Behavioural measures reflect pain-mitigating effects of meloxicam in combination with Tri-Solfen® in mulesed Merino lambs

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Animal (2019), 13:11, pp 2586 – 2593 © The Animal Consortium 2019 doi:10.1017/S1751731119000491 animal Behavioural measures reflect pain-mitigating effects of meloxicam in combination with Tri-Solfen® in mulesed Merino lambs L. Inglis, S. Hancock , M. Laurence and A. Thompson† School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150, Australia (Received 20 March 2018; Accepted 17 January 2019; First published online 2 April 2019) Flystrike costs the Australian industry $173 to 280 M per annum and 70% to 80% of Merino lambs are currently mulesed to reduce the risk of flystrike. To alleviate welfare concerns there has been widespread adoption of analgesics to mitigate the pain associated with mulesing. The objective of this experiment was to determine the effectiveness of Tri-Solfen® and meloxicam (Metacam® 20) at reducing pain-related behavioural responses to mulesing in Merino lambs. One hundred and forty Merino lambs were allocated to one of seven treatment groups: (1) non-mulesed (Control); (2) mulesed with no pain relief; (3) subcutaneous (s.c.) meloxicam administered 15 min before mulesing; (4) Tri-Solfen® administered at time of mulesing; (5) Tri-Solfen® and saline injection (s.c.) 15 min before mulesing; (6) Tri-Solfen® and meloxicam (s.c.) 15 min before mulesing; and (7) meloxicam (s.c.) at time the of mulesing. Behavioural responses such as standing, walking and lying were measured every 15 min for 6 h on the day of marking and for up to 2 h for 4 days thereafter. Standing (hunched v. normal) and walking (stiff v. normal) behaviours were then categorised into pain- and normal-related behaviours while lying remained in its own category. Mulesed lambs with no pain relief displayed significantly more pain-related behaviours than

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elated behaviours while lying remained in its own category. Mulesed lambs with no pain relief displayed significantly more pain-related behaviours than Control lambs during the 6 h post-mulesing (1.22 v. 0.22 out of a total score of 3; RSD = 1.15). Lambs that received a combination of pain relief displayed significantly less pain-related behaviour than mulesed lambs with no pain relief on the day of mulesing (0.85 v. 1.22 out of a total score of 3; RSD = 1.15). Administration of meloxicam or Tri-Solfen® on their own had minimal if any significant effect on pain-related behaviours on the day of mulesing. The results of this experiment support the use of pain-related behaviours to measure the efficacy of analgesics and the use of multimodal analgesia during mulesing of lambs. Keywords: mulesing, analgesia, behaviour, sheep, welfare Implications 2009). Mulesing is a surgical procedure that removes four strips of perineal skin resulting in skin tightening and fibre- This research further supports the use of analgesia during less scar tissue. In Australia, 70% to 80% of Merino lambs mulesing of lambs. Differences in pain-related behaviours are mulesed (reviewed by Phillips, 2009; Lane et al., 2015). supported the use of meloxicam when used in combination Mulesing results in an immediate increase in plasma cortisol with Tri-Solfen® (Bayer, Australia). This may encourage concentration that lasts for up to 24 to 48 h (Shutt et al., producers to also administer a non-steroidal analgesic when 1987; Fell and Shutt, 1989; Chapman et al., 1994; Paull mulesing lambs and where previously they may only have et al., 2008). Behavioural responses to mulesing such as used Tri-Solfen®. hunched standing, stiff walking and reduced lying can persist for up to 3 days (Fell and Shutt,

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ioural responses to mulesing such as used Tri-Solfen®. hunched standing, stiff walking and reduced lying can persist for up to 3 days (Fell and Shutt, 1989; Paull et al., 2008). These postural indicators have been successful in defining Introduction the response in lambs following husbandry painful proce- Flystrike is known to cause substantial physiological and dures (Mellor and Murray, 1989; Molony and Kent, 1997; behavioural stress to affected animals (Lee and Fisher, 2007). Thornton and Waterman-Pearson, 1999; Molony et al., 2002; Associated loss of production, treatment and animal death is Grant, 2004). During the last decade there has been sig- estimated to cost the Australian sheep industry $173 to nificant adoption of post-operative analgesics to reduce the 280 M per annum (Sackett, 2006; Lane et al., 2015). The pain caused by mulesing. incidence of flystrike can be reduced by mulesing (Phillips, Tri-Solfen® is a postoperative analgesic that contains lignocaine (a short-acting local anaesthetic), bupivacaine † E-mail: andrew.thompson@murdoch.edu.au (a long-acting local anaesthetic), centrimide as an 2586 Behavioural effects of pain relief for mulesing antiseptic to cleanse the wound and adrenaline to reduce Material and methods blood loss while enhancing the anaesthetic effects. Paull Animals et al. (2007) and Lomax et al. (2008) reported that The research was performed at Murdoch University’s farm Tri-Solfen® reduced pain-related behaviours in the first 4 to at Whitby Falls in Western Australia (32°17’35”S, 116°00’55”E) 8 h after mulesing. Lomax et al. (2013) concluded that between May and July 2017. A total of 178 pregnant Merino Tri-Solfen® reduced pain-related behaviours for up to 24 h; ewes (122 single bearing and 57 twin bearing)

