . More diffuse and variable responses may occur in neonatal animals however, due to immaturity of neuronal pathways involved with pain processing [73] . Behavioural disturbances have also been ex amined in neonatal piglets following castration . A review of the literature however reveals that in piglets , these behavioural changes may be subtle, transient and/or variably expressed, such that findings are not always reproducible . In some cases , contra dictory results have also been reported (Table s 4 and 5 ). Behavioural assessments usually involve either direct quiet observation and scoring of piglet behaviours by trained blinded observers, or continuous time - lapse video - recording with off - line scoring either using event monitoring software or trained blinded observers. Assessments typically include observations of piglet; (i) posture (lying, standing, sitting etc), (ii) location (under heat, in contact with the sow or pen mates versus in isolation), and (iii) activities, including “ non - specific ” behaviours (sucking, sleeping, walking, playing, exploratory or aggressive behavio u r etc , which may be divided into “active”

chunk 41 · 429 tokens

ing “ non - specific ” behaviours (sucking, sleeping, walking, playing, exploratory or aggressive behavio u r etc , which may be divided into “active” and “inactive” behaviours ) and “pain - specific” behaviours. This latter category , first detailed by Hay et al . [47] based on pain - specific behaviours reported in other species , includes ; “prostration” (standing or sitting with head down below shoulder height), “huddled up” (ventral lying with at least three legs tucked up ), “tremors or trembling”, “spasms ” (localised muscle spasm), “stiffness” ( lying with legs tense and extended ), “tail wagging” and “scratching” (rubbing the rump along the floor or walls, also called “scooting ”). Authors have additionally included standing in “ hunched ” p osture (i.e. with kyphosis) or walking with a stiff or abnormal gait [23,48] . O bservations may be made by “scan sampling” (i.e. recording the general posture, position, and behavioural activity of the piglet, with frequent repetition (e.g. every 1 – 10 min ), over a predetermined time periods (generally 2 - 3 h rs in the morning and afternoon of each assessment day), and / or by “focal assessment” (scoring the presence or absence of “ pain - specific” behaviours at a number of predetermined time points). As incide nces of individual pain - specific behaviours are low, a ggregation of “pain - specific” behaviours is commonly employed to derive a “total” or “global” pain score for each piglet over specific time periods [10,23,47,49] . Using these methods, abnormalities of behaviour have been documented in the early min utes and hours after piglet castration, principally consisting of a low magnitude increase in “pain - specific ” behaviours

chunk 42 · 413 tokens

ocumented in the early min utes and hours after piglet castration, principally consisting of a low magnitude increase in “pain - specific ” behaviours and/or isolation. Although the majority of these behaviours are short - lived (i.e. observed with the greatest frequency in the first 30 min to 1 hr following castration), some particular behaviours such as increased tail wagging and/or scratching tend to develop later in the post - operative period and have been observed to be increased for up to 2 - 5 days post - procedure in some studies [34,38] , although not in others [18,49] . Overall in review, when comparing castrated piglets with sham handled controls, variation in general po stures and non - specific behaviours have been marginal and/or conflicting, and are generally not considered reliable indicators of piglet pain [18,47] . Early studies identified a number of behaviours thought to be indicative of pain in piglets, including ch anges in posture , position and nursing behaviour, with reduced standing and increased lying away from heat, and reduced nursing in the early hours (3 – 6 hrs) following the procedure as compared with uncastrated controls , effects that were ameliorated by u se of lignocaine local anaesthesia prior to castration [36,74,75] . A subsequent study [62] , however, reported differently, documenting decreased lying, increased sitting and increased nursing in piglets post - castration as compared with uncastrated controls. In all cases , however , the authors reported that effects, although Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 43 · 442 tokens

statistically significant, were marginal and/or of low magnitude. Hay et al. [47] introduced a detailed ethogram for behavioural assessment of piglets post - castration. This included recordi ng a range of indices of piglet posture and position, as well as ‘non - specific’ behaviours (such as suckling, walking, running, sleeping, playing , exploring, aggression ) , “pain - specific” behaviours (detailed above) as well as “social cohesion” (isolation a nd desynchronization) . Using this ethogram and scan sampling over 5 days, in a study of piglets 5 days of age (n=84) following castration, increased “ pain - specific” behaviours were documented involving greater incidences of prostration, stiffness, tremblin g , huddled - up posture and tail wagging as well as increased social isolation and de - synchronisation, during the first 2.5 hrs following castration in castrated versus sham - handled piglets. Scratching and tail wagging were increased at later time points and remained elevated for 2 - 4 days. There were no significant changes in other variables, and it was concluded that general postures changes and non - specific activities were not reliable indicators of pain in piglets post - castration [47] . A number of studies have used similar ethograms and / or assessment of “pain - specific” behaviours to investigate post - operative piglet pain since this time (Table 4 and 5 ). These have reported changes in “ pain - specific” behaviours and social isolation, g enerally detectable only during the earl iest assessment periods up to 180 min following castration. A recent study examining shorter time intervals identified significant changes in “ pain - specific” behaviour s were only present over the first 30 min post - c astration [23] . Most

chunk 44 · 448 tokens

time intervals identified significant changes in “ pain - specific” behaviour s were only present over the first 30 min post - c astration [23] . Most studies have reported minimal [47,74] or no significant effect on suckling, and all studies have reported no effects of castration on piglet weight gain when performed on neonatal piglets > 3 days of age (Table 4) . Longer term behaviour al effects have been variably reported. Hay et al. [47] reported scratching was increased with maximum frequency from 24 - 48 hrs post - operatively, and tail wagging was increased for 4 days. Wemelsfelder and van Putten [60] also documented increased tail wagg ing in the days following castration in 4 - week old piglets. However, piglets in both these trials had also undergone prior tail docking, and it was hypothesised that prolonged tail wagging could be related to exacerbation of tail stump hyperalgesia. Viscar di et al. [34,38] recorded a significant increase in tail wagging, peaking at 24 hrs in non - tail - docked piglets, with no significant difference in scratching behaviour. Others have reported no significant differences in scratching or tail wagging in castra ted piglets as compared with non - castrated controls up to four days post - castration [18,49] . Pre - treatment with l ocal anaesthetic or NSAID analgesic has been shown to result in significant differences in certain pain - related behaviour in treated piglets le ss than 2 weeks of age in some trials, [23,25,36] but not others [38,39,57] . McGlone et al. reported that although the changes in behaviour were only minor, piglets castrated without local anaesthetic were observed to display significantly reduced standing, increased lying and reduced nursing behaviours compared to piglets administered lignocaine via

chunk 45 · 446 tokens

ere observed to display significantly reduced standing, increased lying and reduced nursing behaviours compared to piglets administered lignocaine via injection prior to castration [36] . Hansson et al. [19] documented reductions in total “pain - specific” behaviours in piglets administered both lignocaine and meloxicam (but not alone) prior to castration as compared with untreated piglets. Sutherland et al. [25] examined the behavioural responses of piglets aft er castration and found that untreated animals spent significantly more time lying without contact (isolation) compared with piglets given topical anaesthetic via wound instillation during the procedure. In contrast, an alternative study [57] , reported that li gn ocaine injection prior to castration resulted in increased “ pain - specific” behaviour in the first hours after castration as compared with sham or unhandled controls, or NSAID - treated piglets. This was predominantly due to a significant increase in hud dling up in the early hours after the procedure , and a significant increased incidence of tail - wagging evident particularly over the first 3 days. It was hypothesised that either the effect of the lignocaine wore off so quickly that it had no post - operativ e analgesic effects or the sensation of the lignocaine wearing off may have resulted in increased tail - wagging in piglets. Using focal assessment and an amalgamated global “ pain - specific” behaviour score, Keita et al. [10] documented reduced scores at 2 and 4 hr post - castration between Meloxicam - treated piglets versus those without treatment , however, there were not significant effects at 30 mins, 1 hr or 24 hrs. L ittle or no difference in pain - related behavio u r was seen after castration performed with or wi thout

chunk 46 · 72 tokens

ficant effects at 30 mins, 1 hr or 24 hrs. L ittle or no difference in pain - related behavio u r was seen after castration performed with or wi thout general an a esthesia [25] . Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 47 · 446 tokens

It is notable that the majority of studies that have identified changes in “pain - specific” behaviours in the early hours following castration have been performed using direct observation with scan sampling and / focal assessment as opposed to continuous vi deo recording techniques. From a scientific perspective, continuous behavioural observation is generally considered the gold standard for pain evaluation in animals, as it allows detection of deviation in normal behaviour and is considered to have the sens itivity to detect subtle or short duration behaviours [76] . Performed using video recording and off - line analysis, it also avoids the potential for confounding by observer effects on animal behaviour. However, video - recording may be impaired by 2 - dimensiona lity, parallax error and shadowing. Furthermore, behaviours may be missed when animals are grouped, hidden or off - screen, such as may occur frequently in a farrowing pen. Such factors may all contribute to reduce sensitivity of video - recording methods to t he detection of subtle behavioural changes such as are seen in neonatal piglets in the early post - operative period. It is notable that no significant differences in “pain - specific” behaviours between castrated and sham - handled neonatal piglets were evident in the first 2 hrs following castration in trials using video - recording techniques [34,38] as opposed to those using direct observation [19,23,47,49] . Data from these trials suggest that video - recording techniques may have high sensitivity to detect tail - wagging, however, lower sensitivity to detect other “ pain - specific ” behaviours such as tremors, spasms, huddling up, prostration or stiffness in neonatal piglets. Although 2 trials [ 9,35 ] using direct observation

