Impact of topical anaesthesia on pain alleviation and wound healing in lambs after mulesing

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avj_285.fm Page 159 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS Impact of topical anaesthesia on pain alleviation and Blackwell Publishing Asia PRODUCTION ANIMALS wound healing in lambs after mulesing S LOMAX,a M SHEILb and P A WINDSORa,c pain management. Whilst the procedures may be justified for Objective To investigate the impact of using the topical animal preventative health or production management reasons, anaesthetic preparation Tri-Solfen® on pain alleviation and there is growing opposition to them, particularly from animal wound healing in lambs undergoing mulesing. advocacy organisations. Increasingly, the infliction of pain is Design Three separate trials, placebo controlled and/or considered to breech acceptable standards of humane animal randomised, were carried out over a 5 month period on three care. Mulesing, the practice of cutting loose folds of skin from mobs of between 60 and 263 merino lambs undergoing routine the breech area of sheep, is the most important recent example. mulesing. Concern for the welfare of lambs undergoing this procedure is Procedure Wound pain was assessed using 10 and 75 g resulting in international retail boycotts of Australian wool. calibrated Von-Frey monofilaments to determine sensitivity to light touch and pain stimulation over a 4 to 8 h period. The ideal long term solution is to develop painless alternatives to Pain-related behaviour was documented by trained, blinded such procedures. In the interim, the development of effective observers using a numerical rating scale. Wound healing rates pain management must be given high priority to decrease animal were determined using scaled digital photography and image suffering. At present however, there is lack of commercially analysis software to calculate contraction

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ned using scaled digital photography and image suffering. At present however, there is lack of commercially analysis software to calculate contraction in wound surface available pain-alleviating options that meet the practical and area 2 and 4 weeks after mulesing. economical constraints of production animal husbandry. Results There was rapid (3 min) and prolonged (up to 8 h) wound analgesia as shown by pain response scores (P ≤ 0.01), Topical local anaesthesia may be well suited to production with absent or significantly diminished primary and secondary animal farming because of its low cost, practicality and ease of hyperalgesia (P ≤ 0.01) and significant reduction in pain-related application. Local anaesthetic agents generally have poor skin behaviour (P < 0.001) in treated versus untreated lambs. In penetrability, which limits their use for pre-procedural skin addition there was improved wound healing in the treated anaesthesia. However, they are well known to be rapidly and lambs (P ≤ 0.05). highly effective when applied to open wounds or mucosal Conclusion Tri-Solfen® effects rapid and prolonged wound tissues4–9 and may therefore provide an effective means of analgesia, reduction in pain-related behaviour and improved providing analgesia in the immediate and intermediate post- wound healing in lambs undergoing routine mulesing, providing procedural period, which is arguably the time of maximal pain effective alleviation of pain associated with routine mulesing in and stress.1,2,10–12 sheep. Key words: lambs, mulesing, analgesia, welfare Recently, Tri-Solfen® (Bayer Animal Health, Gordon, NSW) a Aust Vet J 2008;86:159–168 doi: 10.1111/j.1751-0813.2008.00285.x spray-on topical anaesthetic and antiseptic gel became commercially available for use on

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t Vet J 2008;86:159–168 doi: 10.1111/j.1751-0813.2008.00285.x spray-on topical anaesthetic and antiseptic gel became commercially available for use on farms in Australia. The product was specifically LT Light touch sensation designed for pain management in sheep undergoing surgical P Pain sensation procedures such as mulesing. It contains lignocaine (as the WSA Wound surface area hydrochloride) 40.6 g/L and bupivacaine (as the hydrochloride) 4.5 g/L, adrenaline (as tartrate) 24.8 mg/L and cetrimide 5.0 g/L. It is sprayed directly on the wound immediately after the procedure. A nimal husbandry procedures such as mulesing, tail docking and castration are currently performed on In this paper we report results from a series of trials investigating between 15 to 20 million merino lambs annually in the impact of using Tri-Solfen® topical anaesthesia on pain Australia. These procedures cause acute pain and stress,1,2 alleviation and wound healing rates, as well as weight gain and resulting in significant disruption to normal behaviour.2,3 mortality in lambs undergoing routine mulesing. Despite this, they are currently performed routinely without Methods Three separate studies were performed on lambs of mixed sex a from commercial flocks aged 6 to 12 weeks undergoing routine Faculty of Veterinary Science, University of Sydney, PMB 3, Camden, New South Wales, 2570 mulesing (study 1) or mulesing and marking (including surgical b Animal Ethics Pty Ltd, PO Box 363, Yarra Glen, Victoria 3775 castration, tail docking and ear notching, plus ear tagging and c author for correspondence: pwindsor@camden.usyd.edu.au vaccination) (studies 2 and 3). Lambs were born in both autumn © 2008 The Authors Australian Veterinary Journal Volume 86, No 5, May 2008 159 Journal compilation © 2008

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ies 2 and 3). Lambs were born in both autumn © 2008 The Authors Australian Veterinary Journal Volume 86, No 5, May 2008 159 Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 160 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS and spring of 2006 and were pastured with their dams on two properties, in the southern highlands and central tablelands PRODUCTION ANIMALS of NSW respectively. General management and mulesing On the day of each trial, lambs from each flock were yarded and drafted into a holding yard. They were then selected at random, weighed, ear-tagged and placed in mulesing cradles. Pre-operative skin sensitivity scoring was performed as required (see below). Lambs were then mulesed by accredited mulesing contractor using standard ‘V’ modified mulesing technique. In Trials 2 and 3, lambs were also surgically tail docked and ram lambs were surgically castrated immediately prior to mulesing. After this, lambs were managed according to one of three trial protocols in which outcomes were measured at different time points and varying conditions, as outlined below. Outcomes measured included skin and wound sensitivity, pain-related behaviour, wound healing, weight change and mortality. Assessment of skin and wound sensitivity Von-Frey monofilaments are used to quantitate sensation. They are calibrated to bend at predetermined pressures to provide repeatable light touch (LT) or pain (P) stimulation. These were used to test skin and wound sensitivity to LT (10N monofilament) and P (75N monofilament) stimulation before mulesing and up to 8 h after mulesing at five predetermined sites on the skin of the breech and nine sites on the mulesing wound (Figure 1A and B). Evidence of local anaesthesia, allodynia (pain from stimuli such as

