The effect of a topical anaesthetic formulation, systemic flunixin and carprofen, singly or in combination, on cortisol and behavioural responses of Merino lambs to mulesing

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PRODUCTION ANIMALS The effect of a topical anaesthetic formulation, systemic flunixin and carprofen, singly or in combination, on cortisol and behavioural responses of Merino lambs to mulesing DR PAULL, C LEE, IG COLDITZ, S$] ATKINSON and AD FISHER CSIRO Livestock Industries, FD McMaster Laboratory, Armidale NSW 2350 Andrew. Fisher@csiro,au } { ulesing is a surgical procedure in which two strips of Objective To determine the pain responses of lambs to mules- skin are cut from the hindquarters of Merino lambs in ing, and the effectiveness of potential analgesic treatments. order to remove wool-bearing wrinkled skin, increase Procedures Merino lambs (n = 64) were allocated at 5 weeks the perineal bare area and reduce the risk of breech strike of age to eight treatment groups: 1) sham mules; 2) conventional throughout life thereafter. The operation is usually performed mules; 3) topical anaesthetic, incorporating lignocaine, bupi- in conjunction with tail docking during the first 12 weeks after vicaine, adrenaline and cetrimide, applied immediately after mulesing; 4) flunixin + topical anaesthetic, with flunixin admini- birth, and is described in the relevant Australian Model Code stered 2.5 mg/kg s.c. 90 min before mulesing; 5) carprofen of Practice for che Welfare of Animals.’ Although, in the years + topical anaesthetic, with carprofen administered 4 mg/kg after its widespread adoption, mulesing was highlighted for its s.c. 90 min before mulesing; 6) carprofen, administered as animal welfare benefits in reducing flystrike,” the practice has above; 7) flunixin, administered as above; and 8) carprofen more recently been the focus of criticism by animal welfare and + flunixin, administered as above. Plasma cortisol was measured animal rights interest groups, at 0, 0.5,

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the focus of criticism by animal welfare and + flunixin, administered as above. Plasma cortisol was measured animal rights interest groups, at 0, 0.5, 6, 12 and 24 h relative to mulesing. Animal behaviour, including posture, was recorded for 12 h after mulesing. Australian wool industry representative organisations announced Results The conventional mules lambs exhibited large increases in 2004 that the current practice of mulesing would cease by in plasma cortisol, reduced lying and increased standing with 2010,° Leading up to this date, there have been ongoing efforts to a hunched back compared with sham mules animals. Topical introduce alternatives to the procedure, and to provide analgesia anaesthetic reduced the cortisol peak to mulesing and hunched in association with the current practice. standing, and increased lying compared with the conventional mules treatment, but generally did not result in values equivalent Previous published peer-reviewed research has examined the to sham mules animals. Carprofen, flunixin, and carprofen + use of analgesics in sheep during castration,*~* tail docking,*"° flunixin treatments did not reduce the cortisol response to and experimental electric shock'"'? but not mulesing. The mulesing but substantially ameliorated some changes in behavioural postures. Flunixin + topical anaesthetic reduced the site-specific injection of local anaesthetic has been shown to cortisol peak following mulesing and substantially ameliorated substantially reduce the pain and stress responses of lambs to most changes in behavioural postures. Carprofe + topical n castration and tail docking.’ Such studies have typically used anaesthetic abolished the cortisol peak following mulesing the local anaesthetic agents lignocaine,“ or bupivicaine.®”

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uch studies have typically used anaesthetic abolished the cortisol peak following mulesing the local anaesthetic agents lignocaine,“ or bupivicaine.®” and substantially ameliorated most changes in behavioural Typically, lignocaine has an effective duration of action of postures. All mulesed animals lost weight in the week after 1.5 to 2h, whereas bupivicaine is effective for 3 to 4 h.*%1415 mulesing regardless of analgesic administration, but there For these local anaesthetics to achieve the upper limits of these were no significant differences in growth rate between any of the eight treatments over the 3 weeks after mulesing. ranges in duration of action, they require the addition of adrenalin to the preparation. This constricts local blood vessels and causes Conclusions Analgesics can moderate the pain response of the drug to remain near the site of administration for longer." lambs to mulesing. The welfare outcome for lambs of mulesing could be improved by use of a combination of local anaesthetic Because of the relatively extensive area of tissue trauma created and long acting non-steroidal anti-inflammatory drug. by mulesing, pre-operative analgesia may not easily be provided Key words: lambs, mulesing, analgesia, non-steroida! anti- through infiltration of local anaesthetics. More generalised topical inflammatory drugs application is an option, and this approach has been used in tail Aust Vet J 2007;85:98-106 doi: 10.1411/.1751-0813.2007.001 15.x docking studies.’ Another option for mulesing analgesia may be NSAID Non-steroidal anti-inflammatory drug the systemic administration of non-steroidal anti-inflammatory drugs (NSAIDs), such as funixin and carprofen. Although there 98 Australian Veterinary Journal Volume 85, No 3, March 2007 © 2007 The Authors

