Topical wound anaesthesia: efficacy to mitigate piglet castration pain

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AUSTRALIA’S PREMIER VETERINARY SCIENCE TEXT PRODUCTION ANIMALS Topical wound anaesthesia: efficacy to mitigate piglet castration pain PRODUCTION ANIMALS ML Sheil,a* M Chambersb and B Sharpeb Piglet castration is well documented to result in acute pain and Objective There is a critical need for safe and effective analge- stress to the animal.2–5 Globally, it is performed on millions of pig- sic treatments to address pain resulting from surgical husbandry lets annually, in most cases without any analgesia what-so-ever.6 procedures in livestock. Piglet castration results in acute pain Injected local anaesthesia and/or general anaesthesia may be effec- and stress to the animal; however, it is performed globally on millions of piglets annually, often without any analgesia what-so- tive to alleviate pain; however, these methods may have welfare ever. Tri-Solfen® (Animal Ethics Pty Ltd, Yarra Glen, Victoria, impacts in and of themselves (e.g. due to pain of administration, Australia) is a combination local anaesthetic and antiseptic for- stress of double handling or sedation, and delayed recovery)7 and/or mulation which, applied topically to wounds, has proven effec- may be logistically or cost prohibitive due to the requirement for tive, and is registered for use to alleviate pain associated with direct veterinary administration. Nonsteroidal anti-inflammatory castration (and other wounds) in lambs and calves in Australia (NSAID) medications (such as meloxicam) provide some analgesia and New Zealand. It is also reported to be effective to reduce 2–4 h following the procedure,8 but do not address the pain of the pain in piglets following castration. procedure itself or during the first minutes and hours following the procedure, when pain is acute.4,7,9–17 Design

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ain in piglets following castration. procedure itself or during the first minutes and hours following the procedure, when pain is acute.4,7,9–17 Design This randomised, blinded, placebo-controlled study examined the safety and efficacy of the formulation, adminis- The use of topical wound anaesthesia is a new and evolving field tered via an adapted wound instillation method, to control pain that shows promise as an alternative or adjunctive means of deliver- both during and following piglet castration. ing effective pain mitigation to farm animals in these settings.1 Tri- Method Piglets received Tri-Solfen or placebo, instilled to the Solfen® is a combination local anaesthetic and antiseptic formula- wound immediately following skin incision. A 30 s wait period tion which, applied topically to wounds, has proven effective and is was then observed prior to completing castration. Pain mitiga- registered for use to alleviate pain resulting from castration, and tion was assessed by grading nociceptive resistance movements other surgical husbandry wounds, in lambs and calves in Australia and piglet vocal response during castration, as well as by grading and New Zealand.18–21 It is a viscous liquid formulation containing response to mechanical sensory stimulation of the wound (von Lignocaine 5%, Bupivacaine 0.5%, Adrenalin 1: 2000 and Cetrimide Frey and needlestick) following castration. 0.5%. It is generally applied topically to coat a wound immediately postcreation. Designed to provide rapid onset wound anaesthesia Results There was a significant reduction in nociceptive motor and vocal response during castration and in response to with prolonged effect, it is applied once-only at the time of the mechanical sensory wound stimulation up to and including 2 h wound

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nd in response to with prolonged effect, it is applied once-only at the time of the mechanical sensory wound stimulation up to and including 2 h wound creation, following which animals are returned to their dams following castration. There were no adverse events. to recover without further interference. Conclusion Administered via this method, Tri-Solfen is effec- Specific to castration, the product is applied immediately postskin inci- tive to mitigate acute peri-operative castration pain in piglets. sion, but immediately prior to clamping and/or cutting the cord and excising the testes. This is to ensure good coating of the remaining cord Keywords animal welfare; castration; local anaesthesia; pain tissue as well as the cut skin edge, which may both otherwise generate management; pigs pain in the postoperative period. Administered in this fashion, it has Abbreviations AUC, area under the dB/time waveform; NSAID, proven effective to reduce pain in piglets up to 4 h following castra- nonsteroidal anti-inflammatory; NRS, numerical rating scale tion.22 (By contrast, simply spraying lignocaine onto the wound pos- Aust Vet J 2020 doi: 10.1111/avj.12930 tcastration is reported to be ineffective to mitigate pain.15) Sutherland et al16 reported that ‘lying without contact’ (considered a behavioural sign of pain in piglets) was reduced in Tri-Solfen- T here is a critical need for safe and effective analgesic treat- treated piglets, as compared with untreated piglets in the first ments to address pain resulting from surgical husbandry 180 min following castration. However, there was no evidence of a procedures in livestock.1 The challenge is to develop significant impact on pain during the procedure itself, based on options that are safe, practical and sustainable for