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on the skin of the breech and nine sites on the mulesing wound (Figure 1A and B). Evidence of local anaesthesia, allodynia (pain from stimuli such as light touch that is not usually painful), and/or primary and secondary hyperalgesia (exaggerated response to a painful stimulus directly in the damaged tissues or in surrounding undamaged tissues respectively), was assessed at each site. Typical LT and P induced involuntary reflexes and motor responses in the rump and head were graded by vigour. Rump response scores were graded as follows: 0 = no response; 1 = minor involuntary motor response such as local skin twitch, subcutaneous muscle twitch Figure 1. Diagram of sites of light touch and pain stimulation sensory or anal contraction; 2 = partial rump withdrawal reflex such as testing. A. On skin of the breech; B. Directly on the mulesing wound sites. multiple subcutaneous muscle group contraction and/or lifting of the tail; 3 = full rump withdrawal reflex with lifting of the rump off the cradle. Facial response scores were graded as follows: 0 = no response; 1 = minor facial ‘awareness’ such as eye observations and reports of behavioural changes in response to widening, blinking or nasal flaring; 2 = partial startle reflex of the mulesing and/or castration.1,2 A trained observer (veterinarian or head such as slight lifting of the snout or partial head rotation; 3 experience sheep handler, ‘blind’ to the treatment) was asked to = full startle reflex of the head, resulting in a major movement observe each lamb for a period of time sufficient to determine such as lifting head off the cradle, full head jerk or full head posture, gait and evoked behaviour, then grade each lamb on rotation. Scores for each site were added to achieve a total score a scale from 0 to 3

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ead posture, gait and evoked behaviour, then grade each lamb on rotation. Scores for each site were added to achieve a total score a scale from 0 to 3 where: 0 = no evidence of pain related for each lamb. Total scores were calculated out of 30 for skin behaviour; 1 = mildly abnormal posture, gait or behaviours such sensitivity around the mulesed area, and out of 54 for direct as mild arching of the back without wide rear leg stance or wound sensitivity. extension of back legs, ventral lying with legs partially extended, mild stiffening of gait without overt limping or leg dragging; 2 = Pain-related behaviour moderate abnormalities of posture, gait and behaviours such as; Pain-related behaviour was assessed using a numerical rating statue standing head down with prominent arching of the back, scale at various different time points after mulesing. The scale prominent extension and/or abduction of hind legs, marked was developed based on a combination of previous field agitation with leg stamping, ventral lying with hind legs fully 160 Australian Veterinary Journal Volume 86, No 5, May 2008 © 2008 The Authors Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 161 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS extended, limping or markedly abnormal gait with hind leg 20 m2 pasture covered yards at a minimum of 40 m from the abduction and/or dragging, anorexia and lack of interest in handling yards, for quiet observation and pain-related behaviour feeding; 3 = extreme abnormalities of posture, gait and/or behaviour PRODUCTION ANIMALS scoring. Lambs were then re-yarded and placed in the mulesing such as; rear leg collapse, dog sitting, lateral lying or lying with cradle for 4-hour wound sensitivity assessment, before being head flat,

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mulesing such as; rear leg collapse, dog sitting, lateral lying or lying with cradle for 4-hour wound sensitivity assessment, before being head flat, prominent tremors and shaking, inability to stand and/ returned to their dams and turned out to pasture. Lambs were or marked and unusual leaning. re-yarded 2 and 4 weeks later when wounds were examined and re-photographed. Wound healing Wound healing was assessed by mapping wound surface area Trial 2 This trial examined wound sensitivity to P stimulation (WSA) using digital photography and Scion Image PC® digital and pain-related behaviour in 80 lambs undergoing both image analysis software (National Institute Health, USA). Digital mulesing and marking. The first 24 lambs were allocated to photographs were taken with a linear scale held against the wool wound sensitivity testing. General management with mulesing immediately above the wound. Using the image analysis was as above. Alternate lambs were treated with Tri-Solfen® or software, pixels per cm2 were calculated, then the wound outline remained untreated. Spinosyn (Extinosad®, Elanco Animal was mapped and surface area was calculated in cm2. Two Health, Macquarie Park, NSW) with blue food dye added to recordings were made for each image by two separate assessors achieve blinding was applied according to recommendations as a and the results were averaged to provide the final surface area flystrike preventative treatment. Lambs remained in the cradle measurement. Wounds were photographed at the time of for 3 min after which skin and wound sensitivity was performed. mulesing (before application of any treatment) and 14 and 28 They were then housed in a mixed treatment group in an indoor days later. Images were identified by lamb tag number and pen and returned to the

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and 28 They were then housed in a mixed treatment group in an indoor days later. Images were identified by lamb tag number and pen and returned to the mulesing cradle at one and 4 h after assessors were blind to treatment protocol at the time of mulesing for repeat skin and wound sensitivity testing, before performing assessments. being returned to their dams and turned out to pasture. The remaining 56 lambs were identified by a coloured number, and Weights used to assess pain-related behaviour. After mulesing lambs were Weights were recorded at time 0, and day 14 and 28 in Trial 3 randomly allocated to receive Tri-Solfen® or remain untreated using digital scales (Rudweigh®) which were calibrated and (28 lambs in each group) and Spinosyn was applied, as above. zeroed prior to each measurement and accurate to 0.1 kg. Lambs were then moved to 3 × 4 m indoor pens for quiet observation and pain-related behaviour scoring. An additional Trial protocols eight lambs served as unmulesed controls. These were placed in the cradle but remained unmulesed and unmarked. Each pen Trial 1 A placebo controlled trial (n = 60) was initiated to thus contained seven lambs, including six mulesed lambs (mixed examine skin and wound sensitivity to LT and P sensation treated and untreated) and one handled but unmulesed control 3 min and 4 h after mulesing in lambs that were undergoing lamb. Behaviour scoring was performed 5 min after return to the mulesing only. Tail docking and castration of wether lambs pen, and at 1 and 4 h. Lambs were then returned to their dams had been previously performed and wounds were fully healed. and turned out to pasture. Pain-related behaviour was measured 1, 2 and 4 hours after mulesing and wound healing was documented as outlined Trial 3 This trial examined