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ammatory drugs (NSAIDs), such as funixin and carprofen. Although there 98 Australian Veterinary Journal Volume 85, No 3, March 2007 © 2007 The Authors Journal compilation © 2007 Australian Veterinary Association PRODUCTION ANIMALS Table 1, Experimental treatments used in the study. Group Treatment descriptor Agent(s) administered Mulesing Dose Route Time of administration relative to mulesing 1. Sham mules Normal saline (Baxter, NSW) No imL 8.c3 Oh 2, Conventional mules Normal saline (Baxter, NSW) Yes TmL 8.03 Oh 3. Topical anaesthetic Topical anaesthetic formulation Yes 8 mL/11-15kg topical Oh (Tri-Solfen, Animal Ethics, Vic): 10 mL/16-20kg 40.6 g/L lignocaine hydrochloride 4.5 g/L bupivacaine hydrochioride 24.8 mg/L adrenaline tartrate 5.0 g/L cetrimide 4, Flunixin + topical Fiunixin meglumine (Finadyne, Yes As per individual As per individual As per individual anaesthetic Schering-Plough, NSW) treatments treatments treatmenis Topical anaesthetic formulation 5. Carprofen + topical Carprofen (Rimady! injection, Yes As per individual As per individual As per individual anaesthetic Pfizer, NSW) treatments treatments treatments Topical anaesthetic formulation 6. Carprofen Carprofen Yes 4.0 mg/kg s.c2 -1.5h 7. Flunixin Fiunixin meglumine Yes 2.5 mg/kg 8.02 -1.5h 8. Carprofen + flunixin Carprofen Yes As per individual 8.0.4 -T.5h Flunixin meglumine treatments ? Subcutaneous, is relatively little published data on the use of NSAIDs in sheep, to acclimatise them to the feed rations and to close handling. both flunixin and carprofen are recommended as analgesics for Lambs were quietly handled for a 5-minute period on two sheep that are undergoing experimental surgery.!° Flunixin has occasions during the week before treatment to reduce the level been shown to be effective

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eep that are undergoing experimental surgery.!° Flunixin has occasions during the week before treatment to reduce the level been shown to be effective in sheep receiving noxious mechanical of stress associated with repeated handling during blood stimuli and to attenuate the development of hyperalgesia.!”"8 There sampling. Two days before mulesing, wool was removed with are also indications that flunixin provides good pain relief follow- Oster clippers from the skin over the jugular vein to facilitate ing orthopaedic surgery in sheep.'? NSAIDs may provide a longer blood sampling. Sheep were fed a pelleted ration of sheep nuts period of analgesia than local anaesthetics; in sheep, carprofen is (Ridley Agriproducts, Tamworth; 22% crude protein DM; cleared slowly, with an elimination half-life of at least 30 h.2° 11.1 MJ/kg DM) at a rate of 0.7 ke/DSE/day. The aim of this study was to evaluate the effectiveness of a To facilitate the collection of biological samples and behavioural commercially available topical local anaesthetic preparation, observations, the experiment was performed in 8 replicates, as well as the NSAIDs flunixin and carprofen, in minimising the blocked across treatments. Thus one lamb from each of the eight physiological and behavioural indicators of pain and stress in groups was treated in the first block, which was replicated on lambs following mulesing. Our hypothesis was that the applica- 7 subsequent occasions over a 2-week period. The day before tion of either the topical anaesthetic or an NSAID would reduce mulesing, one experimental block of lambs and ewes was placed the pain and stress responses to mulesing, and that this effect in a separate but adjoining pen to the main group. This pen was would be enhanced by a combination of the two

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ponses to mulesing, and that this effect in a separate but adjoining pen to the main group. This pen was would be enhanced by a combination of the two classes of drug. further divided in half such that each section (4.5 x 3.0 m) housed four lambs and ewes. At this time, lambs were weighed and individually identified with coloured spray to assist behavioural Materials and methods observations. Ewes and lambs were held in this pen for 24 h after Animals and treatments The experiment was undertaken at CSIRO’s FD McMaster mulesing, during which time blood samples were collected and Laboratory, Armidale, NSW and was approved by the institutional behaviours were recorded on video. The ewes and lambs were then returned to the larger group, Lambs were again weighed at Animal Ethics Committee. Sixty-four mixed sex fine wool Merino lambs at a mean age of 5 weeks were randomised into eight 7 and 21 days post mulesing, All lambs had EMLA topical cream (2.5% lidocaine, 2.5% prilocaine, AstraZeneca, North Ryde) treatment groups of eight animals as shown in Table 1. applied to the site of venepuncture on the neck at the time of The ewes and lambs were group-housed in the animal house for analgesic or saline administration and were given a clostridial 6 a 2-weel period before the commencement of the trial, in order in 1 vaccination (Pfizer, Parkville) and an antibiotic injection © 2007 The Authors Australian Veterinary Journal Volume 85, No 3, March 2007 99 Journal compilation © 2007 Australian Veterinary Association PRODUCTION ANIMALS tail was removed as well as the skin fold on each side of the base of the tail, resulting in a continuous excised area from the tip of the tail to the caudal surface of the hind leg. The mulesing operator was blinded to the experimental treatments

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

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ntibody with corticoster- operation. Hatched areas represent portions of skin removed. one, cortisone, dexamethasone, prednisolone and prednisone were 0.2, < 0.1, < 0.1, 45.3 and 0.3%, respectively. The intra- assay coefficient of variation (CV) for samples containing 176.5, (oxytetracycline at 10 mg/kg) prior to mulesing. Blood samples 81.9 and 34.8 nmol/L cortisol respectively, were 9.3, 8.2, and were taken from the jugular vein via venepuncture into 5 ml EDTA vacutainers at 5 time points over the course of the exper- 5.9%. The inter-assay CVs for the same samples were 7.2, 9.3 and 8.9%, respectively, iment, commencing prior to analgesic treatment and again at 0.5, 6, 12 and 24h post-mulesing. On each occasion, lambs had Behaviour blood samples taken in the sequence in which they had been mulesed. The behaviour of each lamb was captured using four digital video cameras for a total of 12 hours after mulesing, consisting of The lambs received their experimental treatments at the times three periods of 4 hours’ duration (0 to 4, 4 to 8 and 8 to 12h relative to the mules operation described in Table 1. At the time post-mulesing). One operator, blinded to the treatment codes, of mulesing, lambs were caught and placed in a mulesing cradle classified the behaviour of all lambs in the experiment, into the and their tails were docked with a gas fired hot knife. The mules- categories shown in Table 2. The lambs were observed and their ing operator had many years of experience mulesing lambs and behaviours recorded at 5 minute intervals. Lamb postures were had completed accreditation training provided by the Livestock used as the main measure as these were identified in preliminary Contractors Association, Mulesing involved the excision of two research as most indicative of