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ock.1 The challenge is to develop significant impact on pain during the procedure itself, based on options that are safe, practical and sustainable for on-farm use. In vocal response and cortisol levels. This finding is not unexpected as view of the logistics involved, ideally such products (albeit veteri- topical anaesthetics cannot be expected to mitigate procedural pain nary prescribed) are able to be farmer applied. if applied only immediately prior to the procedure. Nevertheless, the question arises whether, if a longer dwell time is provided, Tri- *Corresponding author., baysheil@bigpond.net.au a Solfen may be effective to mitigate the acute procedural pain that Animal Ethics Pty Ltd, Yarra Glen, Victoria, 3775, Australia; baysheil@bigpond. net.au occurs when subsequently severing the cords. Applied topically to b Invetus Pty Ltd, Armidale, New South Wales, 2350, Australia mucosal tissues, lidocaine has an onset time between 20 and 30 s23 Australian Veterinary Journal © 2020 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. 1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. PRODUCTION ANIMALS suggesting that a relatively short increase in Tri-Solfen dwell time the Hypothalamic–Pituitary–Adrenal axis is triggered by surgical may be effective to anaesthetise the cords. This could be highly bene- incision or bleeding regardless of the presence or absence of pain). ficial, as applying traction and severing the cords are reported to be

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ighly bene- incision or bleeding regardless of the presence or absence of pain). ficial, as applying traction and severing the cords are reported to be PRODUCTION ANIMALS They may be further confounded by the administration of adrena- the most painful parts of the procedure based on motor and vocal lin which is commonly co-administered with local anaesthetics responses.11,13 This study thus examines whether, in the on-farm set- when used in surgical settings. ting, topical wound anaesthesia with Tri-Solfen is effective to provide procedural as well as postoperative pain mitigation for piglets under- Based on these findings, nociceptive motor and vocal responses to going castration if a longer wait time (minimum 30 s) is implemented castration were chosen as primary and secondary efficacy variables between applying the dose and severing the spermatic cords. for assessment of pain mitigation during castration, and nociceptive response to wound sensory stimulation was chosen as a primary effi- We hypothesised that topical wound anaesthesia (with Tri-Solfen) cacy variable to assess pain mitigation following castration. A second applied with a 30 s dwell time would be effective to mitigate pain efficacy variable for pain mitigation following the procedure (postop- both during and following subsequent piglet castration. Our aims erative painrelated behaviour) has also been examined (and con- were to examine evidence of peri-operative pain mitigation. firmed) in a large multi-centre complementary field trial which is reported separately. (This was unable to be assessed in the same At present, there is no single validated parameter for measuring pain in cohort of piglets undergoing wound sensory testing, as this involves piglets. It is generally accepted, however, that

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parameter for measuring pain in cohort of piglets undergoing wound sensory testing, as this involves piglets. It is generally accepted, however, that piglets react to pain in a frequent re-handling, and hence disturbance of piglet behaviour.) number of ways including: physiologically, behaviourally and through resistance movements and vocalisation. An extensive literature review was performed to identify the optimal parameters for assessing pain in Materials and methods piglets during and following castration. This identified: The study was conducted in December 2018 and complied with the 1 Scoring of nociceptive motor (resistance) movements during the following national and international standards: VICH GL9 Good procedure (such as by visual analogue scale or numerical rating Clinical Practice (issued June 2000); APVMA Data Guidelines – Effi- scale [NRS]) consistently identifies a marked increase in castrated cacy and target animal safety general guidelines (Part 8, 01 July piglets that may be ameliorated by anaesthetic treatment.24–27 It is 2014), and in compliance with University of New England Animal thus considered to provide a sound method of documenting pain Ethics Committee approval no. 18-100. and pain alleviation during castration. 2 Analysis of vocal response during the procedure is also a useful Study design parameter for pain assessment, albeit that it is an indirect variable The study was a placebo-controlled clinical efficacy study, using a and confounded by piglet vocal response to restraint and han- randomised blocked design based on body weights measured pre- dling. Results are therefore less sensitive and robust. The most treatment. The experimental unit was the individual animal and the consistent results are seen when piglets are studied in

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sitive and robust. The most treatment. The experimental unit was the individual animal and the consistent results are seen when piglets are studied in acoustically statistical animal was the treatment group. Male piglets (commercial separated environments.13,26–28 hybrid) were confirmed suitable for enrolment into the study if they 3 Direct wound sensory testing is reported as a sensitive and repeat- were between 3 and 7 days of age, in good health. These were individ- able measure of postoperative wound pain and pain alleviation ually identified (uniquely numbered via ear tags), weighed and ranked with anaesthetic treatments in livestock,18–21 and the method has (heaviest to lightest) and the heaviest and lightest piglets were been recently validated in piglets postcastration.22 It is thus con- excluded. The remaining piglets (n = 40, mean weight 2.2 kg, range sidered to provide a sound method of documenting pain and pain [1.6–3.0 kg]) were sequentially grouped into blocks of two animals alleviation following the procedure. and randomly allocated to Groups 1 and 2 from within each block, 4 Behavioural analysis is also a well-established variable for docu- such that each of Groups 1 and 2 contained 20 male piglets and had menting postoperative pain; however, postcastration abnormalities similar group mean and range of body weights, (as assessed and con- in piglet behaviour are subtle and short-lived, and results are incon- firmed using Statistix 10.0 [Analytical Software Inc., Tallahassee, FL, sistent between trials. The most consistent results are found when USA 2010]). On Day 0 after treatment, animals numbered 20, 26 and specific ‘pain-associated behaviours’ are conglomerated to give an 41 were removed from the study due to herniation and subsequently