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pasture. Pain-related behaviour was measured 1, 2 and 4 hours after mulesing and wound healing was documented as outlined Trial 3 This trial examined wound sensitivity, wound healing above. Lambs were managed in 4 sequential groups (15 in each and weight change in a mob of 263 lambs undergoing mulesing group): Group 1 = mulesing ‘control’ group (no post mulesing and marking. Wound and skin sensitivity to LT and P sensation treatment applied); Groups 2 to 4 were treatment groups. was assessed in the first 24 lambs with an extra assessment Each treatment group received one of three post-mulesing 8 hours after mulesing. General management with mulesing treatments (A, B or C) which were spray-on gels applied by occurred as above. Alternate lambs were then either treated with metered dose immediately after mulesing. Treatments A and C Tri-Solfen® or remained untreated. Dicyclanil (Clik®, Novartis consisted of Tri-Solfen® and B was placebo gel. They were Animal Health Australasia Pty Ltd, North Ryde, NSW) was used applied directly to the wound using the commercially supplied to prevent flystrike (8 mL applied to wool around the wound). Tri-Solfen® metered dose applicator, in doses of 6 to 12 mLs Lambs remained in the cradle for 3 min after which skin and based on lamb weight, according to manufacturer recommenda- wound sensitivity was assessed. They were then kept in a pasture tions for Tri-Solfen®. The placebo was visually indistinguishable covered holding yard and returned to the mulesing cradle at 4 from Tri-Solfen® and consisted of the blue spray-on gel base and 8 h after mulesing for repeat skin and wound sensitivity of the Tri-Solfen® formulation with no active anaesthetic or testing, before being returned to their dams and turned out to vasoconstrictor

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ensitivity of the Tri-Solfen® formulation with no active anaesthetic or testing, before being returned to their dams and turned out to vasoconstrictor ingredients. The three spray-on gels were pasture. The remaining 239 lambs were weighed, and then each provided from the manufacturer in identical 1 L containers alternate lamb was treated with Tri-Solfen®. Mulesing wounds marked only as A, B or C. After mulesing and wound photography, were photographed prior to application of Tri-Solfen in the first lambs remained in the mulesing cradle for 3 min after which 100 of these lambs. All lambs were treated with Dicyclanil then skin and wound sensitivity scoring was performed. Lambs returned to dams on pasture. Lambs and ewes were re-yarded were then moved in their treatment groups to one of four and drafted 14 and 28 days later. Wounds initially photographed © 2008 The Authors Australian Veterinary Journal Volume 86, No 5, May 2008 161 Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 162 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS were re-photographed and lambs were weighed then returned to pasture. PRODUCTION ANIMALS Statistical analysis Data were analysed using SPSS version 14.0® (SPSS Inc. Chicago, Illinois). Boxplots were examined and one-way analysis of variance was used to measure the short term effects of treatment groups for which there was a reasonable amount of variation. Repeated measures analysis of variance was used to examine within-subject changes over time. Breech skin sensitivity scores before mulesing were adjusted to account for the lower number of testing sites (5) when used for comparison with post mulesing scores from the direct wound assessment sites (9). The average score per site was calculated prior to

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(5) when used for comparison with post mulesing scores from the direct wound assessment sites (9). The average score per site was calculated prior to mulesing and multiplied by 9. Where there were significant differences in baseline measurements ANCOVA/ regression was used to adjust for these differences. Post-hoc pair-wise comparisons are reported for analyses in which the between group comparison was significant at P < 0.05. Multiple linear regression was used to examine relationships between contiguous variables. Results Weather conditions In Trial 1, weather was cool to cold and dry at 5 to17°C with no fly activity. In Trial 2, weather was warm and wet, with heavy rain within 24 h with moderate fly activity. In Trial 3, weather was hot and dry at 27 to 33°C with extremely high fly activity, resulting in seven lambs found with fly-strike prior to mulesing, and these were excluded from the trial. Lamb weights Mean initial body weight in Trial 1 was 13.1 ± 2.5 kg, Trial 2 was 16.8 kg ± 4 kg and Trial 3 was 14.2 ± 2.6 kg. In Trial 3, weights were recorded Day 0, 14 and 28 on the mob of 263 lambs. Seven flyblown lambs on day 0 were excluded from further recordings, and 14 of the remaining 256 lambs either died or were missing Figure 2. Graphs showing mean total response score (± SE) to light touch for one or more follow up recordings. Weight data was therefore of the wound with a 10N Von Frey filament at various time points before and available for 242 lambs, 121 untreated and 121 Tri-Solfen® after mulesing. A. Trial 1, with results to 4 h post mulesing; B. Trial 3 with results to 8 h post mulesing. treated. There was a mean weight gain of 600 g after 2 weeks (both groups), and 1.3 and 1.4 kg after 4 weeks in untreated and Tri-Solfen® treated lambs respectively.

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was a mean weight gain of 600 g after 2 weeks (both groups), and 1.3 and 1.4 kg after 4 weeks in untreated and Tri-Solfen® treated lambs respectively. presumed to have either died or escaped and mixed into a mob Morbidity and mortality in large adjoining paddock. There were no clinical signs consistent with lignocaine-induced neuro or cardiotoxicity in any lambs treated with Tri-Solfen®. Response to LT and P stimulation of the wound and There was zero mortality in Trial 1. In Trial 2, lambs faced an surrounding skin unexpected major Haemonchus contortus burden post mulesing Pre mulesing There was very little response to LT or P stimulation with anaemia and anorexia. Weight and mortality data of intact skin of the breech prior to mulesing. Mean response were therefore not pursued. In Trial 3, 10 of 263 lambs were scores from the five testing sites (maximum possible score of 30) confirmed to have died during the 4 week trial period (mortality were ≤ 0.1 ± 0.4 for LT and ≤ 1.9 ± 3.4 for P, across all three 3.8%). Of these two had been flyblown on day 0, two were Tri- trials (Figures 2, 3 and 4). There were no significant differences Solfen® treated, two were untreated and four were unable to be between groups within each trial. identified due to loss of ear tags. In addition seven lambs were Post mulesing Mulesed, untreated sheep. These demonstrated missing (three treated, three untreated, one flyblown day 0), increasing allodynia and primary hyperalgesia with a significant 162 Australian Veterinary Journal Volume 86, No 5, May 2008 © 2008 The Authors Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 163 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS PRODUCTION ANIMALS Figure 4. Graph showing mean total response score (± SE) to