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the main measure as these were identified in preliminary Contractors Association, Mulesing involved the excision of two research as most indicative of mulesing-induced behavioural pieces of skin from the breech and tail of lambs (Figure 1). The changes in the period after mulesing. mulesing cut commenced near the base of the tail, ran outside the non-wooled perineal area, then back towards the ventral Statistical analyses midline before heading along the caudal surface of the hind Plasma cortisol concentrations were analysed using ASREML leg towards the hock. The cut ceased approximately 2/3 of the statistical software”! in a repeated measures analysis model fitting way along the upper hind leg towards the hock and ended in a the main effects of treatment, time, block and potential interactions, sharp ‘V’ shape. One strip of skin along each lateral side of the with pre-treatment cortisol concentration as a covariate, The 100 Australian Veterinary Journal Volume 85, No 3, March 2007 © 2007 The Authors Journal compilation © 2007 Australian Veterinary Association PRODUCTION ANIMALS Table 2. Categories used to classify behaviour following mulesing. 160 Category Behaviour Sham Mules Carprofey + 120 4 Topical Anaesthetic Normal standing Lamb stood with no obvious postural abnormality. 80 -4 A Straight-backed, with head higher than or level with back. A ap Fe B 40 4 A, oA nn Hunched standing Lamb stood with a rounded, hunched appearance. Back was arched, with head lower than highest point of back. 160 5 5 Normal walking Lamb exhibited a usual gait while waiking, with even Conventional Carprofen steps and no obvious hesitation. 120 + Mules Stiff walking Lamb exhibited stiff or tentative movements while ~ 80 ~ BC B BOC gc B walking. a Lying Lamb lay ventrally (on its

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sitation. 120 + Mules Stiff walking Lamb exhibited stiff or tentative movements while ~ 80 ~ BC B BOC gc B walking. a Lying Lamb lay ventrally (on its sternum and abdomen with all 3 40 4 Nee AB d (ventral and lateral} four legs folded under the body, or lay with one or both hind legs extended laterally) or laterally (on Its side with = 160 = 3 Torical Flunkxin one or both forelegs and both hind legs stretched out 1204 ¢ Anzesthetic laterally). 6 c Feeding Lamb was feeding from the trough. 80 4 \a c Standing other Lamb was standing but the posture was not clearly visible or was unable to be clearly categorised as 40 4 d hunched or straight backed. 160 Standing total Standing, walking and feeding categories combined. D Flunixin + Topical Carprofer + 120 5 , Araesthetic Hlunixin 8o 4 BC c ¢ Bc BC Bc data sets were normally distributed for the parameters measured. Non-significant interactions were omitted, thereby reducing the 40 4 d final model to its simplest components. Least squares means and 0 T Lj T qT T T T T T 7 standard errors are presented in graphs and tables. Pre 0.5 68 12 24 Pre 05 6 12 24 Behavioural data were assessed by analysis of variance using Time (hours) R statistical software (The R Development Core Team, Version Figure 2. Effect of analgesic treatments on plasma cortisol responses 1.6.2).” Results were calculated as the percentage of time a lamb (least squares means, SE = 11.38 nmol/L) to mulesing in Merino lambs. ABCD Means within a sampling time point without a common superscript was observed in each behavioural category, Data in all behavioural are significantly different (P < 0.05). categories were square root transformed for analysis of variance. As there was only one record of lateral lying in the observation of treatment X time (P <

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ories were square root transformed for analysis of variance. As there was only one record of lateral lying in the observation of treatment X time (P < 0.001), on the cortisol response to period, ventral and lateral lying behaviours were pooled into one mulesing. Plasma cortisol concentrations in Conventional Mules category termed lying, There was a significant period x treatment lambs in the absence of an analgesic were elevated 155% above interaction, therefore treatment effects were analysed and pre- Sham Mules animals within 30 min following the procedure sented separately for each period. Where the treatment effect was (Figure 2). Carprofen + topical anaesthetic was the only analgesic significant, a linear model was used for testing the difference treatment to result in similar plasma cortisol concentrations to between the analgesic treatments and the sham mules and con- the sham mules treatment group 30 min post mulesing, Carprofen ventional mules treatments, The standard errors of the mean on + topical anaesthetic blunted the acute rise in cortisol (Figure 2) the observed scale are not presented in the results, as these were and only differed from the sham mules group at 12 and 24h. asymmetrical around the predicted means. Although the experi- Peale plasma cortisol concentrations were not significantly mental animals were in group pens, and could see, hear and smell different (52.9 versus 56.0 nmol/L respectively) between the animals in adjoining pens, pain-induced behavioural changes sham mules and the carprofen + topical anaesthetic groups. in individuals are less likely to be socially transmitted through The topical anaesthetic and flunixin + topical anaesthetic allelomimicry than other behavioural states. With this limitation treatments both resulted in lower