chunk 48 · 444 tokens

c ” behaviours such as tremors, spasms, huddling up, prostration or stiffness in neonatal piglets. Although 2 trials [ 9,35 ] using direct observation methods also failed to detect significant d ifferences in “pain - specific” behaviour in piglets post - castration as compared with sham - handled piglets these trials only examined a narrow range of “pain - specific” behaviours (scooting and huddling up) as compared with the full range detailed by Hay et a l . [47] and involved relatively low piglet numbers per group . This suggests that the studies may have been under powered, and that important pain - specific behaviours such as tremors/trembling, prostration, spasms, stiffness and tail - wagging may have been missed. There are limited validation studies on behavioural m ethodologies to detect piglet pain associated with castration, however, Hay et al. [47] compared 10 - min scan samples to continuous sampling on pain behaviours associated with castration and reported no difference in results when utilizing a scan or continuo us methodology. Additionally, Burkemper [39] has reported low inter - observer error following observer training for direct observation of pain - associated behaviours. On this basis, our group recently examined pre - and post - operative pain - related behaviour i n castrated piglets 3 - 5 days of age with and without wound instillation of topical anaesthesia during the procedure, across two separate trial sites (M. Sheil, unpublished observations). Direct observation using trained blinded observers was used, with s ca n assessments of posture and position (including pain - specific postures and positions, such as prostration, huddled - up, hunched standing, stiffness and isolation ) as well as behaviours (including “non -

chunk 49 · 442 tokens

pecific postures and positions, such as prostration, huddled - up, hunched standing, stiffness and isolation ) as well as behaviours (including “non - specific” and “ pain - specific” behaviours) which were re corded every 10 min for 3 hrs in the morning and 2 hrs in the afternoon; pre - castration and over the first 36 hrs post - castration. In addition, focal assessments of “ pain - specific” behaviours were separately made pre - castration and at 1, 15, 30, 60, 90 min and, 2, 4, 6, 8, 24 and 30 hr post - castration. Our results accord with those of Gottardo et al. [23] , who, using similar methods, reported increased “pain - specific” behaviour evident predominantly in the first 30 min after castration , which was mitigated b y pre - administration of analgesic medication or post - surgical topical anaesthetic medication. Also using similar me thods, Hansson et al. [19] reported reduced total “pain - specific” behaviours in the first 70 min period following castration in neonatal pigle ts administered both NSAID and local anaesthetic prior to castration. These results suggest that this method currently provides the most consistent repeatable method of identifying acute post - operative pain, and documenting pain - mitigation in the early minutes and hours following castration in neonatal piglets. We did not find a difference in “pain - sp ecific” behaviours between groups at later times, based on focal sampling, however, scores at later times were similar to pre - operative values. Using scan and/or focal assessment methods, Keita et al. [10] , Hans s on et al. [19] and Burkemper et al. [39] have p reviously reported relatively increased “pain - specific” behaviour at later time periods following castration in untreated as compared with

chunk 50 · 79 tokens

9] have p reviously reported relatively increased “pain - specific” behaviour at later time periods following castration in untreated as compared with analgesia/anaesthesia - treated piglets, however pre - Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 51 · 448 tokens

operative baseline values were not reported in the piglets under s tudy, nor were sham - handled groups included . Interestingly, we observed that most piglets were sleeping (~55%) or suckling (~20%) during baseline (pre - operative) scan observations. A prominent i ncrease in piglet sleeping was evident the afternoon following castration . A similar finding has been reported by Viscardi et al. [34,38] who similarly compared piglet behaviour pre - and post - castration. A n increase in piglet sleeping has otherwise been infrequently reported as a post - operative behavioural disturbance in piglets although it is, however, a well - documented response to aversive stimulation in neonates [77,78] , and neuroactive steroids such as allopregnanolone, and endogenous opioids such a β - endorphin, released in response to stress, are known t o have potent sedative properties . The majority of previous trials have examined piglet behaviour comparing castrated with sham - handled animals, rather than using a piglet’s pre - castration behaviour as its own control. As handling and restraint are aversiv e to piglets ( resulting in a neuro - endocrine and opiate - neuropeptide stress response), increased sleeping following handling and restraint may be common to both castrated and sham - handled animals . This could explain a lack of difference in sleep between sh am - handled and treatment groups in previous t rials. Kluivers - Poodt et al. [57] , for example reported a large proportion (70 - 75% ) of piglets sleeping during scan assessments the afternoon following castration or sham handling, however there were no t signific ant difference s between castrated and sham - handled piglets. Trends for increased lying, with reduced standing, walking, exploring etc, and /or reduced

chunk 52 · 448 tokens

difference s between castrated and sham - handled piglets. Trends for increased lying, with reduced standing, walking, exploring etc, and /or reduced active behaviours following castration where reported (Table 4 ,5) could all be consequent upon an increa se in piglets sleeping following handling, rather than being indicative of post - castration pain. It is interesting to note that buprenorphine administration prior to handling or castration is reported to significantly reduced the sleep response, and resulted in increased active behaviours in the 8 hrs following castration or sham handling in neonatal piglets [34] . It could be hypothesised that a dministration of exogenous opiates may have blocked the endogenous opiate response and associated sedative effects in this setting. A sedative response to aversive stimulation in piglets , if present, could explain the relatively low proportion of piglets e xhibiting “pain - specific” behaviours over the same period, and contribute to the challenges detecting pain ( and determining the efficacy of pain mitigation strategies) using behavioural observation methods at these later time points. Increased tail - wagging and scratching are the most consistently reported behavioural disturbances evident during later time periods, particularly in docked piglets, however, scratching may not be seen to a significant extent for 24 hrs. It is concluded that the expression of p ain in neonatal piglets is subtle and confounded by behavioural responses to handling stress. Pain assessment is confounded by the lack of a validated assessment method, which has resulted in variability in the methodological approach taken in trials to da te, and in the reported results. This is concerning because of the potential to underestimate both the

chunk 53 · 373 tokens

e methodological approach taken in trials to da te, and in the reported results. This is concerning because of the potential to underestimate both the degree of pain experienced by neonatal piglets, and the ameliorating effects of analgesic medicines. In review, direct observation of piglet behaviour, p re - and post - castration using frequent scan and / or focal assessment and an ethogram that includes and is targeted to observation of known “pain - specific” postures, positions and behaviours, including; tremors/trembling, spasms, prostration, huddled up or hunched posture, stiffness, tail - wagging, scratching, and isolation, currently appears to provide the optimal method to most consistently identify a difference in acute pain - induced behaviour between castrated and non - castrated piglets, and investigate th e potential efficacy of analgesics or anaesthetic medicines in the acute post - operative period. Tail wagging and scratching are the most consistently reported behavioural anomalies at later time points and appear to be equally well documented via continuou s recording with off - line analysis or direct observational methods. These variables may however indicate irritation or itch rather than pain, particularly if present in the absence of other pain indicators (such as hyperalgesia) and appear to be exacerbate d in piglets that are tail - docked. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 54 · 444 tokens

Table 4. Summary of behavioral studies in neonatal piglets following castration 1 Authors Piglets Number, age, Castration experimental groups Measurement method Significant findings (NSE = no significant effect) McGlone and Hellman [36] 20; 14 days CAST; Sham - handled (SHAM); Lidocane (L) Time lapse video recording; 3 hr pre - and 3 hr post - castration. Event recorder monitored general postures, position and feeding behaviour 3 hr post - op  standing;  lying (away from heat);  nursing in CAST piglets (low magnitude no effect on weight gain) McGlone et al. [74] 100; 1, 5, 10, 15 & 20 days CAST; SHAM Time lapse video recording. 24 hours post - op. A digital timing and data summary program [79] was used to measure the duration of each behavior Ethogram based on [36]  standing and  lying and  nursing 6 hr post - castration in CAST piglets (low magnitude, no effect on weight gain. Car r oll et al. [56] 90, 3 - 12 days CAST, SHAM T ime - lapse video recording (WJ - HD500A , 3 - min scan sample immediately after castration for 2 h. O bserved for “ active ” (running walking) , lying, lying under the heat, sitting, sitting under the heat, standing, standing under the heat, and nursing (mutually exclusive). NSE on the time that pigs s pent nursing, lying, standing, or sitting, Trend (P = 0.08) for CAST to be less active than SHAM. O verall age effect (P = 0.01) on the time that pigs spent standing, such that 3 - d - old pigs stood more than 6 - , 9 - , or 12 - d - old pigs. No effect on weight gain. Taylor et al. [62] 84; 3, 10, 17 days CAST; SHAM Time - lapse video recording; Scan sampling. Proportion of total behaviours scored at 10 min intervals Monitored general postures, location nursing and active/inactive behaviours.  standing or sitting and 

chunk 55 · 188 tokens

f total behaviours scored at 10 min intervals Monitored general postures, location nursing and active/inactive behaviours.  standing or sitting and  lying 0 - 2 hr post - castration in CAST piglets;  lying and  nursing in next 22 hrs. No significant effect (NSE) position (all effects low magnitude no effect on weight gain) Hay et al. [47] 84; 5 days CAST; SHAM *Previously taildocked Detailed Ethogram: Posture, location, non - specific and pain - specific activity/behaviours and social F irst 2.5 hr ;  “pain - specific ” behaviours (prostration, huddled up, stiffness & trembling),  tail waagging  isolation and desynchronization  Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 56 · 435 tokens

isolation/desynchronization.Direct observation. Scan sampling every 10 min immediately post - op & 2 hr each morning and evening for 5 days suckling/udder massage,  awake inactive in CAST piglets; 2 - 4 days -  scratching , tail wagging , Through - out -  walking and hud dled up . Low magnitude  kneeling otherwise NSE on postures or weight gain Moya et al. [49] Exp 1 20; 5 - 8hrs post - op CAST; SHAM Direct observation, Scan sampling every 3 min for 3 hr (5 – 8 hr post op ) ; ethogram based on [47]  total “pain - specific ” behaviours (  huddling up ) ;  walking;  udder massage/exploratory activity and scratching (NSE posture or position) Moya et al. [49]Exp 2 20; 4 days CAST; SHAM Direct observation, Scan sampling every 3 min for 2 hr each morning and evening for 4 days; ethogram based on [47]  total “ pain - specific ” behaviours (  huddled up; tremors; spasms) first 0 - 2.5hr; Later time points  sitting and  trend for isolation (tail - wagging not recorded) Keita et al. [10] 150; mean 5 days CAST; Meloxicam (M); Direct observation, Focal assessment (poresence/absence) of “pain - specific” behaviours” based on [47] ( prostration, tremors (trembling), tail movements and isolation ) at 30 min, 1, 2, 4 and 24 hr post - castration; Greater proportion showed total global pain score ‘0’ in M vs CAST at 2 and 4 h rs (NSE 30 min, 1 or 24 hrs) Kluivers - Poodt et al. [ 57 ] 160; 3 – 5 days CAST; SHAM ; unhandled ; Lignocaine ( L ) ; M; L + M *not t ail docked Direct observation, Scan sampling; 12 min intervals for 3.5 hr each morning and afternoon for 4.5 days; Ethogram based on [47] , tail - wagging scored separately from other pain - specific behaviours  “pain - specific ” behaviours (2 - 6hrs) ,  tail -