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avj_285.fm Page 163 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS PRODUCTION ANIMALS Figure 4. Graph showing mean total response score (± SE) to pain stimula- tion of the skin around the mulesed area with a 75N Von Frey filament before mulesing and 4 h and 8 h after mulesing. (P ≤ 0.003, Figure 4). Maximal hyperalgesic responses to P stimulation were elicited from the tail and/or the right and left cut skin edges proximal to the tail in all three trials. The central body of the wound distal to the tail was relatively insensitive with maximum mean P response scores on the right and left ≤ 0.7 at all time points in all three trials. Figure 5 show mean response scores to P stimulation at each testing site, at each of the different time points in Trial 3. Tri-Solfen® treated sheep Primary allodynia, and primary and secondary hyperalgesia were either absent (Trials 2 and 3) or significantly reduced (Trial 1) in Tri-Solfen® treated lambs over the 4 to 8 hour monitored periods. In Trials 2 and 3 there was no significant change in response to LT and P stimulation over time. In Trial 1, LT and P response scores were significantly below those of untreated (P < 0.001 for LT, and P ≤ 0.01 for P) or placebo treated (P < 0.001 and P = 0.002, respectively) lambs (Figures 2 to 4, Table 1). Placebo gel treated sheep (Trial 1) The LT responses were similar to untreated sheep and significantly greater than Tri-Solfen® treated sheep (P < 0.001) (Figures 2 to 4, Table 1). Figure 3. Graphs showing mean total response score (± SE) to pain stimu- lation of the wound with a 75N Von Frey filament at various time points Response scores to P stimulation were lower than in untreated before and after mulesing. A. Trial 1; B. Trial 2 with results to 4 h post sheep (P = 0.014)

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onse scores to P stimulation were lower than in untreated before and after mulesing. A. Trial 1; B. Trial 2 with results to 4 h post sheep (P = 0.014) however were significantly higher than in mulesing; C. Trial 3 up to 8 h post mulesing. Tri-Solfen® treated sheep (P < 0.001). Hyperalgesia to P stimu- lation in the skin around the mulesed area was similar to untreated sheep and significantly higher than Tri-Solfen® increase in response over time to LT (P ≤ 0.001 in Trials 1 and 3, treated sheep (P = 0.002). Figure 2) and P stimulation (P < 0.001, Trials 1 and 3, and P = 0.01 Trial 2, Figure 3). Untreated sheep also demonstrated Pain-related behaviour increasing secondary hyperalgesia, with an increase in response Results are summarised in Figure 6 and Table 2. There was a to P stimulation of intact skin around the mulesed area over time significant increase in pain-related behaviour scores between 1 © 2008 The Authors Australian Veterinary Journal Volume 86, No 5, May 2008 163 Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 164 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS PRODUCTION ANIMALS Figure 5. Colour coded representation of the mean motor response score at each testing site to pain stimulation with a 75N Von Frey monofilament, before and at times up to 8 hours after mulesing in Trial 3 lambs, comparing untreated lambs with those treated with Tri-Solfen®. Table 1. Between subject effects and pairwise comparisons of response to light touch (LT) and pain (P) stimulation of wound and surrounding skin follow- ing mulesing comparing untreated lambs with those treated with Tri-Solfen (Trials 1–3) and placebo gel (Trial 1) Group 1 Group 2 LT response score wound P response score wound P response score Peri-mules skin Mean

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Tri-Solfen (Trials 1–3) and placebo gel (Trial 1) Group 1 Group 2 LT response score wound P response score wound P response score Peri-mules skin Mean Diff (1-2) SE p group Mean Diff (1-2) SE p group Mean Diff (1-2) SE p group Trial 1 mules Tri-Solfen 1.58 0.28 < 0.001 8.10 0.81 <0.001 1.38 0.37 < 0.001 mules placebo 0.54 0.32 0.10 2.36 0.93 0.014 0.2 0.43 0.64 placebo Tri-Solfen 1.04 0.28 < 0.001 5.83 0.81 <0.001 1.18 0.37 0.002 Trial 2 mules Tri-Solfen 8.03 2.59 0.005 Trial 3 mules Tri-Solfen 1.72 0.22 < 0.001 7.73 1.01 0.01 3.8 0.78 0.012 and 2 4 hours after mulesing (Trial 1), and between 5 minutes untreated sheep (P = 0.01, Trial 1), although this was less and 1 hour after mulesing and marking (Trial 2) with a prominent than in Tri-Solfen® treated sheep. significant group effect (P < 0.001 and P = 0.055 Trials 1 and 2 respectively). Tri-Solfen® treated sheep demonstrated significantly Wound healing lower pain–related behaviour scores compared with placebo gel Trial 1 Despite equivalent body weights, mean initial WSA was treated (P = 0.03, Trial 1), and untreated mulesed lambs (P < significantly smaller in untreated than in placebo (P = 0.002) 0.001 Trial 1, P = 0.05, Trial 2) and were not significantly and Tri-Solfen® treated (P = 0.03) lambs (Figure 7). At day 14 different from unmulesed controls (Trial 2). Placebo gel treated mean WSA was not significantly different between the groups. sheep also had lower pain-related behaviour scores than Using repeated measures analysis there was a significant group 164 Australian Veterinary Journal Volume 86, No 5, May 2008 © 2008 The Authors Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 165 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS Table 2. Between subject effects and