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cal anaesthetic and flunixin + topical anaesthetic allelomimicry than other behavioural states. With this limitation treatments both resulted in lower peak cortisol responses than in mind, animal within block was used as the experimental unit mulesing without analgesia, but greater peak cortisol than sham for behavioural analyses,??* mules and carprofen + topical anaesthetic treatments (Table 3), The other analgesic treatments did not differ in peak cortisol Results response from the conventional mules treatment lambs, For both Cortisol the flunixin and the flunixin + topical anaesthetic treatments, there There were significant effects of treatment (P < 0.001), time was a tendency for a rise in cortisol at 12 and 24 h; however, this trend (P < 0.001) and block (P < 0.001), anda significant interaction was not evident in the group receiving topical anaesthetic alone. © 2007 The Authors Australian Veterinary Journal Volume 85, No 3, March 2007 101 Journal compilation © 2007 Australian Veterinary Association PRODUCTION ANIMALS Table 3. Peak plasma cortiso! concentrations (nmol/L) and area under the cortisol curve (AUC, nmol/L x h) during the intervals 0 to 6 h, and 6 to 24 h, 0 to 24 h following mulesing. Treatment: Sham Conventional Topical Flunixin + topical Carprofen + topical Carprofen Flunixin Carprofen + SEM variable mules mules anaesthetic anaesthetic anaesthetic flunixin Peak 52.94 133.1¢ 441.28° 97.68 56.04 142,8° 135,.8° 136.0° 15.79 AUC 0 to 6h 2024 556° 5068 393° 2134 571° 524° 592° 59.4 AUC 6 to 24h 299° 804 4420° 40238° 8o1® 9348 1156° 1214° 147.5 AUC 0 to 24h 5014 13608 4626°° 1416° 1015° 1505°° 1681°° 1805° 175.1 “2° Within row, means without a common superscript are significantly different (P < 0.05), The area under the

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°° 1416° 1015° 1505°° 1681°° 1805° 175.1 “2° Within row, means without a common superscript are significantly different (P < 0.05), The area under the cortisol curve (AUC) is presented in Table 3. For the period 0 to 6 h, there was no significant difference between the sham mules and the carprofen + topical anaesthetic groups. In the subsequent period (6 to 24h), there was a significant cortisol rise in the carprofen + topical anaesthetic treatment group compared to the sham mules controls, For the overall period 0 to 24h, all the analgesic treatments resulted in AUC values that were different from the sham mules lambs, although the AUC for carprofen + topical anaesthetic was the lowest of the seven treatments incorporating mulesing. Adg(vaeri/aldgnye) Growth All mulesed lambs lost weight over the initial period (Day 0 to 7) after mulesing (P < 0.01), compared with the sham mules group (Figure 3), There was a significant block effect on average daily gain/loss during Day 0 to 7 (P < 0.05). For the second period fos) (Day 7 to 21), growth rates did not differ between treatments. When average daily gain was calculated for the 21 day period ~100 4 following mulesing, there was no significant difference between treatments in growth rates (P > 0.05). Day 0-21 100 4 Behaviour The results of the behavioural observations are presented in Table 4 as least squares estimates, back transformed to the observed scale. Overall, conventional mules lambs spent less -100 time lying and more time standing, particularly with a hunched posture than sham mules lambs in all periods. During the -200 + periods 0 to 4h and 4 to 8 h, Conventional mules lambs spent more time walking with a stiff gait than Sham mules lambs. 12 3 4 5 6 7 8 Lambs that received carprofen + topical

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8 h, Conventional mules lambs spent more time walking with a stiff gait than Sham mules lambs. 12 3 4 5 6 7 8 Lambs that received carprofen + topical anaesthetic and flunixin Treatment group + topical anaesthetic differed from conventional mules lambs in all periods, spending more time lying, less time in a hunched Figure 3. Effect of analgesic treatments on average daily gain following mulesing in Merino lambs. 1) sham mules; 2) conventional mules; 3) topical posture and less time standing. These two analgesic treatment anaesthetic; 4) flunixin + topical anaesthetic; 5) carprofen + topical anaesthetic; 6) carprofen; 7) flunixin; 8) carprofen + flunixin. Data plotted groups were not significantly different from the sham mules are least squares means (SEM for Days 0 to 7, 7 to 21 and 0 to 21 were 76.8, control group for the amount of time lying for the three 35.6 and 38.6 g/d, respectively). ‘Swithin a time period, means with a different superscript are significantly assessment periods. Similarly, there were no significant differences different (P < 0.05). between sham mules, carprofen + topical anaesthetic and flunixin + topical anaesthetic treatment groups for the behaviours termed stiff walling and hunched standing. During the period from 0 to Discussion 4 h, all the analgesic treatments, with the exception of flunixin, In this study, there was close alignment between the physiological showed less hunched standing than the conventional mules lambs. and the behavioural responses of lambs to mulesing, identifying 102 Australian Veterinary Journal Volume 85, No 3, March 2007 © 2007 The Authors Journal compilation © 2007 Australian Veterinary Association J PRODUCTION ANIMALS Table 4, Effect of treatment on lamb behaviour. Data presented are back-transform ed least