chunk 57 · 266 tokens

Ethogram based on [47] , tail - wagging scored separately from other pain - specific behaviours  “pain - specific ” behaviours (2 - 6hrs) ,  tail - wagging in L group (3 days) .  sleeping and inactive behaviours in all groups in first 2 - 6 hr post - castration. NSE suckling behaviour Hansson et al . [19] 398 ; 1 – 7 days CAST; L; M; L + M Direct observation scan sampling; each 10 mins for 70 mins . E thogram based on [47,49,60].  total “pain - specific ” behaviours (huddled up, stiffness, prostration, tremors/trembling, spasms, scratching) L+M group day 1 post castration. Gottardo et al. [23] 196; 4 days CAST; SHAM ; 2% topical tetracaine hydrochloride (THCL); 6% THCL; M; Direct observation, scan sampling 1 min intervals for 0 – 30 min & 60 – 90 min post - castration; Ethogram based on [49]  total “ pain - specific” behaviour ( tremors, sc ratching , hunching, tail - wagging) CAST group,  isolation CAST and THCL groups ;  standing Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 58 · 439 tokens

ketoprofen (K); tolfenamic acid inactive all groups except K and SHAM in first 30 mins . NSE 60 – 90 min period Sutherland et al. [25] 36; 3 days CAST; SHAM; topical anaesthetic Direct observation, 1 min scan sampling for 180 min post - castration; ethogram based on [49,79] incl. Lying with or without contact, suckling behavior, general postures and limited “pain - specific ” behaviours (huddled up or scratching)  lying without contac t in the CAST group Sutherland et al. [24] 70; 3 days CAST; SHAM ; General anaesthesia (GA) - (CO 2/ O 2 ); NSAID 1 min scan sampling 0 - 30, 60 - 90 and 120 - 150 minutes post castration ; ethogram as per [25] based on [49,80]  lying without contact; CAST first 30 mins thereafter CAS+CO2 pig let s spent more time lying without contact than other treatments.  total “ pain - specific” behavior (scratching , huddling , hunched ), CAS+CO2 , 0 - 30mins. Viscardi et al. [37] 19; 5 days CAST; M + EMLA® cream, M + Placebo cream, saline+ EMLA® cream, saline + placebo cream, prior to surgical castration , tail docking and i.m. iron injection . Video recording 1 hr pre - ; 0 – 8 hr and 24 hr post - castration; analysed 15mins per hour, ethogram based on [47] , behavio u rs analyzed separately, and grouped into “active” and “inactive” categories  inactive behaviors and  tail - wagging all groups first 6 hours post castration and docking as compared with pre - castration and docking.  isolation in piglets castrated without treatment as compared with treatment groups. (NSE individual “pain - specific ” behaviours other than tail wagging , however small sample si ze). Viscardi and Turner [38] 120; 5 days CAST; SHAM; M; K. *not t ail docked Video recording 1 hr pre - ; 0 – 8 hr and 24 hr post - castration;

chunk 59 · 210 tokens

le si ze). Viscardi and Turner [38] 120; 5 days CAST; SHAM; M; K. *not t ail docked Video recording 1 hr pre - ; 0 – 8 hr and 24 hr post - castration; analysed 15 mins per hour; ethogram adapted from [47] as above . B ehavio u rs analyzed separately, and grouped into “active” “inactive” and “pain” categories . “Pain” included; trembling, stiffness, spasms, tail wagging, and rump scratching At 0hr,  active behaviour s (walking standing); At 5hr ,  suckling; At 7 hr  sleep compared with pre - op, (all groups); At 2, 7 and 24 h post - castration  tail - wagging and “pain” behaviour , CAST, M, and K groups. (Note “pain” category included tail - wagging) . (NSE scratching or other individual pain - specific behaviours ) Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 60 · 437 tokens

2 Viscardi and Turne r [34] 60; 5 days CAST; S HAM (+saline) CAST+buprenorphine; S HAM +buprenorphine *not t ail docked Video recording 1 hr pre - ; 0 – 8 hr and 24 hr post - castration; ethogram based on [47] behaviors analyzed separately, and grouped into “active” and “inactive” and “pain” categories . “Pain” included; trembling, stiffness, spasms, tail wagging, and rump scrat ching  sleeping and  walking, standing and active behaviours 4 - 7 hours as compared with 0hr all groups.  active behaviours Buprenorphine versus other groups 0 - 7 hrs .  tail - wagging and “pain” behaviours 24 h post - castration, CAST versus SHAM group. NB: “pain” category included tail - wagging . Burkemper et al. [39] 235; 3 – 7 days CAST; Lidocane spray (LS); oral M; LS + oral M Direct observation, Scan sampling each 5 min for 5 hr period for 3 days post op; total pain and 5 “ pain - specific” behavio u rs based on [47](t ail wag , t remble , h uddle , p rostrate , s cratch )  total pain - specific behaviours max 0 - 1hr post castration. No significant difference observed in behaviour between treatment groups . (Trend for  pain - specific behaviour in LS group) Langhoff et al., [11] 245;, 4 - 6 days CAST; M, flunixin (F), metamizole (MET) or carprofen, respectively, administered 15 to 30 min before manipulation. post surgical behaviour (0 - 60 min and 180 - 240 min after castration/handling) Tail wagging, drooping the tail and changing the position were reduced in M and F piglets Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 61 · 425 tokens

Table 5 . Detailed summary of statistically significant findings (p<0.05) from behavioral studies examining posture, position, activity & pain - related behaviours 3 in neonatal piglets post - castration. (Arrows indicate statistical significance unless otherwise stated . NSE = No significant effect of treatment ). 4 Authors Posture Position Activity Pain - specific behaviours Weight gain Compared to sham - castrated piglets < 2 weeks of age Time of post - operative assessment Lying Standing Sitting Isolation Heat - lamp/(p osition in crate) Suckling/ nursing Active/inactive behaviours McGlone and Hellman[36] 0 - 3 hrs Minor  Minor  - - Minor  Minor  - - NSE McGlone et al. [74] 0 - 6 hrs Minor  Minor  - - Minor  Minor  - - NSE Taylor et al.[62] 0 - 2hrs 2 - 22hrs Minor  Minor  Minor  NSE Min or  NSE NSE NSE NSE NSE NSE Minor  - - - Car r oll et al. [56] 0 - 2hr NSE NSE - - NSE NSE NSE - NSE Hay et al. [47] 5 days NSE NSE NSE  0 - 2.5 hr NSE  0 - 2.5 hr  awake inactive 0 - 2.5hr  walking through - out  total,  prostration, stiffness, trembling tail - wagging 0 - 2.5 hr;  huddled up  scratching tail - wagging for 2 - 4 days NSE Moya et al. [49] Exp 1 5 - 8hrs NSE NSE NSE  NSE  Trend  walking &  exploratory behaviour  total,  huddled up  scratching NSE Moya et al. [49] Exp 2 4 days NSE NSE  (throu gh - out)  Trend NSE NSE Trend  active behaviours  total,  huddled up, spasms, trembling (0 - 2.5hrs) NSE Keita et al. [10] - - - - - - -  total ( prostration, tremors / trembling, tail movements and isolation ) 2 and 4hr post castration. NSE Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 62 · 382 tokens

5 Kluivers - Poodt et al. [57] NSE NSE NSE NSE NSE NSE  sleeping and inactive behaviours (all groups) Day1pm  total Day 1 pm (2 - 6hrs) (huddled, stiffness, spasms, prostrated, tremors/trembling)  tail wagging Day 5am only (Lidocaine group  tail wagging day 1 - 3, and 5 am). NSE Gottardo et al. [23] NSE NSE NSE  30 min post - op NSE NSE  standing inactive (30 min post - op)  total (tremors, hunching, scratching, tail - wagging) for 30 min post - castration NSE Sutherland et al. [25] - - -  180 min post - op - NSE NSE NSE (limited range = huddled up or scratching) NSE Sutherland et al. [24] - NSE NSE  30 min post - op - NSE NSE NSE ( limited range = huddled up or scratching ) NSE Viscardi et al. [37] Day - 1 – 24hrs NSE NSE NSE  0 - 7hrs (versus Day - 1) - NSE  0 - 7hrs (versus Day - 1)  tail - wagging otherwise NSE (individual) - Viscardi and Turner [34] Day - 1 – 24hrs NSE (various time effects) NSE (various time effects) NSE NSE - NSE  sleeping and lying  walking, standing and active behaviours 4 - 7 hours as compared with 0hr all groups.  total 24 hr (  tail - wagging) NSE Viscardi and Turner [38] Day - 1 – 24hrs Various time effects Various time effects NSE  various - NSE  active behaviours 0 and 24hrs as compared with various other times both groups  total and tail - wagging 2, 7 and 24 hr (Note total “pain” score predominantly increased due to increased tail - wagging) - Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 63 · 447 tokens