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© 2008 Australian Veterinary Association avj_285.fm Page 165 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS Table 2. Between subject effects and pairwise comparisons of pain-related behaviour (mean numerical rating scale scores) following mulesing, comparing untreated mulesed lambs, with: mulesed lambs treated with PRODUCTION ANIMALS Tri-Solfen (Trials 1 and 2); placebo gel (Trial 1); or unmulesed controls (Trial 2) Group 1 Group 2 Numerical rating scale score Mean Difference (1-2) Std Error p value Trial 1 mules placebo gel 0.5 0.19 0.01 mules Tri-Solfen 0.95 0.19 < 0.001 placebo gel Tri-Solfen 0.45 0.19 0.03 Trial 2 mules control 0.87 0.2 0.001 mules Tri-Solfen 0.78 0.2 < 0.001 control Tri-Solfen −0.09 0.2 0.7 Figure 6. Mean numerical rating scale pain-related behaviour scores at time points up to 4 h following mulesing in lambs comparing untreated lambs with: A. lambs treated with Tri-Solfen® or placebo gel (Trial 1); B. lambs treated with Tri-Solfen® and unmulesed controls (Trial 2). effect, with placebo-gel and Tri-Solfen® treated groups demonstrating faster wound contraction than untreated lambs over the first 14 days after mulesing (P = 0.05). By day 28, all three groups had ≥ 90% of wounds fully healed (mean WSA < 0.7 cm2 in all groups). Mean bare area 28 days after mulesing was 20 ± 5 cm2, and was not statistically different between the groups. There was no significant correlation between lamb weight or initial wound size and bare area size at 28 days. There was no clinical evidence of wound infection or flystrike in any of the wounds during the observed period. Trial 3 Mean initial WSA was not significantly different between untreated and Tri-Solfen® treated lambs. There was a significant effect of treatment group on WSA measurements over time

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gnificantly different between untreated and Tri-Solfen® treated lambs. There was a significant effect of treatment group on WSA measurements over time (P = 0.005). At day 14 and day 28 Tri-Solfen® treated sheep had a significantly smaller average WSA than untreated sheep (P = 0.007 and P = 0.02 respectively, Figure 7). There was positive correlation between lamb weight and WSA on day 0 (R Figure 7. Mean (± SE) mulesing wound surface area (cm2) in Tri-Solfen® = 0.46, P = 0.001) indicating larger initial wound size in larger and placebo treated, and untreated lambs immediately after mulesing and lambs. Mean bare area at day 28 was 13.1 ± 5.5 cm2, and was 14 and 28 days later. © 2008 The Authors Australian Veterinary Journal Volume 86, No 5, May 2008 165 Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 166 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS not significantly different between Tri-Solfen® treated and to an acute painful stimulus is an objective, repeatable and readily untreated lambs. Wound contraction was significantly slower in measurable form of assessing pain and allows the assessor to distinguish between various analgesic interventions.24 Our PRODUCTION ANIMALS Trial 3 than in Trial 1 lambs. findings provide new and important information, particularly Discussion regarding the onset, evolution and distribution of pain from Results from our trials indicate that highly significant alleviation mulesing wounds. of pain and improved wound healing can be achieved in lambs after mulesing, with and without tail docking, using a Our results indicate that mild hypersensitivity to LT and P commercially available topical anaesthetic and antiseptic spray-on stimulation is evident within the wound within in the first few gel

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ity to LT and P commercially available topical anaesthetic and antiseptic spray-on stimulation is evident within the wound within in the first few gel preparation. This has a major potential welfare benefit for minutes after mulesing. This is followed by increasing allodynia, lambs having this the procedure. and primary and secondary hyperalgesia which escalates in the ensuing 8 hours. These findings are consistent with published The safety and efficacy profiles of local anaesthetic agents are studies on pain from skin incisions and open wounds.28–31 well described in sheep.13–16 Lignocaine is highly effective as a local anaesthetic agent when administered using infiltrative Local anaesthetic agents act directly on nerve tissue to reversibly techniques in lambs undergoing castration and tail docking,17,18 block conduction of signals responsible for the sensation of pain. however, there is little information regarding the use of topical By blocking the initial nerve fibre signals local anaesthetics not anaesthesia in lambs. Tri-Solfen® contains lignocaine, bupi- only effect wound anaesthesia, but can also prevent or reduce the vacaine and adrenalin in doses equivalent to formulations that subsequent pain escalation response. This occurs even when local have been used to provide effective topical wound anaesthesia anaesthetics are administered after the incision,27,29,32 a finding and haemostasis in humans.4,5 The combination of lignocaine supported by our results. and bupivacaine is designed to provide rapid onset local anaesthesia There appeared to be a moderate pain alleviating effect of the of prolonged duration. The addition of adrenalin is designed to placebo gel. This consisted of the gel base of the Tri-Solfen® achieve haemostasis and to intensify and

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tion. The addition of adrenalin is designed to placebo gel. This consisted of the gel base of the Tri-Solfen® achieve haemostasis and to intensify and prolong the local effect formulation without anaesthetics or adrenalin. It is possible that of the anaesthetic actives by reducing systemic absorption. the gel base had an independent intrinsic analgesic effect by Recently, Paull et al reported a reduction in peak cortisol forming a barrier over the surface of the wound. Barrier gels response and in pain-related behaviour post-mulesing, in lambs and creams have been shown to provide an analgesic effect when treated with Tri-Solfen®,20 providing the first documented applied to open wounds, by coating denuded nerve endings and evidence that topical anaesthesia may provide an effective providing a barrier against on-going environmental exposure and pain-alleviation strategy for lambs undergoing mulesing. These touch stimulation.33,34 results are supported and enhanced by our own findings. Another important finding was that the cut skin edge was more Documenting the efficacy of pain relief in animals can be difficult, sensitive than the body of the wound, particularly in close especially in lambs where behavioural responses to pain can be proximity to the tail. This suggests that the highest nerve fibre subtle and conflicting. We elected to combine behavioural density exists in this area and highlights the importance of ensuring observations with direct wound sensory testing, rather than adequate cover of the area with the anaesthetic agent. measurement of biochemical or physiological responses, as these provide an indirect assessment of pain and are readily We examined pain-related behaviour in lambs using a numerical confounded by non-pain related variables such as