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umbered 20, 26 and specific ‘pain-associated behaviours’ are conglomerated to give an 41 were removed from the study due to herniation and subsequently over-all ‘pain-associated behaviour’ score, and appear to be greatest replaced with animals numbered 61, 62 and 17, respectively. This re- in the first 30 min, and up to 3 h following castration.7–10,12,14–16 allocation was statistically assessed confirming that the group mean 5 Physiological measures (such as an increase in cortisol or Adreno- body weights were similar across treatment groups. corticotrophic hormone levels), despite being frequently investi- gated, do not provide a reliable method of assessing pain or pain alleviation in piglets undergoing castration. These are indirect Investigational veterinary product markers of pain, which result from activation of the Tri-Solfen Batch No.:181031–1. Composition: 50 g/L Lignocaine Hypothalamic–Pituitary–Adrenal – axis, with cortisol and adrena- hydrochloride, 5 g/L Bupivacaine hydrochloride, 0.048 g/L Adrena- lin release and activation of the sympathetic nervous system. line (as acid tartrate), 5 g/L Cetrimide. While useful in some settings, the literature establishes that these are poor indicators of pain associated with piglet Placebo product castration.4,24,29–31 These measures are severely confounded by the Blue food dye was added to 0.9% saline solution (Baxter, Batch: surgical stress response (a reflex response in which activation of W47P5, Expiry: April 2020) at the rate of 2.5 to 250 mL saline 2 Australian Veterinary Journal © 2020 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. PRODUCTION ANIMALS (matching the investigational veterinary

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lished by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. PRODUCTION ANIMALS (matching the investigational veterinary product colour and hence from the snout. Audio recording was time and date stamped to corre- indistinguishable postapplication). late with video data. The recording period commenced 30 s following PRODUCTION ANIMALS treatment application to the castration wound, which was marked Investigational veterinary product and placebo products were admin- with a verbal cue, and finished with the removal of the second testicle istered via a custom designed 1 mL Tri-Solfen applicator with ball- and settling of the piglet. Sound files were analysed off-line by a point tip fitted (Prodigy Instruments Pty Ltd, Mount Kuring-Gai, sound consultant who was blinded to piglet treatment. For analysis, NSW, Aust), which was verified prior to use. Treatment administra- sound files were downloaded from the Zoom recorder and imported tion (total dose) was: piglets 1–<2 kg – 1 mL; piglets >2–4 kg – 2 mL. to Pro Tools® (Avid technology Inc., Burlington, MA, USA). They Study animals were housed in farrowing pens (2.1 m × 2.1 m) with were then placed on the Pro Tools timeline at their time stamp posi- their dam and litter mates, and routine management practices were tion and synchronised with audio from the video recordings to allow followed. Piglets had constant suckling access to their dam and ad precise identification of cutting points. To allow isolation and com- libitum access to potable water. On the day of the study, piglets were parative quantification of vocal output of piglets during the proce- removed from their dams and castration and treatment was per- dure, the time of commencing traction, and time of cord severance

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lets during the proce- removed from their dams and castration and treatment was per- dure, the time of commencing traction, and time of cord severance formed as detailed in the following. Video and sound recording were were annotated from the time stamp (correlated with the video data). performed during piglet castration, and wound sensory testing was Screen shots were generated with the same time duration window performed 1 min following the procedure. Following this, piglets (x axis) and signal scale (y axis). The Area Under the dB/time (wave- were returned to their dams. Clinical observations of animals were form) Curve (AUC) was then calculated (in pixels) using image anal- performed concurrent with postoperative pain assessments at 1 min, ysis software (Image-J® U. S. National Institutes of Health, Bethesda, thence 1, 2, 4, 8, 12 and 24 h posttreatment. MD, USA), for each of the following periods: (1) from commencing traction until severance of the first spermatic cord, (2) from severance Method of castration and treatment: of the first spermatic cord until settled at the end of the procedure, and (3) total recorded procedure (Figure 1). 1 Restrain piglet gently but firmly in Kerbl piglet castration cradle (Albert Kerbl GmbH, Buchbach, Germany); 2 When settled, incise scrotum (including tunica) with one single Wound sensitivity testing transverse incision using a sterile scalpel to expose and exteriorise The response of each piglet to sensory testing of the wound site was each testis; assessed with a von Frey filament (300 g filament) and thence pin- 3 Immediately apply treatment to wound (40% total dose to each prick (18G, 1.5 inch needle) at 1 min, thence 1, 2, 4, 8, 12 and 24 h side) to coat the exposed spermatic cords; posttreatment, by trained staff,

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each prick (18G, 1.5 inch needle) at 1 min, thence 1, 2, 4, 8, 12 and 24 h side) to coat the exposed spermatic cords; posttreatment, by trained staff, blind to piglet treatment. Each assess- 4 Wait 30 s; ment was scored on a scale of 0–3, based on Lomax et al22: 0 = no 5 Then, remove testes by severing the cords as per routine procedure; motor response; 1 = there was a mild local motor response – including 6 Apply the remaining 20% of the total dose to coat the cut skin a local muscle twitch, flick of the tail or puckering of the anus; 2 = there edge of the wound. was a partial lower body withdrawal response – including lifting the rump from the cradle and/or prominent movements (e.g. extension or escape movements) of the rear legs; 3 = there was a full body with- Video recording during castration A video-camera recording device (iPhone 8 – Apple Inc., Cupertino, drawal response – including lifting the rump from the cradle and tho- CA, USA) was fixed in position above the cradle, and used to record racic movements and/or vigorous movement of both the front and from time of application of the topical wound treatment to approxi- rear legs. Animals were restrained similarly to that used for the castra- mately 5 s following the severance of the second spermatic cord tion procedure in the piglet cradle for these assessments. Points of (i.e. completion of the castration procedure). Each piglet was clearly application of the von-Frey or needle tip for each site included: Cut identified by placing a number label next to the piglet cradle within edge; both lateral aspects of castration wound – 2 sites. Intact skin: the camera view. The behavioural response to castration was assessed dorsal and ventral aspects of the castration wound ~3 mm from cut off-line, by a