6.0 Post - operative mechanical nociceptive testing. 6 Quantitative Sensory testing is a long established and validated method of assessing the efficacy 7 of local anaesthesia and wound analgesia in laboratory research and clinical settings [81] . The flexion 8 reflex, or nociceptive withdrawal reflex, is a reflex response to a nociceptive stim ulus resulting in 9 withdrawal of a limb or body part from a painful stimulus, which may be abolished by effective local 10 anaesthesia or analgesia . I n the setting of tissue injury, the release of chemical mediators such as SP, 11 prostaglandins and bradykinin in volved in the inflammatory response, increase sensitization of 12 neurons to nociceptive signals resulting in the development of hyperalgesia and a reduction in the 13 threshold for the nociceptive reflex response [82] . Afferent nerve sensitisation resulting in 14 h yperalgesia is considered the primary pathological mechanism underlying the development of post - 15 operative inflammatory pain [7] . The threshold for eliciting the flexion reflex may be clearly 16 measured, including in rats [83] , and pigs [84] and used to assess t he development of hyperalgesia and 17 the efficacy of anaesthetic or analgesic interventions. The reflex is evoked by stimulation of small 18 calibre A6 or C fibre primary afferents which transmit noxious information. The absence of the reflex 19 response and/or a measurable change in the reflex threshold may be detected using a variety of 20 stimuli including needlestick, heat pads, calibrated or electronic Von Frey Filaments and/or Pressure 21 Algometry. 22 Von Frey filaments or ‘hairs’ are a set of calibrated filaments t hat bend when a certain pressure 23 is reached, allowing a reproducible mechanical stimulus to be delivered ,

chunk 64 · 446 tokens

irs’ are a set of calibrated filaments t hat bend when a certain pressure 23 is reached, allowing a reproducible mechanical stimulus to be delivered , graduating from that 24 inducing a light - touch sensation through to a pain - weighted stimulation of skin or tissues. Electronic 25 versions are also availabl e. U sing an electronic von Frey anesthesiometer , Herskin and 26 Rasmussen [85] have described thresholds of mechanical nociception in the pelvic limb of pigs , using 27 four categories of behavio u ral response ( from slight leg movements to kicking ) to detect and g rade 28 the threshold response. In addition to laboratory studies in humans, pigs and experimental animals, 29 modified techniques have been developed for use “in the field” for assessment of pain and pain - 30 alleviation in association with surgical husbandry wound s in livestock species. Applied to skin in 31 proximity to a wound at time points before and after surgery, an animals response to a fixed light 32 touch and pain - weighted von - Frey filament stimuli can be graded (via NRS) from a nil response (0) 33 through to; a lo cal twitch (1), or partial (2) or full body (3) nociceptive withdrawal response. The 34 development of hyperalgesia lowers the threshold for a response, resulting in a greater response score 35 to application of the same stimulus. Th is method ha s provided a sensitive, consistent and repeatable 36 method of documenting the development of post - operative wound hyperalgesia and assessing the 37 efficacy of topical or local anaesthetic - induced wound anaesthesia / analgesia in a range of livestock 38 species fol lowing surgical husbandry procedures, including mulesing, tail docking and/or castration 39 in lambs [71,86,87] , castration and dehorning in calves

chunk 65 · 448 tokens

l lowing surgical husbandry procedures, including mulesing, tail docking and/or castration 39 in lambs [71,86,87] , castration and dehorning in calves [72,88,89] . Using th is technique, a heightened 40 nociceptive motor response to stimulation of a surgical husbandry wound has been documented in 41 the minutes and hours following the procedure , in lambs, calves and piglets, as compared with sham 42 handled animals, and / or with pre - operative assessments , indicative of the development of post - 43 operative hyperalgesia . Pre - oper ative use of injected local anaesthetic ( lignocaine ) and / or immediate 44 post - operative use of topical local anaesthetic applied to the wound has result ed in a significant 45 reduction in nociceptive withdrawal responses evident within 1 - 3 min of application , and continuing 46 in the minutes and hours following the procedure, indicative of significant wound anaesthesia or 47 hypoaesthesia . Where present, this has been associated with evidence of reduced post - operative pain - 48 related behaviour in treated animals over th e same period. 49 In pigs, this method has been shown to elicit similar and measurable responses to those reported 50 in human studies, and is sensitive to the effects of local anaesthetic agents [84] (Table 6) . Von Frey 51 filaments have been employed in studies to assess the efficacy of pain mitigation in piglets following 52 surgical castration [30,35] . W ound sensitivity testing involved the use of von Frey monofilaments of 53 weights 4g and 300 g and an 18 - gauge needle to stimulate the wound and surrounding skin at 54 pred etermined sites prior to treatment and then at defined periods of time afterwards. Involuntary 55 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020

chunk 66 · 46 tokens

to treatment and then at defined periods of time afterwards. Involuntary 55 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 67 · 448 tokens

nociceptive motor responses were scored using an NRS as above . Topical anaesthe sia using a 56 lignocaine, bupivacaine adrenalin combination formulation was found to provide rapid wound 57 anaesthesia and subsequent effective wound ana lgesia , with treated pigs displaying significantly 58 reduced responses compared to untreated animals [30,35] within one minute and continuing 2 - 4 59 hours post operatively, and showing similar re sponses to wound stimulation as sham - treated 60 piglets [35] . Pre - operative lidocaine injection (scrotal and intra - testicular), also induced early wound 61 hypoaesthesia, with reduced responses as compared with untreated piglets for up to 1 hour following 62 castrat ion. 63 As an alternative to von Frey filaments and needlestick stimulation, pressure algometry 64 involves applying a force to a point and measuring the pressure at which a withdrawal response is 65 elicited using a pressure algometer. Both A and C fibers mediat e pain induced by pressure 66 stimulation [81] . Acute pain in piglets following castration and the impact of local and topical 67 anaesthesia (tetracaine) has also been assessed by pressure algometry [23] . Efficacy of pain relief was 68 assessed prior to and during a 300 min period after castration by scrotal skin pressure sensitivity, 69 amongst other methods. Increasing pressure was applied to a designated point on the skin of the 70 scrotum adjacent to the incision site and the pressure point by which a physical or vocal isation 71 response was elicited was recorded. Results were consistent with behavioural results in which 72 reduced pain related behaviours documented in the first 30 min following the procedure were more 73 prominent in NSAID than topical tetracaine - treated piglet s. While one study

chunk 68 · 449 tokens

urs documented in the first 30 min following the procedure were more 73 prominent in NSAID than topical tetracaine - treated piglet s. While one study investigating wound 74 sensitivity in calves found a good agreement between both Von Frey filament stimulation and 75 pressure algometry [72] , other comparative studies in piglets (M. Sheil, unpublished observations) 76 found pressure algometers w ere relatively insensitive due to the soft nature of the scrotal tissues. The 77 pressure device induced discernible indents or trauma to the soft tissues at the site without 78 consistently eliciting a response. Janczak et al. [90] examined factors affecting mechanical 79 (nociceptive) thresholds in piglets and the stability and repeatability of measures of mechanical 80 (nociceptive) thresholds in piglets when using a hand held algometer to examine potentially 81 confounding factors. These investigators reported that mechanical (nociceptive) thresholds can be 82 used both for testing the efficacy of anaesthetics and analgesics, and for assessing hyperalgesia in 83 chronic pain states in research and clinical settings, however identified that in pi glets age and weight 84 affected responses to pressure algometry, particularly in the first week of life. 85 Whilst the number of reports of quantitative nociceptive response testing in neonatal piglets post 86 castration are limited, d irect sensory testing using needlestick and von Frey stimulation with NRS 87 grading of the nociceptive withdrawal reflex response , has thus to date proven consistent, repeatable, 88 sensitive and specific to the pathophysiological process generating pain, and is concluded to provide 89 the o ptimal method currently available for assessing post - operative hyperalgesia secondary to 90 peripheral

chunk 69 · 232 tokens

ng pain, and is concluded to provide 89 the o ptimal method currently available for assessing post - operative hyperalgesia secondary to 90 peripheral afferent nerve sensitisation following castration in neonatal piglets. 91 Quantitative sensory testing allows assessme nt of an animal ’s response to noxious stimuli, as an 92 indicator of the peripheral afferent nerve sensitisation that underlies the development of post - 93 operative pain but does not necessarily indicate the more complex experience of spontaneous pain. 94 C ombining the use of QST with assessment of spontaneous pain - related behavio u r is recommended 95 when assess ing pain mitigation strategies , such as to provide evidence of reduced experience of pain, 96 as well as reduction in its primary underlying pathophysiological mec hanism. 97 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 70 · 364 tokens

Table 6 . Summary of studies assessing wound sensitivity after castration 98 99 Authors Piglets Age, number Castration experimental groups Measurement method Significant findings Lomax et al.[35] 40; 3 – 5 days Castration without anaesthesia (CAST); sham - castrated (SHAM); topical anaesthetic von Frey filaments (4g and 300g) and 18G needle; testing immediately after, 1 min, & every 30 min up to 4 hr; grading on NRS for involuntary motor response Significantly  NRS scores up to 4 hr post - castration Gottardo et al. [23] 196; 4 days CAST; SHAM; local anaesthesia - 2% topical tetracaine (THCL) hydrochloride & 6% THCL; analgesia – M & ketoprofen (KET) & tolfenamic acid Pressure algometry (Pressure Rate Onset Device) with pressure ranging betwee 0.1 - 20 kg/cm 2 ; testing 300 min post - castration  sensitivity in i njectable analgesia - treated piglets vs other treatments Sheil et al. [30] 40; 3 – 7 days Topical anaesthetic; CAST von Frey filament (300g) and pin - prick; testing 1 min & 1, 2, 4, 8, 12 and 24 hr post - castration; grading on NRS for involuntary nociceptive response based on [35] Statistically significant difference between treated and CAST groups at 1 min and up to 2 hr post - castration Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 71 · 448 tokens