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rect assessment of pain and are readily We examined pain-related behaviour in lambs using a numerical confounded by non-pain related variables such as handling, rating scale. These are subjective and can lack sensitivity, but are stress and wounding. Cortisol levels, for example, rise during commonly used for grading pain behaviour35–37 and have been surgical procedures even when pain is completely abolished by used in many pain trials.38–41 We attempted to limit subjectivity general anaesthesia.21,22 This is because cortisol plays an and reduce potential bias by using a single observer blinded to important role in maintaining blood volume, mediating the treatment protocol and having a clearly defined scale based on inflammatory response and facilitating wound healing so that abnormal postures and behaviours that have previously been levels may rise even if pain is absent.23 This may explain the defined in lambs after mulesing.1,2,11,20 In addition we examined findings of Paull et al,20 who reported that treatment of lambs lambs grouped according to treatment (Trial 1) and in mixed with non-steroidal inflammatory drugs resulted in a significant treatment groups (Trial 2), and included placebo gel and reduction in post-mulesing pain-related behaviour, but not in a unmulesed control groups. Our results, indicating that Tri-Solfen® corresponding reduction in cortisol response. treatment significantly reduced or abolished wound pain and pain related behaviour in the first 4 h after mulesing concurs Similar issues limit the reliability of parameters such as heart with and supports the recent findings of Paull et al.20 Our findings rate, blood pressure or endorphin release, for estimating pain of absent or significantly reduced wound pain in treated lambs alleviation in

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findings rate, blood pressure or endorphin release, for estimating pain of absent or significantly reduced wound pain in treated lambs alleviation in this setting. In addition Tri-Solfen® contains 8 hours after mulesing are also consistent with the findings of adrenalin, which can have important and confounding effects on Paull et al20 who reported a significant reduction in pain related such physiological parameters. behaviour (specifically less stiff walking and less standing with Quantitative sensory testing is a validated technique that is hunched posture compared with untreated lambs, and normal widely used in scientific literature. Observation of reflex responses feeding) at a similar time point (4 to 8 h post mulesing). 166 Australian Veterinary Journal Volume 86, No 5, May 2008 © 2008 The Authors Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 167 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS In our trials, pain-related behaviour was greater, and earlier in being mulesed and tail-docked, than in older weaned lambs onset, in Trial 2 than Trial 1 lambs. Observation conditions may being mulesed only.45 Our findings appear to be the reverse. In PRODUCTION ANIMALS have played a role in this finding, as pain-related behaviours may our trials, lambs being mulesed only (Trial 1) had markedly be heightened where animals are held in relatively confined faster wound healing rates than those undergoing tail docking indoor pens as occurred in Trial 2, rather than open paddocks and mulesing (Trial 3), despite larger initial wound size. We removed from visual and auditory stressors as occurred in Trial 1. suspect that seasonal conditions, particularly high fly activity, However, the finding is most likely to reflect that Trial 2 lambs may

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ed in Trial 1. suspect that seasonal conditions, particularly high fly activity, However, the finding is most likely to reflect that Trial 2 lambs may be an important factor in this discrepancy. The flocks that were also undergoing tail docking and castration. Castration is exhibited the most delayed wound healing, both in our trial and commonly performed at the time of mulesing and generates that of Dun and Donnelly,45 were mulesed during hot conditions significant pain unrelated to the mulesing wound. This pain was with high fly activity compared with their counterparts. Of note, not addressed in our trial, and may explain the increased variability slower healing was associated with higher mortality in Trial 3. in post-mulesing pain-related behaviour scores in Trial 2. To These observations suggest that production and welfare benefits minimise the suffering associated with mulesing and marking may be achieved by controlling the seasonal conditions under it will be necessary to develop strategies to deal with castration- which mulesing is performed, and suggest the need for further related pain in wether lambs. Studies investigating the efficacy of study of this important observation. topical anaesthesia for alleviation of castration-related pain have Acute weight loss of up to 10% body weight has been reported begun. in lambs in the first week after mulesing.1,11 This weight loss Wound healing is a critical outcome of the mulesing procedure. was prevented in a group of 21 weaned lambs treated with Wound contraction results in reduction of wrinkle and enlargement Tri-Solfen® (unreported data Thompson, Sheil 2005), but not of the bare area of the breech, which are believed to be the principle in unweaned lambs treated with Tri-Solfen® in the trial by Paull factors

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ut not of the bare area of the breech, which are believed to be the principle in unweaned lambs treated with Tri-Solfen® in the trial by Paull factors that reduce susceptibility to fly-strike. Despite this, there et al21 We showed a mean weight gain 2 and 4 weeks after is currently very little information documenting wound healing mulesing, with no significant difference between treated and patterns after mulesing and our findings contribute important untreated lambs, but no weights were recorded in the first 13 new information. days after mulesing due to the need to minimise wound trauma. Initial wound surface area had an important impact on wound Our finding of absence of clinical cardio and neurotoxicity healing rates with larger wounds taking significantly longer to heal concurs with other trials reporting the safety of relatively high within each trial group. Interestingly, and contrary to expectations, dose topical lignocaine application to peripheral wound sites, initial wound size did not appear to correlate with resulting bare particularly when administered in combination with adrenalin.6–9,46,47 area size in untreated lambs. Together these findings suggest that This is further supported by unpublished data (Thompson, Sheil limiting initial mulesing wound size may result in production 2005) that peak plasma lignocaine levels in Tri-Solfen® treated and welfare benefits without necessarily having a negative effect lambs occurred 30 to 60 minutes after mulesing and remained on bare area enlargement. This may be an important area for 100 times below toxic thresholds despite application of up to future study. twice the recommended therapeutic dose (n = 12). Studies suggest that local anaesthetic infiltration can have delete- In conclusion, the topical