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2007 Australian Veterinary Association J PRODUCTION ANIMALS Table 4, Effect of treatment on lamb behaviour. Data presented are back-transform ed least square means representing the percentage of time spent in each behaviour.’ Treatment/ Variable Sham Conventional Topical Flunixin + topical Carprofen + topical Carprofen Flunixin Carprofen + 4 mules mules anaesthetic anaesthetic anaesthetic flunixin = < Period 1 (0 to 4 h) 3 Lying 28M 0.3° 7.5 om te" 5.4° 17" 14¥ 5) Normal walking 7.0 4.3 9.0 6.4 8.6 4.1° 4,3° 2.5 8 Stiff walking oM 1.3° 03 0.3 0.1" 0.1! oM oM ee Upright standing 33 38 38 29 44 48 40 53° Hunched standing oM 16° 1.8! 0.1" om 1.4TM 3.8 0.1 Feeding 23! 40° 16 26" 18 11° 16 15 Standing unclassified 1.0" 17° 10° 3.6" 5.7" 17° 13¢ 6.7" Standing total 67s 98° 87° 78! 79" 88° Bio g2oM Period 2 (4 to 8h) Lying 45M 4,8° 5.1° 46" 34! 11° 16° 2iM Normal walking 5.6" 0.1° 1.7e¢ 5.9! 4.2m 0.6° 1.4° 1.2° Stiff walking o” 4.1° 0.6° oM oM o” oM oM Upright standing 32 24 4iTM 26 38 58M 49M 44M Hunched standing oM 22° 7.9% 0.1" 0.2" 2.3" 1.4" 1.5M Feeding 9.7 3.9 6.0 4.6 3.9 3.2 6.4 5.4 Standing unclassified 1,2 28° 140M 7.7 490" 420M 14oM 41 Standing total 52 age gee 52 64 g3° 78° 69° Period 3 (8 to 12 h) Lying 6g! 8.0° 75° 54" 4gM 22° 13° 12° Normal walking 2.3! 0° 0,2° 241" 0,2° 0.2° 1.0! 0.8 Stiff walking 0 1.2 0.8 0.4 0.4 0.6 0.2 0.2 Upright standing 20 16 4yom 25 28" 4gom 50° 470" Hunched standing oM 2i1¢ 8° oM 0.4 5.0TM 2.2m 2.0 Feeding 0.4 14 13 0.9 2.7 11 2.6 2.4 Standing unclassified 0.6" 33° 9.3 4,3" 9,8 10% 19° 10°" Standing total 26" 83° 83° 39M 4gos 73° 81° 73° ° Means within a row are significantly different to ihe sham mules (P < 0,05), “ Means within a row are significantly different to the

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gos 73° 81° 73° ° Means within a row are significantly different to ihe sham mules (P < 0,05), “ Means within a row are significantly different to the conventional mules treatment (P < 0.05). * Means may not sum to exactly 100 because they are back-transformed. pain responses and the effects of analgesia. No analgesic drug exhibited a large increase in plasma cortisol at 30 min, and main- when used alone was effective in controlling both the acute and tained elevated concentrations compared with sham mules more chronic physiological and behavioural responses to mulesing animals at 24h after mulesing. In addition, the conventional and tail docking. However, a combination of a topical anaesthetic mules lambs exhibited less lying and a greater amount of time formulation and a long-acting NSAID (carprofen) provided a in a hunched standing position than the sham mules group, substantial reduction in the indicators of pain associated with with these behavioural changes persisting throughout the 12 h mulesing. When used alone, the topical anaesthetic formulation of observation. Similarly, in a previous study, plasma cortisol caused a significant but moderate decrease in peak cortisol concentrations rose rapidly after mulesing, peaking at 15 min, response to mulesing. When the topical anaesthetic formulation and were still increased over basal levels at 24 h.° Behavioural was combined with flunixin or carprofen, greater decreases in studies showed that mulesed lambs exhibited shade-seeking, peak cortisol response were observed. hunched standing and reduced grazing and lying, with the greatest The physiological and behavioural changes in response to effects seen 2 to 5 h after mulesing.”” mulesing in the conventional mules group were consistent with Both cortisol

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behavioural changes in response to effects seen 2 to 5 h after mulesing.”” mulesing in the conventional mules group were consistent with Both cortisol increases and changes in behaviour, particularly the results of previous studies, The conventional mules lambs the adoption of abnormal postures, are indicative of the pain © 2007 The Authors Australian Veterinary Journal Volume 85, No 3, March 2007 103 Journal compilation © 2007 Australian Veterinary Association PRODUCTION ANIMALS responses of lambs to acute surgical procedures,®!38 For lamb effective in ablating the cortisol response to surgical castration in castration and tail docking, the use of analgesics has been shown calves, whereas local anaesthesia alone was only partially effec- to be able to reduce cortisol responses to levels close to those of tive.” Similarly, Earley and Crowe reported that ketoprofen by control animals.’* In the present study, the topical anaesthetic itself, or in combination with local anaesthetic, was better than formulation resulted in a lower peak cortisol response than that local anaesthetic alone in minimising the cortisol response in for the conventional mules lambs, but did not reduce the inte- calves to surgical castration.*® The common finding of these two grated response (AUC). Furthermore, all measures of cortisol previous studies with calves and the current study with lambs response were greater for the topical anaesthetic treatment than is that a local anaesthetic and NSAID combination produces for the sham mules lambs. This discrepancy with the results of a minimised cortisol response to surgery. The difference in the the previous studies may be due to mulesing causing a greater present study is that this was achieved with local anaesthetic applied area of