7.0 Other measures of pain Several alternative methods to assess perioperative pain in piglets have also been described. A piglet grimace scale (PGS) was recently proposed as an alternative method to assess cast ration and tail docking pain in piglets [37] . Similar methodologies have previously been developed and validated for a variety of livestock species, including sheep [91] and horses [92] . The piglet PGS was developed following analysis and comparison between s till images of piglet faces captured at various stages after surgical castration and the concurrent presence/absence of behaviours indicative of piglet pain. This initial study reported a strong correlation between PGS score and behavioural activity in ani mals in the first several hours after castration [37] . Some doubts about the robustness of this method to consistently detect pain in neonatal piglets currently exist though. In a follow - up study applying the PGS, there were not significant differences between sham handled and castrated piglets, and a potential cofounder in the form of piglet body weight was identified, suggesting that facial grimacing may also in dicate weakness or stress related to lower body weight rather than pain [34] . It was also documented that administration of buprenorphine significantly reduced facial grimace scores as compared with both sham - handled and untreated castrated piglets. As bup renorphine also reduced sleep and increased the activity state of both sham handled and castrated piglets, this suggests the possibility that piglet activity state (as opposed to pain) may also impact facial grimace scores. The second issue relates to inte r - user operability with one study [23] revealing that the PGS method was too unreliable for use in comparative evaluation of

chunk 72 · 448 tokens

d issue relates to inte r - user operability with one study [23] revealing that the PGS method was too unreliable for use in comparative evaluation of piglet pain . I t failed to show consistent inter - observer reliability in scoring in 2 of the measures while the 3rd measure, orbital tightening, did not differentiate the positive and negative control. This is therefore considered to be a promising new development however further experience and validation is needed for use in in - field trials of piglet castration pain and analgesic effic acy . Infra - red thermography (IRT) measurement of skin temperature has also been used as a non - invasive method to assess pain responses in piglets with conflicting results reported [16,19, 20 , 48, 93] . Animals in pain lose heat from the body’s periphery, measur able by IRT, due to activation of the SNS causing vasoconstriction and redirection of blood flow to the internal organs [94] . Thus, piglets experiencing significant pain via surgical castration should display quantifiably lower skin temperatures than sham - castrated piglets or piglets treated with effective pain mitigation strategies. Consistent with this hypothesis, skin temper ature dropped to a greater extent immediately following castration in untreated piglets as compared with sham - handled animals and those administered both lidocaine and meloxicam prior to castration [ 20 ] , and cranial temperatures in castrated and tail - docked piglets of meloxicam - treated sows, were found to be significantly higher than temperatures recorded in piglets from placebo - treated sows up to 60 hrs after castration [16] . However, there were not significant differences between groups in IRT values at oth er sites (ear or snout - tip ) , and t hese results conflict with an

chunk 73 · 410 tokens

wever, there were not significant differences between groups in IRT values at oth er sites (ear or snout - tip ) , and t hese results conflict with an earlier study that found ear temperatures were increased in untreated piglets compared to piglets treated with meloxicam and / or intra - testicular lidocaine prior to castration [19] . Skin tempe rature measured using IRT at the wound site did not differ significantly between groups. Similarly, a second report examining effect of NSAID treatment , (administered to the sow prior to husbandry procedures in piglets ) found decreased skin temperatures in piglets of sows treated with an NSAID compared with piglets from placebo - treated sows at 2 and 4 h rs post - procedure, with no difference between groups at 1 h r , or from 7 - 24 h rs following the procedure [ 93 ] . This conflicted with eye tem perature recordings in the same cohort which were increased at 1 h r in the NSAID versus the placebo group, but not significantly different between groups from 2 and 4 h rs or up to 30 hrs following procedures. These investigators also identified significant temperature differences between male and female piglets, and a seasonal variation in skin and eye temperature recordings. A confounder to IRT measurements in this setting is that body temperature is also affected by the post - surgical inflammatory respon se (i.e. not only the SNS response to pain). Lonardi et al. [48] , examined rectal temperature and eye temperature in castrated versus sham handled piglets and Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 74 · 448 tokens

documented that there was an increase in both rectal and eye temperature over time following castration or sham handling and, although some values were numerically higher in castrated animals, there were no significant differences between the two group s. The increase in eye temperature correlated with the increase in rectal temperature. It was noted that body temperature is reported to increase in response to anxiogenic or stress - inducing stimuli or injury (surgery and trauma) secondary to endogenous in flammatory activation [95 - 97] . Inflammatory mediators such as TNF - α and IL - 1β are considered the main endogenous pyrogens [95] . These endogenous pyrogens are increased in piglets 3 h r after castration or sham handling [49] . It was considered that t his may ex plain the tardive hyperthermia observed in the study in both castrated and handled piglets, although other external factors interfering with body temperature such as exposure to heat lamps or time from milk intake c ould not be excluded . NSAIDs have anti - in flammatory and associated direct anti - pyretic effects and thus may have a lowering effect on temperature that may confound assessment of any effect due to mitigation of the SNS response to pain . General anaesthetics may also have direct effects on body tem perature and peripheral vasodilation. Local anaesthetics generally do not have significant direct anti - pyretic effects, however, are commonly administered with adrenalin, which may cause peripheral vasoconstriction and similarly confound skin temperature a ssessment. Yet another confounder is the relationship between the body’s temperature and circadian rhythms with day/night cycles influencing body temperature results in meloxicam - treated and untreated castrated piglets [16] . In

chunk 75 · 447 tokens

ture and circadian rhythms with day/night cycles influencing body temperature results in meloxicam - treated and untreated castrated piglets [16] . In view of the lack of consistency in results to date , and multiple confounders, thermography does not currently appear to provide a reliable indicator of pain in neonatal piglets’ post - castration, particularly following administration of local anaesthesia with adrenalin . Thermo graphy may be more reliable for assessment of pain or pain mitigation in non - surgical settings. 8.0 Conclusion Sensitive, specific and well validated methods of assessing pain provide the cornerstone for developing effective analgesic medications. Unfort unately, there are few such methods available for assessing pain associated with castration in neonatal piglets. This is confounded by the neonatal piglet’s physiological response to restraint, handling and surgical stress due to tissue trauma, and the see mingly subtle, and short - lived expression of pain in the post - operative period. An understanding of the strengths and weaknesses of currently available methods for pain assessment is critical to identifying and develop ing effective pain mitigation strategi es in neonatal piglets. Employing methodologies that lack specificity or reliability risks underestimating both piglet pain, and the efficacy of pain - relieving medications, and creates welfare concerns associated with unproductive or counter - productive res earch. In the absence of a validated “gold standard ” method of assessment, different methods are required and, indeed, this is a foundational requirement for any treatment method seeking regulatory approval. This review has discussed the potential strength s and weaknesses of a range of currently available methods of

chunk 76 · 292 tokens

ent method seeking regulatory approval. This review has discussed the potential strength s and weaknesses of a range of currently available methods of pain assessment in the context of examining the efficacy of different anaesthetic and/or analgesic treatment options in field trial settings . Based on the detailed review of different methods for assessing perioperative pain associated with surgical castration of piglet s, this review concludes that: • There is a relatively short - lived (0 - 3hr) physiological response to castration in neonatal piglets, however physiological parameters lack specificity for pain , and may be significantly confounded by the surgical stress respon se as well as response to restraint and handling . They do not provide a reliable method for assessment of pain - alleviating efficacy of general or local anaesthetic interventions. Due to differences in mechanisms of action, these parameters may however prov ide a more reliable method to assess efficacy of NSAIDs where confounding variables are adequately controlled . Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 77 · 446 tokens

• pain control during piglet castration may be evidenced most consistently and reliably by a reduction in spontaneous nociceptive motor response d uring the procedure such as by NRS or VAS scoring of intensity of motor response. • measurement of piglet vocal response to castration provides a second method for assessing pain control in piglets during the procedure. Variables includ ing; peak dB, total vo cal (dB/time) response, the frequency (Hz) of call with the highest intensity (dB(A)), and the rate of high frequency calls (>1000Hz) , or stress calls as documented by Stremodo  , appear to provide the most consistent or reliable parameters for detection of a significant reduction in vocal response. • for both nociceptive motor and vocal response assessments care should be taken to ensure piglets are settled prior to commencing procedures and recordings to provide a consistent baseline. It is also suggested t hat measures be adopted to minimise confounding factors (such as piglet responses to restraint and / or extraneous environmental stimulation) by targeting / limiting the assessment period as closely as possible to the time of acute pain generation. This is considered particularly important if studies are required in the field situation as opposed to acoustically separated environments. • Post - operative pain control is most effectively evidenced by documenting a combination of reduced peripheral afferent ner ve sensitisation with an associated reduction in pain - related behaviour. • Peripheral nerve sensitization (hyperalgesia) is currently most reliably and consistently documented in neonatal piglets using nociceptive threshold testing with Von Frey and needles tick as opposed to pressure algometry. • Post - operative pain - related behaviour may

chunk 78 · 435 tokens

s using nociceptive threshold testing with Von Frey and needles tick as opposed to pressure algometry. • Post - operative pain - related behaviour may be variable, subtle and short - lived. C areful planning of variables and time points to be measured as well as power is required . The most consistently reported behavioural c hanges indicative of acute pain in piglets post castration include ; “huddling up”, “ prostration ” , “ hunching ” , “ stiffness ” (lying or of gait) , “spasms”, “ tremors /trembling” , “ isolation ” , “tail - wagging” and “scratching” (as defined above), which are most evident in the first 30 min to 1 hr following castration. The most consistently reported abnormalities of “pain - specific” behaviour at later timer points are tail - wagging and “scratching”. It is noted however that both tail - wagging and scrat ching may indicate itch or irritation as opposed to pain, particularly if present in the absence of other indicators of pain (such as presence of hyperalgesia) at these later time points. They may be exacerbated in piglets that are also tail docked. • Other methods in development such as facial grimace scores and thermography, hold promise in many situations however do not currently appear to provide a reliable or consistent method of documenting pain or pain mitigation in neonatal piglets following castratio n. It is hoped that this review may assist the future development of more standardized methods of assessing pain mitigation in neonatal piglets, assist investigators to optimise (reduce and refine) future analgesic efficacy trials in this field, and supp ort the development and evaluation of innovative effective and practical approaches to improve piglet welfare where surgical

chunk 79 · 82 tokens

s in this field, and supp ort the development and evaluation of innovative effective and practical approaches to improve piglet welfare where surgical castration is still utilised in commercial pig facilities worldwide. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 80 · 131 tokens