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the camera view. The behavioural response to castration was assessed dorsal and ventral aspects of the castration wound ~3 mm from cut off-line, by a blinded assessor, using an NRS intensity scale of 0 to edge – 2 sites. This gave a maximum score of 12 for each test method 2 at each of four time points: (1) traction on first testis, (2) cutting (von Frey or needle) at each time point. first spermatic cord, (3) traction on second testis and (4) cutting sec- ond spermatic cord. The nociceptive motor response was graded as: Statistical methods 0 = no motor response, 1 = mild motor response, such as a short- Raw data were entered into Microsoft EXCEL 2016 using double-entry lived leg extension or kicking but no major body resistance movement techniques. Summary tables were prepared in EXCEL while summary in the cradle, 2 = marked motor response, such as prolonged leg figures were prepared using EXCEL and TIBCO Spotfire S+ 8.2, 2010® movements and/or marked body resistance movement in the cradle. (TIBCO software Inc. Palo Alto, CA, USA). Video (nociceptive motor Assessment thus documented the handling of each teste (traction) and response NRS scores) and Audio AUC data were compared using both cutting of each spermatic cord, giving a score (0–2) for each event, parametric t-tests and the equivalent nonparametric test (Wilcoxon and a combined total score (0–8) for the castration procedure. rank-sum tests); statistical comparisons were performed using S+®. Mechanical sensory response score data were collated by treatment, Audio recording during castration location (cut edge and intact skin), time point and method of stimulus Sound was recorded with a Zoom H2n Handy Recorder (Zoom (von Frey filaments and 18-gauge needles). Total scores for von Frey, North America,

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f stimulus Sound was recorded with a Zoom H2n Handy Recorder (Zoom (von Frey filaments and 18-gauge needles). Total scores for von Frey, North America, Hauppauge, NY, USA), mounted on a stand 50 cm needle-stick and overall were calculated. Scores to 4 h posttreatment Australian Veterinary Journal 3 © 2020 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. PRODUCTION ANIMALS PRODUCTION ANIMALS Figure 1. Example screenshot of dB/time waveform output used for image analysis and calculation of ‘area under the curve’ (AUC) piglet vocal response to castration. AUC was calculated for the following time periods: (1) Commencement of traction on the first spermatic cord until sever- ance of the cord, (2) severance of the first spermatic cord until sound returned to baseline at the end of the procedure, and (3) total of 1 + 2. Table 1. Group mean nociceptive motor response scores from video assessment (numerical rating scale) of motor response to castration procedures in Tri-Solfen or placebo-treated piglets Group Treatment Traction on Cut first Traction on Cut second Total Motor first testis (/2) spermatic cord (/2) 2nd testis (/2) spermatic cord (/2) Response score (/8) 1 Placebo 1.5 2.0 1.6 1.8 6.9 2 Tri-Solfen 0.4 1.4 0.4 1.4 3.7 Figure 3. Box plot of vocal response (area under the dB/time waveform or ‘AUC’) during time period 1 (from commencing traction until sever- Figure 2. Box plot of total nociceptive motor response to castration, ance of the first spermatic cord), showing a significant reduction in showing a significant reduction (P < 0.01) in Tri-Solfen versus placebo- vocal response of Tri-Solfen as compared with placebo-treated pig- treated piglets. lets (P < 0.01).

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eduction (P < 0.01) in Tri-Solfen versus placebo- vocal response of Tri-Solfen as compared with placebo-treated pig- treated piglets. lets (P < 0.01). 4 Australian Veterinary Journal © 2020 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. PRODUCTION ANIMALS the responses to traction on testes (combined) and cutting of sper- matic cords (combined) were significantly lower in Tri-Solfen- PRODUCTION ANIMALS treated piglets (P = 0.000; P = 0.000; P = 0.004, respectively) than in placebo piglets (Figure 2). Audio recording during castration Treatment with Tri-Solfen resulted in a significant reduction in vocal response of piglets to castration as compared with placebo treatment as measured by AUC (mean SD of 5070 5667 versus 12,109 9270 pxls, respectively) during the first recorded time period (from commencing traction until severance of the first sper- matic cord), (P = 0.007, Figure 3). Numerically lower mean AUC values were recorded in Tri-Solfen-treated piglets, throughout the remainder, or whole of the procedure; however, these differences were not statistically significant, due to wider variability (Figure 4). (A sample size of 20 per group predicted a significant [P < 0.05] Figure 4. Box plot of vocal response (area under the dB/time waveform reduction in phase 1 AUC with 83% power, versus 10% power for or ‘AUC’) during time period 2 (from severance of the first spermatic cord until the end of the castration procedure). There is wide variability the whole of procedure AUC assessment.) and results are not significantly different between groups. Wound sensitivity testing were compared by treatment, method and over time using Repeated- Group mean wound sensitivity scores