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nd July 2017. A total of 178 pregnant Merino Tri-Solfen® reduced pain-related behaviours for up to 24 h; ewes (122 single bearing and 57 twin bearing) lambed between however, this study involved very low numbers of animals. May and June 2017. Seven days before mulesing, all lambs aged Other than this single study, there is limited evidence to 6 to 10 weeks were weighed, ear tagged, paint-branded on both support the premise that Tri-Solfen® offers pain relief sides with a unique number and their dam was recorded. Two beyond 4 to 8 h post-mulesing and its duration of action days before mulesing, lambs were weighed again with an may be much less. average weight of 12 kg. One hundred and forty Merino lambs Non-steroidal anti-inflammatory drugs (NSAIDs) provide were allocated to one of seven treatments (n = 20 lambs per another option for mulesing analgesia. They provide long treatment) based on live weight, sex and rear type (number of acting pain relief through inhibition of the COX-1 and lambs reared per ewe). Lambs were then allocated into four COX-2 pathways in prostanoid production in soft tissues. plots (n = 5 lambs/treatment per plot). Twin siblings were allo- This results in relief from pain and inflammation (Mathews, cated to the same treatment and plot. The four plots (50 × 50 m) 2002; Ricciotti and FitzGerald, 2011). Non-steroidal anti- were adjacent to the yards where the lambs were treated. Ewes inflammatory drugs may therefore provide a longer period were supplemented with 1 kg of oaten hay (9.0 MJ Metaboli- of pain relief following mulesing than Tri-Solfen®. Non- sable Energy per kg Dry Matter; 6.0% Crude Protein) and 600 g steroidal anti-inflammatory drugs have been shown to be of lupins (13.1 MJ Metabolisable Energy per kg Dry Matter; effective at

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rude Protein) and 600 g steroidal anti-inflammatory drugs have been shown to be of lupins (13.1 MJ Metabolisable Energy per kg Dry Matter; effective at mitigating pain in a range of species following 31.3% Crude Protein) per day per ewe for the experimental docking, dehorning and castration (Zöls et al., 2005; period, which ceased 10 days post-mulesing. Lambs were Coetzee and Smith, 2010; Keita et al., 2010; Small et al., weighed 10 days post-mulesing. The average weight of the 2014), however, there has been less work on their effec- lambs that were not mulesed were 15.6 kg and those that were tiveness to reduce pain associated with mulesing. Paull mulesed, regardless of pain relief administered, were 13.5 kg. et al. (2008) found no differences in peak cortisol con- centrations or pain-related behaviours between lambs that received meloxicam at the time of mulesing or no pain Experimental design relief. In this study dose rates and time of administration The treatments were implemented at mulesing (day 0) which may have limited the effectiveness of meloxicam in miti- consisted of lambs that were not mulesed (Control), lambs gating pain in response to mulesing. Furthermore, no that were mulesed and administered a saline injection as attempt was made to determine if meloxicam could opposed to pain relief (Placebo), lambs mulesed and admi- enhance or extend relief from pain provided by Tri-Solfen® nistered meloxicam (Metacam® 20, Boehringer Ingelheim, following mulesing. Sydney, Australia) 15 min before mulesing (MC-15), lambs The use of multimodal analgesia was considered pre- mulesed and administerd Tri-Solfen® only (TS), lambs mulesed viously by Paull et al. (2007) who found that treatment with a and administered a combination of meloxicam 15 min before topical

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y (TS), lambs mulesed viously by Paull et al. (2007) who found that treatment with a and administered a combination of meloxicam 15 min before topical analgesic in combination with the NSAIDs, carprofen mulesing and Tri-Solfen® (MC-15 + TS) and lambs mulesed or flunixin, reduced peak cortisol concentrations following and administered meloxicam in the cradle (MC0). mulesing and reduced pain-related behaviours compared to At marking, all lambs were placed in dorsal recumbency lambs treated with only a topical analgesic. They postulated into a cradle that restrains the animal around the hocks of all that the enhanced effectiveness of combining local anaes- four limbs. All lambs, except for the Control (un-mulesed and thetic and NSAIDs might be due to their complementary untreated) were ear marked, vaccinated with ScabiGard® durations and mechanisms of action. However, there were (Zoetis, Sydney, Australia) via a scratch to the skin on the inner no significant differences in plasma cortisol concentrations thigh and Glanvac® (Zoetis, Sydney, Australia) via a sub- between these treatments 24 h after mulesing and there was cutaneous injection, both per manufacturer’s instructions. Tails no attempt to measure impacts on lamb behaviours beyond were docked using a gas heated knife and mulesed. Females 12 h post-mulesing. were also vaccinated with Gudair® (Zoetis, Sydney, Australia) The current study tested the hypotheses that (i) pain- via subcutaneous injection high on the neck skin and in related behaviours in Merino lambs could be used to accordance with manufacturer’s instructions. Males were assess the efficacy of pain mitigation treatments following castrated using rubber rings. Mulesing was performed by a mulesing; (ii) a combination of meloxicam and