Funding: Funding for this review was provided by Animal Et hics Pty Ltd. Yarra Glen, Victoria, NSW Australia. Acknowledgments: No acknowledgements Conflicts of Interest: Dr Meredith Sheil is founding Director of Animal Ethics Pty Ltd, and inventor of Tri - Solfen  , topical anaesthetic formulation. Dr Polkin g horne was funded by Animal Ethics Pty Ltd for this project. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 81 · 448 tokens

References 1. Rault, J. - L.; Lay, D.C.; Marchant - Forde, J.N. Castration Induced Pain in Pigs and Other Livestock. Appl Anim Behav Sci 2011 , 135 , 214 - 225. 2. Andresen, O. Boar tain related compounds: androstenone/skatole/other substances. Acta Veterinaria Scandinavica 200 6 , 48 , S5. 3. Windsor, P.A.; Lomax, S.; White, P. Progress in pain management to improve small ruminant farm welfare. Small Ruminant Research 2016 , 142 , 55 - 57. 4. Prunier, A.; Bonneau, M.; Von Borell, E.H.; Cinotti, S.; Gunn, M.; Fredriksen, B.; Giersing, M.; Morton, D.B.; Tuyttens, F.A.M.; Velarde, A. A review of the welfare consequences of surgical castration in piglets and the evaluation of non - surgical methods. Animal Welfare 2006 , 15 , 277 - 289. 5. Fredriksen, B.; Font, I.F.M.; Lundstrom, K.; Migdal, W.; Prunier, A.; Tuyttens, F.A.; Bonneau, M. Practice on castration of piglets in Europe. Animal 2009 , 3 , 1480 - 1487, doi:10.1017/S1751731109004674. 6. von Borell, E.; Baumgartner, J.; Giersing, M.; Jaggin, N.; Prunier, A.; Tuyttens, F.A.; Edwards, S.A. Animal welfare implications of surgical castration and its alternatives in pigs. Animal 2009 , 3 , 1488 - 1496, doi:10.1017/s1751731109004728. 7. Amaya, F.; I zumi, Y.; Matsuda, M.; Sasaki, M. Tissue injury and related mediators of pain exacerbation. Curr Neuropharmacol 2013 , 11 , 592 - 597, doi:10.2174/1570159X11311060003. 8. American Veterinary Medical Association. Literature Review on the Welfare Implications of Swine Castration. Availabe online: https://www.avma.org/resources - tools/literature - reviews/welfare - implications - swine - castration (accessed on 8th June 2020). 9. Marsalek, P.; Svoboda, M.; Smutna, M.; Blahova, J.; Vecerek, V. Neopterin and biopterin as bio markers of immune system activation associated with

chunk 82 · 448 tokens

9. Marsalek, P.; Svoboda, M.; Smutna, M.; Blahova, J.; Vecerek, V. Neopterin and biopterin as bio markers of immune system activation associated with castration in piglets. J Anim Sci 2011 , 89 , 1758 - 1762, doi:10.2527/jas.2010 - 3157. 10. Keita, A.; Pagot, E.; Prunier, A.; Guidarini, C. Pre - emptive meloxicam for postoperative analgesia in piglets undergoi ng surgical castration. Vet Anaesth Analg 2010 , 37 , 367 - 374, doi:10.1111/j.1467 - 2995.2010.00546.x. 11. Langhoff, R.; Zöls , S.; Barz, A.; Palzer, A.; Ritzmann, M.; Heinritzi, K. Untersuchungen uber den Einsatz von Schmerzmitteln zur Reduktion kastrationsbed ingter Schmerzen beim Saugferkel [Investigation about the use of analgesics for the reduction of castration - induced pain in suckling piglets]. . Berl Munch Tierarztl Wochenschr. 2009 , 122 , 325 - 332. 12. Reiner, G.; Schollasch, F.; Hillen, S.; Willems, H.; P iechotta, M.; Failing, K. Effects of meloxicam and flunixin on pain, stress and discomfort in male piglets during and after surgical castration. . Berl Munch Tierarztl Wochenschr 2012 , 125 , 305 - 314. 13. Zöls, S.; Ritzmann, M.; Heinritzi, K. Einfluss von Sc hmerzmitteln bei der Kastration männlicher Ferkel [Effect of analgesics on the castration of male piglets]. Berl Munch Tierarztl Wochenschr. 2006 , 119 , 193 - 196. 14. Schwab, S.; Follrich, B.; Kurtev, V.; Keita, A. Ketoprofen - practical use and efficacy for post - surgical analgesia in piglet castration. Tierartzliche Umschau 2012 , 67 , 207 - 213. 15. Wavreille, J.; Danard, M.; Servais, V.; Art, T.; Nicks, B.; Laitat, M. Effect of preoperative meloxicam or tolfenamic acid administration on stress and pain induced by surgical castration in piglets Journees Recherche Porcine 2012 , 44 , 275 - 276. Preprints (www.preprints.org) | NOT

chunk 83 · 56 tokens

ion on stress and pain induced by surgical castration in piglets Journees Recherche Porcine 2012 , 44 , 275 - 276. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 84 · 448 tokens

16. Bates, J.L.; Karriker, L.A.; Stock, M.L.; Pertzborn, K.M.; Baldwin, L.G.; Wulf, L.W.; Lee, C.J.; Wang, C.; Coetzee, J.F. Impact of transmammary - delivered meloxicam on biom arkers of pain and distress in piglets after castration and tail docking. PLoS One 2014 , 9 , e113678, doi:10.1371/journal.pone.0113678. 17. Courboulay, V.; Hemonic, A.; Gadonna, M.; Prunier, A. Effect of local anesthesia or anti - inflammatory treatment on pa in associated with piglet castration and on labour demand. Journess de la recherche Porcine en France 2010 , 42 , 27 - 34. 18. Kluivers - Poodt, M.; Hopster, H.; Spoolder, H.A.M. Castration under anesthesia and/or analgesia in commercial pig production. Report: Animal Sciences Group van Wageningen UR Postbus 65, 8200 AB Lelystad; 2007 https://edepot.wur.nl/45006 19. Hansson, M.; Lundeheim, N.; Nyman, G.; Johansson, G. Effect of local anaesthesia and/or analgesia on pain responses induced by piglet castration. Acta Vet Scand 2011 , 53 , 34, doi:10.1186/1751 - 0147 - 53 - 34. 20. Bonastre, C.; Mitjana, O.; Tejedor, M.T.; Calavi a, M.; Yuste, A.G.; Ubeda, J.L.; Falceto, M.V. Acute physiological responses to castration - related pain in piglets: the effect of two local anesthetics with or without meloxicam. Animal 2016 , 10 , 1474 - 1481, doi:10.1017/S1751731116000586. 21. Nyborg, P.Y.; Sørig, A.; Lykkegaard, K.; Svendsen, O. Nociception after castration of juvenile pigs determined by quantitative estimation of c - Fos expressing neurons in the spinal cord dorsal horn. Dansk Veterinærtidsskrift 2000 , 83 , 16 - 17. 22. Svendsen, O. Castration o f piglets under carbon dioxide (CO2) anaesthesia. Journal of Veterinary Pharmacology and Therapeutics 2006 , 29 , 54 - 55. 23. Gottardo, F.; Scollo, A.; Contiero, B.; Ravagnani, A.; Tavella, G.;

chunk 85 · 449 tokens

esia. Journal of Veterinary Pharmacology and Therapeutics 2006 , 29 , 54 - 55. 23. Gottardo, F.; Scollo, A.; Contiero, B.; Ravagnani, A.; Tavella, G.; Bernardini, D.; De Benedictis, G.M.; Edwards, S.A. Pain alleviati on during castration of piglets: a comparative study of different farm options. J Anim Sci 2016 , 94 , 5077 - 5088, doi:10.2527/jas.2016 - 0843. 24. Sutherland, M.A.; Davis, B.L.; Brooks, T.A.; Coetzee, J.F. The physiological and behavioral response of pigs cast rated with and without anesthesia or analgesia. J Anim Sci 2012 , 90 , 2211 - 2221, doi:10.2527/jas.2011 - 4260. 25. Sutherland, M.A.; Davis, B.L.; Brooks, T.A.; McGlone, J.J. Physiology and behavior of pigs before and after castration: effects of two topical an esthetics. Animal 2010 , 4 , 2071 - 2079, doi:10.1017/S1751731110001291. 26. Walker, B.; Jaggin, N.; Doherr, M.; Schatzmann, U. Inhalation anaesthesia for castration of newborn piglets: experiences with isoflurane and isoflurane/NO. J Vet Med A Physiol Pathol Clin Med 2004 , 51 , 150 - 154, doi:10.1111/j.1439 - 0442.2004.00617.x. 27. Kohler, I.; Moens, Y.; Busato, A.; Blum, J.; Schatzmann, U. Inhalation anaesthesia for the castration of piglets: CO2 compared to halothane. Zentralbl Veterinarmed A 1998 , 45 , 625 - 633. 28. Horn, T.; Marx, G.; von Borell, E. [Behavior of piglets during cast ration with and without local anesthesia]. Dtsch Tierarztl Wochenschr 1999 , 106 , 271 - 274. 29. Leidig, M.S.; Hertrampf, B.; Failing, K.; Schumann, A.; Reiner, G. Pain and discomfort in male piglets during surgical castration with and without local anaesthes ia as determined by vocalization and defence behavior. Appl Anim Behav Sci 2009 , 116 , 174 - 178. 30. Sheil, M.L.; Chambers, M.; Sharpe, B. Topical wound anaesthesia: efficacy to mitigate piglet

chunk 86 · 148 tokens

behavior. Appl Anim Behav Sci 2009 , 116 , 174 - 178. 30. Sheil, M.L.; Chambers, M.; Sharpe, B. Topical wound anaesthesia: efficacy to mitigate piglet castration pain. Aust Vet J 2020 . 31. White, R.G.; DeShazer, J.A .; Tressler, C.J.; Borcher, G.M.; Davey, S.; Waninge, A.; Parkhurst, A.M.; Milanuk, M.J.; Clemens, E.T. Vocalization and physiological response of pigs during castration with or without a local anesthetic. J Anim Sci 1995 , 73 , 381 - 386, doi:10.2527/1995.732 381x. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 87 · 449 tokens