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fferent between groups. Wound sensitivity testing were compared by treatment, method and over time using Repeated- Group mean wound sensitivity scores by test and time point are Measures Analysis of Variance and Statistix 10.0® (2013, Analytical presented in Table 2 and Figure 5. Placebo-treated animals were Software, Tallahassee, FL, USA). Suitability of the statistical model was more sensitive to both the von Frey filament and needle and at both checked via sphericity assumptions, assumptions of covariance and the cut skin edge and intact skin adjacent to the wound, at 1 min fol- residuals plots. Sphericity assumptions were met and residual plots lowing treatment through to and including 2 h posttreatment. These were generally acceptable although assumptions of covariance were differences were statistically significant at 1 min and 1 h as shown in generally not met. Data were therefore also aggregated up to ‘response Table 3. At 2 h posttreatment, a greater proportion of placebo- to stimuli/no response to stimuli’ based on total score criteria (>2, >3, treated piglets had wound pain response scores greater than >4 and >5) and the proportion of piglets responding to stimuli at each 4 (P = 0.04) or greater than 5 (P = 0.01) than Tri-Solfen-treated pig- time calculated to 8 h posttreatment. Proportions were then compared lets. There were no significant differences between groups at 4–24 h using Chi-Squared tests and Statistix 10.0. following treatment. Clinical observations and adverse effects Results Intestinal herniation (inguinal hernia) occurred in three piglets Video recording during castration requiring their removal from the trial as previously noted. One Tri-Solfen-treated piglets demonstrated lower nociceptive motor placebo-treated piglet was found

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their removal from the trial as previously noted. One Tri-Solfen-treated piglets demonstrated lower nociceptive motor placebo-treated piglet was found dead 24 h posttreatment. Postmor- response scores associated with traction on each teste and cutting of tem examination revealed this to be secondary to haemorrhage from each spermatic cord (Table 1). The total motor response score and the testicular artery stump. There were no other adverse events. Table 2. Group mean response scores from piglets castrated with Tri-Solfen or placebo treatment to stimulation with von-Frey filament (300 g) or needlestick, by test, site and time point Site Test Group Treatment 1 min 1h 2h 4h 8h 12 h 24 h Cut edge Von Frey 1 Placebo 0.41 0.48 0.85 1.05 0.85 0.45 1.05 2 Tri-Solfen 0.05 0.00 0.55 0.85 1.35 0.65 1.10 Needle 1 Placebo 0.91 1.38 1.35 2.90 2.55 1.70 2.63 2 Tri-Solfen 0.19 0.30 0.90 2.45 2.95 2.00 2.25 Intact skin Von Frey 1 Placebo 0.73 0.43 0.50 1.05 0.60 0.60 0.89 2 Tri-Solfen 0.05 0.10 0.25 1.10 1.20 0.80 0.90 Needle 1 Placebo 1.64 1.33 1.95 2.60 2.55 1.40 2.42 2 Tri-Solfen 0.62 0.55 0.95 2.50 2.70 1.90 2.30 Australian Veterinary Journal 5 © 2020 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. PRODUCTION ANIMALS PRODUCTION ANIMALS Figure 5. Group mean total wound stimulation response scores (von- Frey filament [300 g] and needle- stick) from piglets castrated with Tri- Solfen or placebo treatment, by site and time point. Table 3. Statistical analysis of response to von-Frey (300 g) and needle- The results obtained in this trial are considered to indicate that in stick stimulation of the wound following castration in piglets adminis- piglets 3–7 days of age undergoing castration in an

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idered to indicate that in stick stimulation of the wound following castration in piglets adminis- piglets 3–7 days of age undergoing castration in an on-farm setting, tered Tri-Solfen or placebo during castration topical wound anaesthesia with Tri-Solfen, administered to the Total all P-value wound immediately postskin incision, and followed by a minimum 30 s wait period, effects significant pain mitigation during the subse- Group <0.001 quent castration procedure, as well as in the minutes and early hours Time <0.001 following the procedure. Group × time 0.277 The application was able to be incorporated into standard castration Sphericity assumptions √ procedure with only a minimally prolonged handling time of 30 s Covariance assumptions X per piglet. There were no drug-related adverse events reported, con- Group Time point Mean Statistical comparison sistent with previous reports.16,22 (The use of two or more cradles, (values with different with staggered loading and piglet treatment, would negate any addi- letters differ tional handling time for operators). significantly [P < 0.05]) 1. PLACEBO 0 3.79 C In this study, we analysed piglet nociceptive motor and vocalisation 2.TRISOLFEN 0 0.85 D responses as evidence of castration-related pain and pain mitigation. 1. PLACEBO 1 3.63 C The literature identifies a reduction in nociceptive motor response as a 2.TRISOLFEN 1 0.95 D robust and repeatable method of documenting pain alleviation due to 1. PLACEBO 2 4.68 BC local anaesthesia in piglets. Piglet castration without anaesthesia 2.TRISOLFEN 2 2.65 CD induces protracted violent struggling and escape behaviour from pig- lets.24 This motor response, variously termed ‘escape attempts’,32 1. PLACEBO 4 7.84 A ‘defence behaviour’26 or ‘resistance