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tudy. twice the recommended therapeutic dose (n = 12). Studies suggest that local anaesthetic infiltration can have delete- In conclusion, the topical anaesthetic and antiseptic formula- rious effects on wound healing after surgical incisions,42 but tion Tri-Solfen®, is effective at alleviating pain and enhancing impaired wound healing has generally not been apparent in wound healing in lambs post-mulesing. These results suggest clinical trials.43 We have documented a significant improvement that if widely adopted, the use of topical anaesthesia has the in wound contraction rates in lambs treated with Tri-Solfen®. In capacity to dramatically reduce the burden of acute animal Trial 1, there was an unanticipated and inadvertent discrepancy husbandry related pain and suffering in young lambs through-out in initial wound size between groups. Nevertheless improved Australia. wound healing with Tri-Solfen® was suggested by finding a sig- nificant group effect over the first 14 day evaluation period. This Acknowledgements This research program was approved by the Animal Ethics was confirmed in the second larger trial in which initial wound Committee of the University of Sydney and conforms to the size bias was eliminated. These results support the conclusions of provisions of the Declaration of Helsinki (2000). The support of Eroglu et al,44 that topical anaesthesia does not impair, and may Mr Steven Burgun and staff at Arthursleigh and Mr Geoff improve wound healing. However, it is probable that actives Moore and staff at Yerilla, the statistical support of Associate other than the local anaesthetic agents in Tri-Solfen®, such as Professor Jennifer Peat PhD and funds for handling equipment the antiseptic and the gel base may be responsible for this effect, from the

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en®, such as Professor Jennifer Peat PhD and funds for handling equipment the antiseptic and the gel base may be responsible for this effect, from the McGarvie Smith Institute is gratefully acknowledged. as improved healing was also documented in placebo (gel base with antiseptic) treated lambs in Trial 1. References 1. Fell LR, Shutt DA. Behavioural and hormonal responses to acute surgical The between-trial wound healing results are interesting in that stress in sheep. Appl Anim Behav Sci 1989;22:283–294. they appear to conflict with results from a previous study which 2. Grant C. Behavioural responses of lambs to common painful husbandry reported improved wound healing in younger unweaned lambs procedures. Appl Anim Behav Sci 2004;87:255–273. © 2008 The Authors Australian Veterinary Journal Volume 86, No 5, May 2008 167 Journal compilation © 2008 Australian Veterinary Association avj_285.fm Page 168 Thursday, April 17, 2008 5:08 PM PRODUCTION ANIMALS 3. Lee C, Fisher AD. The Welfare Consequences of Mulesing Sheep. Aust Vet J 24. Duarte A, Pospisilova E, Reilly E, et al. Reduction of postincisional allodynia 2007;85:89–93. by subcutaneous bupivacaine: findings with a new model in the hairy skin of the 4. Bush S. Is cocaine needed in topical anaesthesia? J Emerg Med rat. Anesthesiology 2005;103:113–125. PRODUCTION ANIMALS 2002;19:418–422. 25. Fu KY, Light AR, Maixer W. Relationship between nociceptor activity, 5. Smith GA, Strausbaugh SD, Harbeck-Weber C, et al. Comparison of topical peripheral edema, spinal microglial activation and long-term hyperalgesia anesthetics without cocaine to tetracaine-adrenaline-cocaine and lidocaine infil- induced by formalin. Neuroscience 2000;101:1127–1135. tration during repair of lacerations: bupivacaine-norepinephrine

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ne-cocaine and lidocaine infil- induced by formalin. Neuroscience 2000;101:1127–1135. tration during repair of lacerations: bupivacaine-norepinephrine is an effective 26. Brennan TJ, Umali EF, Zahn PK. Comparison of pre-versus post-incision new topical anesthetic agent. Pediatrics 1996;97:301–307. administration of intrathecal bupivacaine and intrathecal morphine in a rat model 6. Argov S, Levandovsky O. Local anaesthesia in anal surgery: a simple, safe of postoperative pain. Anesthesiology 1997;87:1517–1528. procedure. Am J Surg 2006;191:111–113. 27. Dahl JB, Brennum J, Arendt-Nielsen L, Jensen TS, Kehlet H. The effect of 7. Brofeldt B, Cornwell P, Doherty D, Batra K, Gunther R. Topical lidocaine in the pre-verses post-injury infiltration with lidocaine on thermal and mechanical treatment of partial-thickness burns. J Burn Care Rehabil 1989;10:63–68. hyperalgaesia after heat injury to the skin. Pain 1993;53:43–51. 8. Jellish WS, Gamelli RL, Furry PA, McGill VL, Fluder EM. Effect of topical local 28. Hardie EM. Recognition of pain behaviour in animals. In: Hellebrekers L, anesthetic application to skin harvest sites for pain management in burn patients editor. Animal Pain. Van der wees, Utrecht, Netherlands, 2000:51–69. undergoing skin-grafting procedures. Ann Surg 1999;229:115–120. 29. Pogatzki EM, Vandermeulen EP, Brennan TJ. Effect of plantar local anes- 9. Johnstone RE, Wax MK, Bishop DJ, Chafin JB. Large doses of topical thetic injection on dorsal horn neuron activity and pain behaviours caused by lidocaine during microvascular surgery are not associated with toxic blood incision. Pain 2002;97:151–161. concentrations. Anesthesiology 1995;82:593–596. 30. Meyer RA, Ringkamp M, Campbell JN, Raja SN. Neural mechanisms of 10. Shutt DA, Fell LR,