32. Marx, G.; Horn, T.; Thielebein, J.; Knubel, B.; von Borell, E. Analysis of pain - related vocalization in young pigs. Jounral of Sound and Vibration 2003 , 266 , 687 - 698. 33. Chenery, A.L.; Alhallaf, R.; Agha, Z.; Ajendra, J.; Parkinson, J.E.; Cooper , M.M.; Chan, B.H.K.; Eichenberger, R.M.; Dent, L.A.; Robertson, A.A.B., et al. Inflammasome - Independent Role for NLRP3 in Controlling Innate Antihelminth Immunity and Tissue Repair in the Lung. J Immunol 2019 , 203 , 2724 - 2734, doi:10.4049/jimmunol.1900640. 34. Viscardi, A.V.; Turner, P.V. Efficacy of buprenorphine for management of surgical castration pain in piglets. BMC Vet Res 2018 , 14 , 318, doi:10.1186/s12917 - 018 - 1643 - 5. 35. Lomax, S.; Harris, C.; Windsor, P.A.; White, P.J. Topical anaesthesia reduces s ensitivity of castration wounds in neonatal piglets. PLoS One 2017 , 12 , e0187988, doi:10.1371/journal.pone.0187988. 36. McGlone, J.J.; Hellman, J.M. Local and general anesthetic effects on behavior and performance of two - and seven - week - old castrated and u ncastrated piglets. J Anim Sci 1988 , 66 , 3049 - 3058, doi:10.2527/jas1988.66123049x. 37. Viscardi, A.V.; Hunniford, M.; Lawlis, P.; Leach, M.; Turner, P.V. Development of a Piglet Grimace Scale to Evaluate Piglet Pain Using Facial Expressions Following Castration and Tail Docking: A Pilot Study. Front Vet Sci 2017 , 4 , 51, doi:10.3389/fvets.201 7.00051. 38. Viscardi, A.V.; Turner, P.V. Use of Meloxicam or Ketoprofen for Piglet Pain Control Following Surgical Castration. Front Vet Sci 2018 , 5 , 299, doi:10.3389/fvets.2018.00299. 39. Burkemper, M.C.; Pairis - Garcia, M.D.; Moraes, L.E.; Park, R.M.; Mo eller, S.J. Effects of Oral Meloxicam and Topical Lidocaine on Pain associated Behaviors of Piglets Undergoing Surgical Castration. J Appl

chunk 88 · 450 tokens

k, R.M.; Mo eller, S.J. Effects of Oral Meloxicam and Topical Lidocaine on Pain associated Behaviors of Piglets Undergoing Surgical Castration. J Appl Anim Welf Sci 2019 , 10.1080/10888705.2019.1590717, 1 - 10, doi:10.1080/10888705.2019.1590717. 40. Sutherland, M.A. Welf are implications of invasive piglet husbandry procedures, methods of alleviation and alternatives: a review. N Z Vet J 2015 , 63 , 52 - 57, doi:10.1080/00480169.2014.961990. 41. Dzikamunhenga, R.S.; Anthony, R.; Coetzee, J.; Gould, S.; Johnson, A.; Karriker, L .; McKean, J.; Millman, S.T.; Niekamp, S.R.; O'Connor, A.M. Pain management in the neonatal piglet during routine management procedures. Part 1: a systematic review of randomized and non - randomized intervention studies. Anim Health Res Rev 2014 , 15 , 14 - 38, doi:10.1017/S1466252314000061. 42. O'Connor, A.; Anthony, R.; Bergamasco, L.; Coetzee, J.; Gould, S.; Johnson, A.K.; Karriker, L.A.; Marchant - Forde, J.N.; Martineau, G.S.; McKean, J., et al. Pain management in the neonatal piglet during routine management procedures. Part 2: grading the quality of evidence and the strength of recommendations. Anim Health Res Rev 2014 , 15 , 39 - 62, doi:10.1017/S1466252314000073. 43. Lykkegaard, K.; Lauritzen, B.; Tessem, L.; Weikop, P.; Svendsen, O. Local anaesthetics attenua tes spinal nociception and HPA - axis activation during experimental laparotomy in pigs. Res Vet Sci 2005 , 79 , 245 - 251, doi:10.1016/j.rvsc.2004.11.017. 44. Kehlet, H. Surgical stress: the role of pain and analgesia. Br J Anaesth 1989 , 63 , 189 - 195, doi:10.109 3/bja/63.2.189. 45. Molina, P.E. Opiate modulation of hemodynamic, hormonal, and cytokine responses to hemorrhage. Shock 2001 , 15 , 471 - 478, doi:10.1097/00024382 - 200115060 - 00011. 46. Marchant - Forde, J.N.; Lay,

chunk 89 · 129 tokens

ormonal, and cytokine responses to hemorrhage. Shock 2001 , 15 , 471 - 478, doi:10.1097/00024382 - 200115060 - 00011. 46. Marchant - Forde, J.N.; Lay, D.C., Jr.; McMunn, K.A.; Cheng, H.W.; Pajor, E. A.; Marchant - Forde, R.M. Postnatal piglet husbandry practices and well - being: the effects of alternative techniques delivered separately. J Anim Sci 2009 , 87 , 1479 - 1492, doi:10.2527/jas.2008 - 1080. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 90 · 449 tokens

47. Hay, M.; Vulin, A.; Génin, S.; Sales, P.; Prunier, A. A ssessment of pain induced by castration in piglets: behavioral and physiological responses over the subsequent 5 days. Appl Anim Behav Sci 2003 , 82 , 201 - 218. 48. Lonardi, C.; Scollo, A.; Normando, S.; Brscic, M.; Gottardo, F. Can novel methods be useful fo r pain assessment of castrated piglets? Animals (Basel) 2015 , 9 , 871 - 877. 49. Moya, S.L.; Boyle, L.A.; Lynch, P.B.; Arkins, S. Effect of surgical castration on the behavioral and acute phase responses of 5 - day - old piglets. Applied Animal Behavior Science 2 008 , 111 , 113 - 145. 50. O'Connor, T.M.; O'Connell, J.; O'Brien, D.I.; Goode, T.; Bredin, C.P.; Shanahan, F. The role of substance P in inflammatory disease. J Cell Physiol 2004 , 201 , 167 - 180, doi:10.1002/jcp.20061. 51. Aloisi, A.M.; Buonocore, M.; Merlo, L. ; Galandra, C.; Sotgiu, A.; Bacchella, L.; Ungaretti, M.; Demartini, L.; Bonezzi, C. Chronic pain therapy and hypothalamic - pituitary - adrenal axis impairment. Psychoneuroendocrinology 2011 , 36 , 1032 - 1039, doi:10.1016/j.psyneuen.2010.12.017. 52. Zacharieva, S.; Borissova, A.M.; Andonova, K.; Stoeva, I.; Matrozov, P. Role of prostaglandin E2 (PGE2) on the corticotropin - releasing hormone (CRH) - induced ACTH release in healthy men. Horm Metab Res 1992 , 24 , 336 - 338, doi:10.1055/s - 2007 - 1003327. 53. Perez - Pedraza, E .; Mota - Rojas, D.; Ramirez - Necoechea, R.; Guerrero - Legarreta, I.; Martinez - Burnes, J.; Lezama - Garcia, K.; Mora - Medina, P.; Rosas, M.; Martinez, V.; Gonzalez - Lozano, M. Effect of the number of incisions and use of local anesthesia on the physiological indic ators of surgically - castrated piglets. Int J Vet Sci Med 2018 , 6 , 159 - 164, doi:10.1016/j.ijvsm.2018.10.002. 54. Harris, J.A. Using c -

chunk 91 · 449 tokens

cal indic ators of surgically - castrated piglets. Int J Vet Sci Med 2018 , 6 , 159 - 164, doi:10.1016/j.ijvsm.2018.10.002. 54. Harris, J.A. Using c - fos as a neural marker of pain. Brain Res Bull 1998 , 45 , 1 - 8, doi:10.1016/s0361 - 9230(97)00277 - 3. 55. Prunier, A.; Mounier, M.; Hay, M. Effects of castration, tooth resection, or tail docking on plasma metabolites and stress hormones in young pigs. Journal of Animal Scienceq 2005 , 83 , 216 - 222. 56. Carroll, J.A.; Berg, E.L.; Strauch, T.A.; Roberts, M.P.; Kattesh, H.G. Hormonal profiles, behavioral responses, and short - term growth performance after castration of pigs at three, six, nine or twelve days of age. . Journal of Animal Scienceq 2006 , 84 , 127 1 - 1278. 57. Kluivers - Poodt, M.; Zonderland, J.J.; Verbraak, J.; Lambooij, E.; Hellebrekers, L.J. Pain behaviour after castration of piglets; effect of pain relief with lidocaine and/or meloxicam. Animal 2013 , 7 , 1158 - 1162, doi:10.1017/S1751731113000086. 58 . Dobromylskyj, P.; Flecknell, B.D.; Lascelles, B.D.; Livingston, A.; Taylor, P.; Waterman - Pearson, A. Pain assessment. In Pain management in animals , Flecknell, P., Waterman - Pearson, A., Eds. Wiley - Blackwell Saunders: China, 2001; pp. 53 - 76. 59. Adriani, J.; Zepernick, R.; Arens, J.; Authement, E. The Comparative Potency and Effectiveness of Topical Anesthetics in Man. Clin Pharmacol Ther 1964 , 5 , 49 - 62, doi:10.1002/cpt19645149. 60. Wemelsfelder, F.; van Putten, G. Behavior as a possible indicator for pain in piglets ; 1985. 61. Weary, D.M.; Braithwaite, L.; Fraser, D. Vocal response to pain in piglets. Applied Animal Behavior Science 1998 , 56 , 161 - 172. 62. Taylor, A.A.; Weary, D.M.; Lessard, M.; Braithwaite, L. Behavioural responses of piglets to castration : the effect of piglet

chunk 92 · 165 tokens

98 , 56 , 161 - 172. 62. Taylor, A.A.; Weary, D.M.; Lessard, M.; Braithwaite, L. Behavioural responses of piglets to castration : the effect of piglet age. Appl Anim Behav Sci 2001 , 73 , 35 - 43. 63. Marx, G.; Horn, T.; Thielebein, J.; Knubel, B.; von Borell, E. Analysis of pain - related vocalization in young pigs. Journal of Sound and Vibration 2003 , 266 , 687 - 698. 64. Schon, P.C.; Pup pe, B.; Manteuffel, G. Automated recording of stress vocalisations as a tool to documented impaired welfare in pigs. Animal Welfare 2004 , 13 , 105 - 110. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 93 · 449 tokens