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escape behaviour from pig- lets.24 This motor response, variously termed ‘escape attempts’,32 1. PLACEBO 4 7.84 A ‘defence behaviour’26 or ‘resistance movements’,27 is usually accompa- 2.TRISOLFEN 4 6.90 AB nied by a loud vocal response and is attributable to the nociceptive withdrawal response to acute pain induced during the procedure. Measured using focal assessment, Visual Analogue Scale or NRS, stud- Discussion ies have consistently reported that piglet motor response to castration is significantly increased in piglets undergoing castration as compared The direct application of topical local anaesthesia to wounds, to pro- with sham-handled controls,32 and/or is significantly ameliorated vide rapid onset wound anaesthesia and postoperative pain mitigation, through the use of general or injected local anaesthetics, indicative of is a new and evolving field. It is particularly relevant to livestock significant pain alleviation.25–27 Our results, confirming a marked and industries, where there is an acute need for safe and effective analgesic highly significant reduction of nociceptive motor response in piglets products that may be administered by farmers to address pain associ- treated with local anaesthetics (as present in Tri-Solfen) applied topi- ated with surgical husbandry procedures in their animals. cally during castration, are consistent with these previous reports, and 6 Australian Veterinary Journal © 2020 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. PRODUCTION ANIMALS indicate that significant procedural pain mitigation is achieved wound anaesthesia or hypoanaesthesia. This has been associated through the use of Tri-Solfen administered via this

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cedural pain mitigation is achieved wound anaesthesia or hypoanaesthesia. This has been associated through the use of Tri-Solfen administered via this method. with reduced postoperative pain-related behaviour in treated animals over the same period.18–22 Most recently, this method has also been PRODUCTION ANIMALS Piglet vocalisation is also a useful indicator of pain. Although piglets validated as a method of pain assessment in piglets postcastration, commonly vocalise when they are handled, and particularly when including efficacy of local anaesthetic (injected and topical) adminis- restrained, the literature shows that, during castration, piglets may tration. The authors concluded: ‘This method provides a direct mea- squeal more often, more loudly and/or at a higher frequency than sure for wound pain and presence or absence of anaesthesia’.22 Our piglets that are only being held.9,11–13,26,33 Pulling and severing of the results indicating relative hypoanaesthesia in Tri-Solfen-treated pig- spermatic cords lead to the greatest vocalisation response.11,13 Piglets lets as compared with placebo-treated control piglets from 1 min up castrated without local anaesthesia produce a higher number of to and including 2 h following application are consistent with those screams with higher frequencies compared with piglets castrated of Lomax et al22 and indicative of significant mitigation of wound with anaesthesia.13,25–28 A caveat, however, is that in most cases, pain in this period. Lomax et al documented a longer duration of these findings have been recorded in rooms acoustically isolated effect (4 h) than was evident in our study (2 h). The dose was the from the farrowing pens and using a variety of different methods. In same in both studies; however, there was

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ent in our study (2 h). The dose was the from the farrowing pens and using a variety of different methods. In same in both studies; however, there was a slight difference in castra- most piggeries, piglet castration is performed in the farrowing pens tion procedure in that Lomax et al used the two (smaller) incision with rapid through-put to minimise handling times and return pig- techniques versus the single (larger) incision technique utilised in lets to the sow as quickly as possible. Piglet vocal response to each our study. It is not known if this may have an impact on postopera- part of the handling and surgical procedures may overlap in this set- tive wound sensitivity, which may be an area for future study. ting, and piglet vocal responses may be impacted by proximity to other piglets and the sow, as well as being confounded by extraneous Postoperative pain-related behaviour was unable to be assessed in noise, including squeals of nearby piglets. Our study was designed to this study, as previously discussed. An additional linked multicentre assess efficacy ‘in-real practice’ and was thus performed in the safety and efficacy trial is reported separately. farrowing room. The method of analysis utilised (area under the dB/time waveform measured in pixels) provided a comparable mea- We did not include a ‘sham’ group in this study. Others have dem- sure of ‘total vocal response’ from each piglet over the selected time onstrated motor, vocal and wound sensory responses in castrated periods. Our result identifying a significant reduction in vocal piglets versus sham animals as documented earlier. This study was response during the time from first traction on the spermatic cord designed to investigate amelioration of pain in response to noxious until

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study was response during the time from first traction on the spermatic cord designed to investigate amelioration of pain in response to noxious until cord severance in Tri-Solfen-treated piglets is consistent with procedures, (cord traction, severing, and so on) which would not be previous reports indicating a reduction in vocal response to castra- assessable in piglets that did not undergo the procedures. Further- tion (and specifically cord traction) in local anaesthetic-treated pig- more, assessments would not be able to be blinded. lets, considered indicative of significant pain mitigation. While we Similarly, we have not compared the use of topical wound anaesthesia did not find the same significant difference when comparing vocal with injected local anaesthesia. Others have demonstrated motor, vocal responses during the remainder or whole of the procedure, this may and wound sensory responses in piglets castrated with and without reflect wider variability in the duration of the total procedure, and/or injected local anaesthesia as documented earlier. The intent of this piglet response to handling over the longer period, and greater study was to investigate topical wound anaesthesia as a stand-alone opportunity for confounding. treatment (such as for use in situations where injected anaesthesia is Direct wound sensory testing was used to assess evidence of pain not available). As both methods of administration result in a significant alleviation following the procedure. Direct sensory testing (such as reduction in vocal and motor responses, it may be concluded that large using von-Frey or needlestick) is a long established and validated numbers would be required to detect a significant difference between method of assessing the efficacy of local