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7:151–161. concentrations. Anesthesiology 1995;82:593–596. 30. Meyer RA, Ringkamp M, Campbell JN, Raja SN. Neural mechanisms of 10. Shutt DA, Fell LR, Connell R, et al. Stress-induced changes in plasma hyperalgesia after tissue injury. J Hopkins Apl Tech D 2005;26:56–66. concentrations of immunoreactive beta-endorphin and cortisol in response to 31. Wall PD. The painful consequence of peripheral injury. J Hand Surg routine surgical procedures in lambs. Aust J Biol Sci 1987;40:97–103. 1984;9:37–39. 11. Chapman RE, Fell LR, Shutt DA. A comparison of stress in surgically and 32. Lam KW, Pun TC, Ng EH, Wong KS. Efficacy of preemptive analgesia for non-surgically mulesed sheep. Aust Vet J 1994;71:243–247. wound pain after laproscopic operations in infertile women: a randomised, 12. Jongman EC, Morris JP, Barnett JL, Hemsworth PH. EEG changes in 4- double blind and placebo control study. BJOG 2004;111:340–344. week-old lambs in response to castration, tail docking and mulesing. Aust Vet J 33. Emflorgo CA. The assessment and treatment of wound pain. J Wound Care 2000;78:339–343. 1999;8:384–385. 13. Morishima HO, Pedersen H, Finster M, et al. Toxicity of lidocaine in adult, 34. Bose B. Burn wound dressing with human amniotic membrane. Ann Roy Coll newborn, and fetal sheep. Anesthesiology 1981;55:57–61. Surg 1979;61:444–447. 14. Rutten AJ, Nancarrow C, Mather LE, et al. Hemodynamic and central 35. Mathews K. Pain Assessment and General Approach to Management. Vet nervous system effects of intravenous bolus doses of lidocaine, bupivacaine, and Clin N Am–Small Anim Pract 2000;30:734–755. ropivacaine in sheep. Anesth Analg 1989;69:291–299. 36. Anil S, Anil L, Deen J. Challenges of pain assessment in domestic animals. J 15. Feldman HS, Dvoskin S, Halldin

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eep. Anesth Analg 1989;69:291–299. 36. Anil S, Anil L, Deen J. Challenges of pain assessment in domestic animals. J 15. Feldman HS, Dvoskin S, Halldin MH, Ask AL, Doucette AM. Comparative Am Vet Med Assoc 2002;220:313–318. local anesthetic efficacy and pharmacokinetics of epidurally administered 37. Hartrick C, Kovan J, Shapiro S. The numeric rating scale for clinical pain ropivacaine and bupivacaine in the sheep. Region Anesth 1997;22:451–460. measurement: A ratio measure? Pain Pract 2003;3:310–316. 16. Huang YF, Pryor ME, Mather LE, Veering BT. Cardiovascular and central 38. Kent JE, Meikle L, Molony V, McKendrick IJ. Qualitative versus quantitative nervous system effects of intravenous levobupivacaine and bupivacaine in assessment of an acute pain in lambs. Proc of Meetings: Sheep Vet Soc sheep. Anesth Analg 86(4):797–804, 1998 Apr 1998;86:797–804. 2001;25:65–66. 17. Wood GN, Molony V, Fleetwood-Walker SM, Hodgson JC, Mellor DJ. Effects 39. Kent JE, Thrusfield MV, Molony V, Hosie BD, Sheppard BW. A randomised, of local anesthesia and intravenous naloxone on the changes in behaviour and controlled field trial of two new techniques for castration and tail docking of lambs plasma concentrations of cortisol produced by castration and tail docking with less than two days of age. Vet Rec 2004;154:193–200. tight rubber rings in young lambs. Res Vet Sci 1991;51:193–199. 40. Carpenter R, Wilson D, Evans A. Evaluation of intraperitoneal and incisional 18. Dinniss AS, Mellor DJ, Stafford KJ, Bruce RA, Ward RN. Acute cortisol lidocaine or bupivacaine for analgesia following ovariohysterectomy in the dog. responses of lambs to castration using a rubber ring and/or a castration clamp Vet Anaesth Analg 2004;31:46–52. with or without local anaesthetic. NZ Vet J

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nses of lambs to castration using a rubber ring and/or a castration clamp Vet Anaesth Analg 2004;31:46–52. with or without local anaesthetic. NZ Vet J 1997;45:114–121. 41. Thornton PD, Waterman-Pearson AE. Quantification of the pain and distress 19. Mellor DJ, Stafford KJ. Acute castration and/or tailing distress and its allevia- response to castration in young lambs. Res Vet Sci 1999;66:107–118. tion in lambs. NZ Vet J 2000;48:33–43. 42. Brower MC, Johnson ME. Adverse effects of local anesthetic infilitration on 20. Paull DR, Lee C, Colditz IG, Atkinson SJ, Fisher AD. The effect of a topical wound healing. Region Anesth Pain M 2003;28:233–240. anaesthetic formulation, systemic flunixin and carprofen, singly or in combination, 43. Dahl JB, SM, Kehlet H. Wound infiltration with local anesthetics for postoper- on cortisol and behavioural responses of Merino lambs to mulesing. Aust Vet J ative pain relief (review). Acta Anaesthesiol Scand 1994;38:7–14. 2007;85:98–106. 44. Eroglu E, Eroglu F, Agalar F, et al. The effect of lidocaine/prilocaine cream on 21. Hughan SC, Loose JM, Caddy DJ, et al. Combined xylazine and ketamine as an experimental wound healing model. Eur J Emerg Med 2001;8:199–201. an analgesic regimen in sheep. Aust Vet J 2001;79:207–211. 45. Dun RB, Donnelly FB. Effectiveness of the Mules operation at lamb marking. 22. Fox SM, Mellor DJ, Firth EC, Hodge H, Lawoko CR. Changes in plasma Aust J Exp Agric Anim Husb 1965;5:6–10. cortisol concentrations before, during and after analgesia, anaesthesia and 46. Karatassas A, Morris G, Slavotinek AH. The relationship between regional anaesthesia plus ovariohysterectomy in bitches. Res Vet Sci 1994;57:110– blood flow and absorption of lignocaine. Aust NZ J Surg 1993;63:766–771. 118. 47. Palve H,

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lus ovariohysterectomy in bitches. Res Vet Sci 1994;57:110– blood flow and absorption of lignocaine. Aust NZ J Surg 1993;63:766–771. 118. 47. Palve H, Kirvela O, Olin H, Syvalahti E, Kanto J. Maximum recommended 23. Grose R, Werner S, Kessler D, et al. A role for endogenous glucocorticoids doses of lignocaine are not toxic. Br J Anaesth 1995;74:704 –705. in wound repair. EMBO Rep 2002;3:575–582. (Accepted for publication 11 March 2008) 168 Australian Veterinary Journal Volume 86, No 5, May 2008 © 2008 The Authors Journal compilation © 2008 Australian Veterinary Association