65. Puppe, B.; Schon, P.C.; Tuchscherer, A.; Manteuffel, G. Castration - induced vocalisation in domestic p iglets, Sus scrofa : Complex and specific alterations of the vocal quality. Applied Animal Behavior Science 2005 , 95 , 67 - 78. 66. Schon, P.C.; Puppe, B. Linear prediction coding analysis and self - organizing feature map as tools to classify stress calls of do mestic pigs ( Sus scrofa ). J Accoust Soc Am 2001 , 110 , 1425 - 1431. 67. Taylor, A.A.; Weary, D.M. Vocal responses of piglets to castration: identifying procedural sources of pain. Appl Anim Behav Sci 2000 , 70 , 17 - 26, doi:10.1016/s0168 - 1591(00)00143 - x. 68. Gut zwiller, A. Castration of piglets under local anaesthesia. Agrarforschung (Switzerland) 2003 , 10 , 10 - 13. 69. Molony, V.; Kent, J.E. Assessment of acute pain in farm animals using behavioral and physiological measurements. J Anim Sci 1997 , 75 , 266 - 272, doi: 10.2527/1997.751266x. 70. Mellor, D.J.; Stafford, K.J. Acute castration and/or tailing distress and its alleviation in lambs. N Z Vet J 2000 , 48 , 33 - 43, doi:10.1080/00480169.2000.36156. 71. Lomax, S.; Dickson, H.; Sheil, M.; Windsor, P.A. Topical anaesthes ia alleviates short - term pain of castration and tail docking in lambs. Aust Vet J 2010 , 88 , 67 - 74, doi:10.1111/j.1751 - 0813.2009.00546.x. 72. Lomax, S.; Windsor, P.A. Topical anesthesia mitigates the pain of castration in beef calves. J Anim Sci 2013 , 91 , 4945 - 4952, doi:10.2527/jas.2012 - 5984. 73. Hatfield, L.A. Neonatal pain: What's age got to do with it? Surg Neurol Int 2014 , 5 , S479 - 489, doi:1 0.4103/2152 - 7806.144630. 74. McGlone, J.J.; Nicholson, R.I.; Hellman, J.M.; Herzog, D.N. The development of pain in young pigs associated with castration and attempts to prevent castration - induced behavioral

chunk 94 · 450 tokens

; Hellman, J.M.; Herzog, D.N. The development of pain in young pigs associated with castration and attempts to prevent castration - induced behavioral changes. J Anim Sci 1993 , 71 , 1441 - 1446, doi:10. 2527/1993.7161441x. 75. Ison, S.H.; Clutton, R.E.; Di Giminiani, P.; Rutherford, K.M. A Review of Pain Assessment in Pigs. Front Vet Sci 2016 , 3 , 108, doi:10.3389/fvets.2016.00108. 76. Anil, L.; Anil, S.S.; Deen, J. Pain detection and amelioration in anima ls on the farm: issues and options. J Appl Anim Welf Sci 2005 , 8 , 261 - 278, doi:10.1207/s15327604jaws0804_3. 77. Emde, R.N.; Harmon, R.J.; Metcalf, D.; Koenig, K.L.; Wagonfeld, S. Stress and neonatal sleep. Psychosom Med 1971 , 33 , 491 - 497, doi:10.1097/00006 842 - 197111000 - 00002. 78. Gunnar, M.R.; Malone, S.; Vance, G.; Fisch, R.O. Coping with aversive stimulation in the neonatal period: quiet sleep and plasma cortisol levels during recovery from circumcision. Child Dev 1985 , 56 , 824 - 834. 79. McGlone, J.J. A qu antitative ethogram of aggressive and submissive behaviors in recently regrouped pigs. J Anim Sci 1985 , 61 , 559 - 565. 80. Sutherland, M.A.; Bryer, P.J.; Krebs, N.; McGlone, J.J. Tail docking in pigs; acute physological and behavioral responses. Animals (Bas el) 2008 , 2 , 292 - 297. 81. Curatolo, M.; Petersen - Felix, S.; Arendt - Nielsen, L. Sensory assessment of regional analgesia in humans: a review of methods and applications. Anesthesiology 2000 , 93 , 1517 - 1530, doi:10.1097/00000542 - 200012000 - 00025. 82. Ren, K.; Dubner, R. Inflammatory Models of Pain and Hyperalgesia. ILAR J 1999 , 40 , 111 - 118, doi:10.1093/ilar.40.3.111. 83. Fitzgerald, M.; King, A.E.; Thompson, S.W.; Woolf, C.J. The postnatal development of the ventral root reflex in the rat; a comparative in vivo and in vitro

chunk 95 · 85 tokens

erald, M.; King, A.E.; Thompson, S.W.; Woolf, C.J. The postnatal development of the ventral root reflex in the rat; a comparative in vivo and in vitro study. Neurosci Lett 1987 , 78 , 41 - 45, doi:10.1016/0304 - 3940(87)90558 - 1. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1

chunk 96 · 449 tokens

84. Castel, D.; Sabbag, I.; Meilin, S. The effect of local/topical analgesics on incisional pain in a pig model. J Pain Res 2017 , 10 , 2169 - 2175, doi:10.2147/JPR.S144949. 85. Herskin, M.; Rasmussen, J. Pigs in pain - porcine behavioural responses towards mechanical nociceptive stimula tion directed at the hind legs. Journal of Pain 2010 , 1 , 175 - 176. 86. Lomax, S.; Sheil, M.; Windsor, P.A. Impact of topical anaesthesia on pain alleviation and wound healing in lambs after mulesing. Aust Vet J 2008 , 86 , 159 - 168; quiz CE151, doi:10.1111/j.1 751 - 0813.2008.00285.x. 87. Lomax, S.; Sheil, M.; Windsor, P.A. Duration of action of a topical anaesthetic formulation for pain management of mulesing in sheep. Aust Vet J 2013 , 91 , 160 - 167, doi:10.1111/avj.12031. 88. Espinoza, C.; Lomax, S.; Windsor, P. T he effect of a topical anesthetic on the sensitivity of calf dehorning wounds. J Dairy Sci 2013 , 96 , 2894 - 2902, doi:10.3168/jds.2012 - 5954. 89. Espinoza, C.A.; McCarthy, D.; White, P.J.; Windsor, P.A.; Lomax, S.H. Evaluating the Efficacy of a Topical Anesth etic Formulation and Ketoprofen, Alone and in Combination, on the Pain Sensitivity of Dehorning Wounds in Holstein - Friesian Calves. Animal Production Science 2016 , 56 , 1512. 90. Janczak, A.M.; Ranheim, B.; Fosse, T.K.; Hild, S.; Nordgreen, J.; Moe, R.O.; Z anella, A.J. Factors affecting mechanical (nociceptive) thresholds in piglets. Vet Anaesth Analg 2012 , 39 , 628 - 635, doi:10.1111/j.1467 - 2995.2012.00737.x. 91. Guesgen, M.J.; Beausoleil, N.J.; Leach, M.; Minot, E.O.; Stewart, M.; Stafford, K.J. Coding and quantification of a facial expression for pain in lambs. Behav Processes 2016 , 132 , 49 - 56, doi:10.1016/j.beproc.2016.09.010. 92. Dalla Costa, E.; Minero, M.; Le belt, D.; Stucke, D.; Canali, E.;

chunk 97 · 374 tokens

n lambs. Behav Processes 2016 , 132 , 49 - 56, doi:10.1016/j.beproc.2016.09.010. 92. Dalla Costa, E.; Minero, M.; Le belt, D.; Stucke, D.; Canali, E.; Leach, M.C. Development of the Horse Grimace Scale (HGS) as a pain assessment tool in horses undergoing routine castration. PLoS One 2014 , 9 , e92281, doi:10.1371/journal.pone.0092281. 93. Coetzee, J.F. Evaluation of the tr ansmammary delivery of Firocoxib in sows to alleviate pain assocated with piglet castration, teeth clipping and tail docking. ; National Pork Board Iowa: Iowa, USA, 2019. 94. McCafferty, D.J. The value of infrared thermography for research on mammals: previ os applications and future directions. Mammal Review 2007 , 37 , 207 - 233. 95. Roth, J.; Rummel, C.; Barth, S.W.; Gerstberger, R.; Hubschle, T. Molecular aspects of fever and hyperthermia. Immunol Allergy Clin North Am 2009 , 29 , 229 - 245, doi:10.1016/j.iac.200 9.02.005. 96. Olivier, B.; Zethof, T.; Pattij, T.; van Boogaert, M.; van Oorschot, R.; Leahy, C.; Oosting, R.; Bouwknecht, A.; Veening, J.; van der Gugten, J., et al. Stress - induced hyperthermia and anxiety: pharmacological validation. Eur J Pharmacol 2003 , 463 , 117 - 132, doi:10.1016/s0014 - 2999(03)01326 - 8. 97. Takakazu, O.; Kae, O.; Testsuro, H. Mechanisms and mediators of pschological stress - indcued rise in core temperature. Psychosomatic Medicine 2001 , 64 , 476 - 486. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 August 2020 doi:10.20944/preprints202008.0090.v1