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tick) is a long established and validated numbers would be required to detect a significant difference between method of assessing the efficacy of local anaesthesia and wound the two treatments for these variables. While this may be of interest, analgesia in laboratory research and clinical settings,34 including in ultimately, the greatest point of difference between injected local anaes- pigs. Castel et al35 reported the results of studies using von Frey fila- thesia and topical wound anaesthesia, (such as via application of Tri- ment assessment of nociceptive threshold to assess incision pain in Solfen) stems from the disadvantages of each method of administration. pigs and impact of local anaesthetic infiltration, and reported that Injected local anaesthesia provides preoperative skin anaesthesia; how- the method was repeatable, and sensitive to the effects of local anaes- ever, it requires veterinary presence, induces pain of injection and may thetic agents. Modified techniques have been developed for use in pose risks due to inadvertent intravascular administration. It also livestock species ‘in the field’. In this setting, the nociceptive reflex requires a 3–15 min wait time resulting in the need for, and stress of, response to needlestick and von Frey Filament application to the double handling and separation from the sow. Topical wound anaes- wound has been assessed by grading the vigour of the nociceptive thesia avoids the need for painful injection, double handling and veteri- withdrawal response at different time points following the procedure. nary presence; however, it does not provide anaesthesia for skin Heightened nociceptive motor response has been documented in the incision. Nevertheless, skin incision (performed in a matter of milli-sec-

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a for skin Heightened nociceptive motor response has been documented in the incision. Nevertheless, skin incision (performed in a matter of milli-sec- minutes and hours following castration (and other husbandry proce- onds), is considered the least painful part of the procedure, and, with dures) in lambs and calves, as compared with sham-handled ani- topical wound anaesthesia effective within 30 s, pain is very short-lived. mals, and/or with preoperative assessments). A significant reduction True comparator studies would need to examine the impact of these in nociceptive responses has been documented following Tri-Solfen differences in total, (preprocedure and postprocedure) to gain a true application to wounds, evident within 1–3 min of application, and understanding of comparative piglet welfare. Again, this is considered persisting for hours following procedures, indicative of significant an area for future study. Australian Veterinary Journal 7 © 2020 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. PRODUCTION ANIMALS Additionally, there is the potential for topical wound anaesthesia to 11. Taylor AA, Weary DM Vocal responses of piglets to castration: identifying be used as part of a multimodal approach to pain mitigation in pig- procedural sources of pain. Appl Anim Behav Sci 2000;70:17–26. 12. Taylor AA, Weary DM, Lessard M et al. Behavioural responses of piglets to PRODUCTION ANIMALS lets. Synergistic analgesic efficacy has been reported when Tri-Solfen castration: the effect of piglet age. Appl Anim Behav Sci 2001;73:35–43. is combined with NSAID medications in other species,36 and topical 13. Marx G, Horn T, Thielebein J et al. Analysis of pain-related

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Sci 2001;73:35–43. is combined with NSAID medications in other species,36 and topical 13. Marx G, Horn T, Thielebein J et al. Analysis of pain-related vocalization in wound anaesthesia may provide enhanced postoperative analgesia young pigs. J Sound Vib 2003;266:687–698. 14. Hay M, Vulin A, Génin S et al. Assessment of pain induced by castration in when used in combination with NSAIDs or with general anaesthesia. piglets: behavioral and physiological responses over the subsequent 5 days. These are also areas for future study. Appl Anim Behav Sci 2003;82:201–218. 15. Burkemper MC, Pairis-Garcia MD, Moraes LE et al. Effects of oral meloxicam It is concluded that in piglets 3–7 days of age undergoing castration and topical lidocaine on pain associated behaviors of piglets undergoing surgi- in an on-farm setting, topical wound anaesthesia with Tri-Solfen, cal castration. J Appl Anim Welf Sci 2019:1–10. https://doi.org/10. administered immediately postskin incision followed by a minimum 1080/10888705.2019.1590717. 16. Sutherland MA, Davis BL, Brooks TA et al. Physiology and behavior of pigs before 30 s wait period, effects highly significant pain mitigation during the and after castration: effects of two topical anesthetics. Animal 2010;4:2071–2079. subsequent castration procedure, as well as in the minutes and early 17. Gottardo F, Scollo A, Contiero B et al. Pain alleviation during castration of hours following the procedure. Significant pain control is thus piglets: a comparative study of different farm options. J Anim Sci 2016;94: achieved during the time-periods associated with maximum pain in 5077–5088. 18. Lomax S, Sheil M, Windsor PA Impact of topical anaesthesia on pain allevia- piglets undergoing this procedure. tion and wound healing in lambs