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ce in the the previous studies may be due to mulesing causing a greater present study is that this was achieved with local anaesthetic applied area of tissue trauma than castration or tail docking, and the fact topically post-surgery, rather than injected pre-operatively.°>*® that local anaesthetic was administered before castration and tail Possible reasons for the effectiveness of combined local anaes- docking commenced, thetic and NSAID analgesia include complementary durations The failure of either carprofen or flunixin to reduce cortisol of action and mechanisms of action. Whereas local anaesthesia responses compared with the conventional mules treatment was may be present for 1.5 to 4 hours after administration, depending unexpected, given that both were administered 90 min before on the agent used, many NSAIDs typically have a longer dura- mulesing in order to maximise the accumulation of NSAIDs tion of action. Carprofen, one of the longest-acting NSAIDs, has into damaged and inflamed tissues.?**° The cortisol response a plasma half-life of 33 h in sheep.”° Secondly, local anaesthetics of lambs to castration was reduced by the intramuscular admin- such as lignocaine and bupivicaine are usually administered to istration of the NSAID diclofenac 20 min beforehand, with regional tissues and thus act locally to inhibit action potentials in a corresponding reduction in abnormal postures.‘ Similarly, nerve cells by inhibiting sodium influx through the nerve cell diclofenac administration reduced the cortisol response of lambs membrane. In contrast, the systemic administration of NSAIDs to the application of rubber rings for tail docking.? Flunixin and has been shown to act both centrally and peripherally, with carprofen were chosen for this study because they

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er rings for tail docking.? Flunixin and has been shown to act both centrally and peripherally, with carprofen were chosen for this study because they are among the central actions in sheep shown to be related to supraspinal effects recommended treatments for pain relief in experimental sheep causing inhibition of spinal transmission of nociceptive inputs.°” post surgery,'° and both had shown promise in a pilot study prior The analysis of the behavioural data, which comprised approxi- to the start of this experiment. mately nine thousand observations, provided a clear picture of It is possible that the effectiveness of flunixin as an analgesic in normal postures and modified behaviour in response to the pain sheep may vary with the type of nociception produced by the of mulesing. Conventional mules lambs without analgesic treat- injury. Although flunixin was effective in providing analgesia ment spent a greater percentage of time standing, standing against mechanical stimulation pain in sheep,!”"8 it was not hunched or walking abnormally and less time lying, In the initial effective in a study which used electrical stimulation.2! In a 4 h observation period, the conventional mules lambs also spent separate study, both carprofen and flunixin attenuated the less time eating but this was not evident during the subsequent development of mechanical hyperalgesia in sheep.” In dogs, two 4h periods, Analgesic treatments generally increased the carprofen has been demonstrated to be effective in controlling amount of time spent lying compared with the conventional pain for up to 24 h following orthopaedic surgery,”® and carpro- mules animals. Evidently, conventional mules lambs found it fen reduced the cortisol response to castration in cattle.34 painful to lie down

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d carpro- mules animals. Evidently, conventional mules lambs found it fen reduced the cortisol response to castration in cattle.34 painful to lie down in the first 6 to 12 hours after mulesing and Given the rather modest benefits conferred by the topical anaes- hence spent a greater proportion of their time standing, They thetic treatment in reducing the plasma cortisol response to also assumed a hunched posture, walking was restricted and mulesing, and the failure of the carprofen treatment to show any when undertaken, occurred with less flexion of the hock joint, benefit, it is interesting that the carprofen + topical anaesthetic thereby producing a stiff gait. These postural changes may have combination was effective in reducing the cortisol response to assisted the animal to cope with the pain being experienced at mulesing, and resulted in cortisol values that were not different the time. to those of the sham mules lambs for 6 h after mulesing. This For the topical anaesthetic, carprofen and carprofen + topical effect could be due in part to a shift in the cortisol response curve anaesthetic treatments, there appeared to be close alignment to the right, It is noteworthy however that the AUC for cortisol between the cortisol and behavioural results, The topical anaesthetic in the interval 6 to 24 h was as low or lower for the carprofen + treatment had moderate benefits, especially during the first 4 h, topical anaesthetic combination than any other analgesic treat- reducing the amount of hunched standing, and providing a level ment. For the 24 hour AUC, the combination was significantly of lying intermediate between sham mules and conventional lower than any other analgesic treatment. Studies in calves have mules animals, The carprofen + topical anaesthetic

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etween sham mules and conventional lower than any other analgesic treatment. Studies in calves have mules animals, The carprofen + topical anaesthetic treatment yielded similar benefits from combined administration of local resulted in behaviour that was not different from that of the anaesthetic and NSAIDs of improved analgesia for surgical sham mules animals, apart from less normal walking and more husbandry procedures. Stafford and colleagues found the com- standing from 8 to 12 h after mulesing. Furthermore, during bination of local anaesthesia and the NSAID ketoprofen to be the first 4 h, for every postural category in which the sham mules 104 Australian Veterinary Journal Volume 85, No 3, March 2007 © 2007 The Authors Journal compilation © 2007 Australian Veterinary Association PRODUCTION ANIMALS lambs differed from the conventional mules animals, the carprofen treatment. Regulatory requirements for use of carprofen in sheep + topical anaesthetic lambs also differed from the conventional in Australia also need to be established. mules treatment. Analgesic treatments incorporating flunixin, either alone or in combination, appeared more effective at Acknowledgments ameliorating behavioural responses to mulesing than in reducing The authors wish to thank Sue Belson and Andrew Eichorn for plasma cortisol responses. Given that there is no absolute posi- their valuable contributions to the conduct of this study, tion agreed by researchers on the relative merits of cortisol and behavioural changes in assessing the responses of farm animals References to painful procedures,” it is our assumption that a potential 1. Primary Industries Standing Committee. Model Code of Practice for the analgesic treatment should demonstrate both physiological and Welfare of Animals.