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88. 18. Lomax S, Sheil M, Windsor PA Impact of topical anaesthesia on pain allevia- piglets undergoing this procedure. tion and wound healing in lambs after mulesing. Aust Vet J 2008;86:159–168. 19. Lomax S, Dickson H, Sheil M et al. Topical anaesthesia alleviates short-term pain of castration and tail docking in lambs. Aust Vet J 2010;88:67–74. Acknowledgments 20. Lomax S, Windsor PA Topical anesthesia mitigates the pain of castration in beef calves. J Anim Sci 2013;91:4945–4952. The authors thank Leonora Pearson, DipRQA (Quality Assurance) 21. Cuttance E, Mason W, Yang D et al. Effects of a topically applied anaes- Invetus Pty Ltd, Armidale, NSW 2350, Timothy Pearce, Cert. Audio thetic on the behaviour, pain sensitivity and weight gain of dairy calves fol- Eng. (Sound Analysis), Annandale, Sydney 2038 and BettaPork Pty lowing thermocautery disbudding with a local anaesthetic. N Z Vet J 2019;67: 295–305. Ltd, Valentine Plains Queensland, Australia. 22. Lomax S, Harris C, Windsor PA et al. Topical anaesthesia reduces sensitivity of castration wounds in neonatal piglets. PLoS One 2017;12:e0187988. 23. Adriani J, Zepernick R, Arens J et al. The comparative potency and effective- Conflicts of interest and sources of funding ness of topical anesthetics in man. Clin Pharmacol Ther 1964;5:49–62. 24. Walker B, Jäggin N, Doherr M et al. Inhalation anaesthesia for castration of Research was sponsored by Animal Ethics Pty Ltd, and carried out newborn piglets: experiences with isoflurane and isoflurane/NO. J Vet Med A by independent veterinary research company Invetus Pty Ltd, to Physiol Pathol Clin Med 2004;51:150–154. 25. Horn T, Marx G, von Borell E Behavior of piglets during castration with and GCP and VICH standards, to address national and international vet-

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54. 25. Horn T, Marx G, von Borell E Behavior of piglets during castration with and GCP and VICH standards, to address national and international vet- without local anesthesia. DTW Dtsch Tierarztl Wochenschr 1999;106:271–274. erinary medicines regulatory approval requirements. Dr Sheil is an 26. Leidig MS, Hertrampf B, Failing K et al. Pain and discomfort in male inventor of Tri-Solfen®, a Founding Director and indirect Share- piglets during surgical castration with and without local anaesthesia as holder of Animal Ethics Pty Ltd. determined by vocalisation and defence behaviour. Appl Anim Behav Sci 2009;116:174–178. 27. Hansson M, Lundeheim N, Nyman G et al. Effect of local anaesthesia and/or References analgesia on pain responses induced by piglet castration. Acta Vet Scand 2011; 53:34. 28. White RG, DeShazer JA, Tressler CJ et al. Vocalization and physiological 1. Windsor PA, Lomax S, White P Progress in pain management to improve response of pigs during castration with or without a local anesthetic. J Anim Sci small ruminant farm welfare. Small Ruminant Res 2016;142:55–57. 1995;73:381–386. 2. Prunier A, Bonneau M, Von Borell EH et al. A review of the welfare conse- 29. Kluivers-Poodt M, Hopster H, Spoolder HAM. Castration under anaesthesia quences of surgical castration in piglets and the evaluation of non-surgical and/or analgesia in commercial pig production. Lelystad, Netherlands, Report methods. Anim Welfare 2006;15:277. Animal Sciences Group van Wageningen, 2007;92. http://www.asg.wur.nl. 3. von Borell E, Baumgartner J, Giersing M et al. Animal welfare implications of 30. Bonastre C, Mitjana O, Tejedor MT et al. Acute physiological responses to surgical castration and its alternatives in pigs. Animal 2009;3:1488–1496. castration-related pain in

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jedor MT et al. Acute physiological responses to surgical castration and its alternatives in pigs. Animal 2009;3:1488–1496. castration-related pain in piglets: the effect of two local anesthetics with or 4. Rault J-L, Lay DC, Marchant-Forde JN Castration induced pain in pigs and without meloxicam. Animal 2016;10:1474–1481. other livestock. Appl Anim Behav Sci 2011;135:214–225. 31. Pérez-Pedraza E, Mota-Rojas D, Ramírez-Necoechea R et al. Effect of the 5. Leaders MA, Calculator VS, Network ACA. Welfare implications of swine cas- number of incisions and use of local anesthesia on the physiological indicators tration. Backgrounder, 2013. Available at: https://www.avma.org/KB/Resources/ of surgically-castrated piglets. Int J Vet Sci Med 2018;6:159–164. LiteratureReviews/Pages/Welfare-Implications-of-Swine-Castration.aspx?mode= 32. Marchant-Forde JN, Lay DC, McMunn KA et al. Postnatal piglet husbandry mobile&PF=1 Accessed 3 May 2017]. practices and well-being: the effects of alternative techniques delivered sepa- 6. Fredriksen B, Font I, Furnols M et al. Practice on castration of piglets in rately. J Anim Sci 2009;87:1479–1492. Europe. Animal 2009;3:1480–1487. 33. Weary DM, Braithwaite LA, Fraser D Vocal response to pain in piglets. Appl 7. McGlone JJ, Hellman JM Local and general anesthetic effects on behavior Anim Behav Sci 1998;56:161–172. and performance of two- and seven-week-old castrated and uncastrated pig- 34. Curatolo M, Petersen-Felix S, Arendt-Nielsen L Sensory assessment of lets. J Anim Sci 1988;66:3049–3058. regional analgesia in humans. A review of methods and applications. Anesthesi- 8. Keita A, Pagot E, Prunier A et al. Pre–emptive meloxicam for postoperative anal- ology 2000;93:1517–1530. gesia in piglets undergoing surgical

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