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rimary Industries Standing Committee. Model Code of Practice for the analgesic treatment should demonstrate both physiological and Welfare of Animals. The Sheep. Second Edition. PISC Report 89. CSIRO Publications, Collingwood, 2006. behavioural benefits in order to conclude that a clear analgesic 2, Beveridge WIB. The origin and early history of the Mules operation. Aust Vet effect is occurring, especially when other treatments are fulfilling J 1984;61:161-163. this criterion in the same study. A previous study on pain 3. Australian Sheep and Wool Industry Taskforce. Industry agreement on mules- responses in lambs to castration and tail docking also indicated ing http://www.nff.org.au/pages/policies_printfiles/NR_TF_08_11_04_PETA.pdf. 2004. Retrieved 7 March 2006. a need to interpret behavioural changes in conjunction with 4, Dinniss AS, Mellor DJ, Stafford KJ, Bruce RA, Ward RN. Acute cortiso! cortisol responses.” responses of lambs to castration using a rubber ring and/or a castrating clamp with or without local anaesthetic. NZ Vet J 1997;45:114~121. The growth of the lambs was significantly reduced by mulesing 5, Dinniss AS, Stafford KJ, Mellor DJ, Bruce RA, Ward RN. The behaviour during the experimental period, at least in the short term, pattern of lambs after castration using a rubber ring and/or castrating clamp Despite the administration of analgesics, all mulesed lambs lost with or without focal anaesthetic. NZ Vet J 1899;:47:198-203., weight from mulesing to Day 7, Subsequently, all lambs resumed 6. Molony V, Kent JE, Hosie BD, Graham MJ. Reduction in pain suffered by lambs at castration. VetJ 1997;153:205-213. a. positive average daily gain through to the completion of the 7. Sutherland MA, Mellor DJ, Stafford KJ et al. Reduction in the acute cortisol

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5-213. a. positive average daily gain through to the completion of the 7. Sutherland MA, Mellor DJ, Stafford KJ et al. Reduction in the acute cortisol experiment on Day 21, and there were no significant differences response of lambs to ring castration plus tailing by injecting lignocaine into between the treatment groups. During Day 0 to 7, despite the the scrotum or testes at the time of ring application. Aust Vet J 1999:77:738— 741, use of analgesics, mulesing either reduced feed intake, or reduced 8. Kent JE, Molony V, Graham MJ. Comparison of methods for the reduction of growth through altering the utilisation or partitioning of the acute pain produced by rubber ring castration or tail docking of week-old lambs. available nutrients, Weight loss in the first week after mulesing VetJ 1998;155:39~51, could be due to catabolic effects following activation of the HPA 9. Graham Mu, Kent JE, Molony V. Effects of four analgesic treatments on the behavioural and cortisol! responses of 3-week-old lambs to tail docking. axis or due to independent catabolic effects of tissue damage. VetJ 1997;153:87—97, There is good evidence for inflammatory events acting through 10. Wood GN, Moiony V, Fleetwood-Walker SM, Hodgson JC, Mellor Du. Effects pro-inflammatory cytokines to alter partitioning of nutrients, of local anaesthesia and intravenous naloxone on the changes in behaviour and plasma concentrations of cortisol produced by castration and tail docking with restricting skeletal muscle accretion and lipogenesis within the tight rubber rings in young lambs. Res Vet Sci 1991;15:193—-199. body and thereby impacting on growth.” Such nutrient parti- 11. Grant C, Upton RN, Kuchel TR. Efficacy of intra-muscular analgesics for tioning provides substrates for the inflammatory and

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Such nutrient parti- 11. Grant C, Upton RN, Kuchel TR. Efficacy of intra-muscular analgesics for tioning provides substrates for the inflammatory and healing acute pain In sheep. Aust VetJ 1996;73:129-132. response to a surgical procedure. Therefore, a decrease in growth 12. Grant C, Upton RN. Comparison of the analgesic effects of xylazine in sheep via three different administration routes. Aust Vet J 2004;82:304-307. may be unavoidable in the short term even in the absence of pain 13. Mellor DJ, Stafford KJ. Acute castration and/or tailing distress and its allevi- and activation of the HPA axis. The measurement of individual ation in lambs. NZ Vet J 2000;48:33-43, feed intake may provide additional insight into these effects. 14. Hail LW, Clarke KW, Trim CM. General principles of local analgesia. in: Previous research in calves has suggested that analgesia for Veterinary Anaesthesia. 10th edn. WB Saunders, London, 2001:225—246., 15, Reich! M, Quinton D. Comparison of 1% lignocaine with 0.5% bupivacaine in castration may not alter reductions in individual feed intake in digital ring blocks. J Hand Surg 1987;12:375-376. the week after surgery.“ 16. Dobromylskyj, P, Flecknell PA, Lascelles BD et al. Management of post- operative and other acute pain. In: Flecknell PA, Waterman-Pearson A, editors. Conclusions Pain Management in animals, WB Saunders, London, 2000:81-145. 17. Chambers JP, Waterman AE, Livingston A. The effects of opioid and alpha Analgesics can moderate the pain response of lambs to mutlesing. 2 adrenergic blockade on non-steroidal anti-inflammatory drug analgesia in Greatest pain relief, as indicated by the behavioural and cortisol sheep. J Vet Pharmacol Ther 1995;18:161-166. tesponses to mulesing, was provided by the combined administration 18. Welsh

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the behavioural and cortisol sheep. J Vet Pharmacol Ther 1995;18:161-166. tesponses to mulesing, was provided by the combined administration 18. Welsh EM, Nolan AM. Effect of flunixin meglumine on the thresholds to of a topical local anaesthetic formulation and the long acting mechanical stimulation in healthy and lame sheep. Res Vet Sci 1995;58:61—66. 19. Taylor PM. Anaesthesia in sheep and goats. In Pract 1991;13:31-36, NSAID carprofen. The administration of the topical anaesthetic 20, Welsh EM, Baxter P, Nolan AM. Pharmacokinetics of carprofen adminis- alone had some benefits in terms of reduced peak cortisol and tered intravenously to sheep. Res Vet Sci 1992;53:264-—266, less hunched standing compared with no treatment, and the 21. Gilmour AR, Goge! BJ, Cullis BR, Welham SJ and Thompson R. ASRemi user guide release 1.0. 2002 VSN International Ltd, Hemel Hempstead, HP1 administration of NSAIDs alone provided some behavioural 1ES, UK. benefits but did little to reduce plasma cortisol. The results 22. R Development Core Team. R: A language and environment for statistical suggest that the welfare outcome for lambs that are mulesed computing, Vienna, Austria. ISBN 3-900051-00-3. 2004. could be improved by use of a combined analgesic strategy. 23. Phillips CJC. The use of individual dairy cows as replicates in the statistical analysis of their behaviour at pasture. App/ Anim Behav Sci 1998:60:365—369. Further work is needed to identify the influence of time of 24, Phillips CJC. Further aspects of the use of individual animals as replicates in administration of the NSAID on the efficacy of this combination statistical analysis. App! Anim Behav Sci 2000;69:85-88. © 2007 The Authors Australian Veterinary Journal Volume 85, No 3, March 2007 105 Journal compilation ©

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al analysis. App! Anim Behav Sci 2000;69:85-88. © 2007 The Authors Australian Veterinary Journal Volume 85, No 3, March 2007 105 Journal compilation © 2007 Australian Veterinary Association PRODUCTION ANIMALS 25. Phillips CJC. Further aspects of the use of individual animals as replicates in interferon-y production, acute-phase proteins, feed intake, and growth. J Anim Statistical analysis. App! Anim Behav Sci 2002;75:265~268, Se/ 2006;84:351-359, 26. Shutt DA, Fell LR, Connell R et al. Stress-induced changes in plasma con- 35. Stafford KL, Mellor DJ, Todd SE, Bruce RA, Ward RN. Effects of local anaes- centrations of immunoreactive beta-endorphin and cortisol in response to routine thesia or local anaesthesia plus a non-steroidal anti-inflammatory drug on the surgical procedures in lambs. AustJ Biol Sci 1987;:40:97-103. acute cortisol response of calves to five different methods of castration. Res Vet 27. Fell LR, Shutt DA. Behavioural and hormonal responses to acute surgical Scj 2002;73:61-70. stress in sheep. App/ Anim Behav Sci 1989;22:283-294, 36. Earley B, Crowe MA. Effects of ketoprofen alone or in combination with local 28, Grant C. Behavioural responses of lambs to common painful husbandry anesthesia during the castration of bull calves on plasma cortisol, immunological procedures. App/ Anim Behav Sci 2004;87:255-273, and inflammatory responses. J Anim Sci 2002;80:1044—1052, 29, Toutain PL, Lees P. Integration and modelling of pharmacokine tic and 37. Lizarraga |, Chambers JP. Involvement of opioidergic and «2-adrenergic pharmacodynamic data to optimize dosage regimens in veterinary medicine, mechanisms in the central analgesic effects of non-steroidal anti-inflammatory d Vet Pharmacol Therap 2004;27:467-477. drugs in sheep. Res Vet Sci, 2006;80:194-200.,

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he central analgesic effects of non-steroidal anti-inflammatory d Vet Pharmacol Therap 2004;27:467-477. drugs in sheep. Res Vet Sci, 2006;80:194-200., 30. Sidhu PK, Landoni MF, Lees P. Pharmacokinetic and pharmacodyna mic 388. Mellor DJ, Cook CJ, Stafford KJ. Quantifying some responses to pain as a interactions of tolfenamic acid and marbofloxacin in goats. Res Vet Sci 2006; stressor. In: Moberg GP, Mench JA, editors. The biology of animal stress: basic 80:79-90. principles and implications for animal welfare. CAB International, Wallingford, 31. Grant C, Upton RN, Kuchel TR. Efficacy of intra-muscular analgesics for 2000:171-198. acute pain in sheep. Aust Vet U 1996;73:129--132, 39. Lester SJ, Mellor DJ, Holmes RJ, Ward RN, Stafford KJ, Behavioural and 32. Weish EM, Nolan, AM. Effects of non-steroidal anti-inflammatory drugs on cortisol responses of lambs to castration and tailing using different methods. the hyperalgesia to noxious mechanical stimulation induced by the application of NZ Vet J 1996;44:45-54, a tourniquet to a forelimb of sheep. Res Vet Sci 1994:57:285—291, 40. Johnson RW. Inhibition of growth by pro-inflammatory cytokines: an 33. Laredo FG, Belda E, Murciano J et al. Comparison of the analgesic effects integrated view. J Anim Sci 1997;75:1244—1255. of meloxicam and carprofen administered preoperatively to dogs undergoing 41. Fisher AD, Crowe MA, Alonso de la Varga ME, Enright WJ. Effect of castration orthopaedic surgery. Vet Rec 2004;155:667-671. method and the provision of local anesthesia on plasma cortisol, scrotal circum- 34, Pang WY, Earley B, Sweeney T, Crowe MA. Effect of carprofen administra- ference, growth and feed intake of bull calves. J Anim Sci 1996;74:2336-—2343. tion during banding or burdizzo castration of bulls on plasma

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ministra- ference, growth and feed intake of bull calves. J Anim Sci 1996;74:2336-—2343. tion during banding or burdizzo castration of bulls on plasma cortisol, in vitro (Acceptedfor publication 5 November 2006) 106 Australian Veterinary Journal Volume 85, No 3, March 2007 © 2007 The Authors Journal compilation © 2007 Australian Veterinary Association