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fficacy of pain mitigation treatments following castrated using rubber rings. Mulesing was performed by a mulesing; (ii) a combination of meloxicam and Tri-Solfen® professional contractor accredited by the Livestock Contractors would be the most effective treatment in mitigating pain Association of Australia. Mulesing involved the removal of two in response to mulesing in Merino lambs, and (iii) strips of skin from the breech area outside the non-wooled administration of meloxicam 15 min before mulesing perineal area and one strip of skin along each lateral side of the would be more effective in mitigating pain in response to tail. Lambs allocated to the Control treatment were placed in mulesing than administration of meloxicam at the time of the cradle for 60 s (average time of marking) before being mulesing. released to their allocated plots. 2587 Inglis, Hancock, Laurence and Thompson Table 1 Categories used to classify behaviour of Merino lambs post-mulesing Category Behaviour Normal Normal standing Lamb stood with no obvious postural abnormality; straight-backed, with head higher than or level with back Normal walking Lamb exhibited a usual gait while walking, with even steps and no obvious hesitation Pain-related Abnormal standing Lamb stood head lower than highest point of back Hunched standing Lamb stood with a rounded, hunched appearance; back was arched, with head lower than highest point of back Stiff walking Lamb exhibited stiff or tentative movements while walking Lying Ventral Lamb laid on its sternum and abdomen with all four legs folded under the body Lateral Lamb laid on its side with one or both forelegs and both hind legs stretched out laterally Ventral/lateral Lamb laid on its sternum and abdomen with one or both hind legs extended laterally The

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legs and both hind legs stretched out laterally Ventral/lateral Lamb laid on its sternum and abdomen with one or both hind legs extended laterally The live weight of each lamb measured two days prior to Statistical analyses mulesing was rounded to the nearest 5 kg and meloxicam All statistical analyses were performed using GENSTAT (18th was administered subcutaneously based on the manu- edition; VSN International, 2017, Hemel Hempstead, UK). All facturers recommended dose of 1 ml/20 kg live weight – data was analysed using the method of restricted maximum 1 mg/kg (0.5 to 1 ml per lamb). The dose of saline adminis- likelihood. Comparisons of lamb behaviour were made tered to lambs in the Placebo treatment was also based on between the three composite traits; total pain, total normal the recommended doses for Metacam® 20. Tri-Solfen® was and lying. Data were analysed over time for each hour and administered immediately after mulesing at 8 ml for lambs each day, and for day 0 and day 1 combined, and the values between 11 and 15 kg and 10 ml for lambs between 16 and reported are from a total score of 3. A number of correlated 20 kg. structures were examined, with a split plot in time structure considered appropriate for all analyses. The fixed effects Assessment of lamb behaviour were treatment, time of measurement, rear type, sex and On day 0, four observers were allocated five or 10 lambs per interactions thereof. Random effects included plot, lamb plot to record the behaviours categorised in Table 1 and were (nested within plot) and measurement period (nested within blinded to the treatments. All observers were experienced in lamb within plot) along with observer and ewe source. For assessing sheep behaviour having been trained before the live weight change of the

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ienced in lamb within plot) along with observer and ewe source. For assessing sheep behaviour having been trained before the live weight change of the lambs between days −7 and 10, commencement of the experiment to standardise their treatment, sex, rear type and interactions thereof were fitted observations for comparability. Live observations were made as fixed effects while plot and lamb (nested within plot) 5 min after the lamb was released from the marking cradle along with ewe source were fitted as random effects. For all and then every 15 min for the following 6 h. Behaviours analyses there were no significant interactions and so the recorded were standing, walking and lying; standing and final models only included main effects of the fixed terms. walking were then further categorised into normal and Main effects and interactions have been considered sig- abnormal behaviours as described by Small et al. (2014) nificant at the 5% level. Difference between means were (Table 1). Comparisons of lamb behaviour were made assessed using LSD (P = 0.05). There were generally no sig- between three composite traits; total pain (hunched and nificant differences in behavioural measures between Tri- abnormal standing and stiff walking), total normal (normal Solfen® and Tri-Solfen® and placebo injection hence only standing and walking) and lying (ventral, lateral and ventral/ data for the Tri-Solfen® treatment are reported. lateral lying). The lambs were observed for a total of 15 s at each time point and the most dominant behaviour expressed Results was recorded for each 5-s interval. The data were then scored as 0 (not observed during 15 s), 1 (dominant behaviour in Effect of mulesing on lamb behaviour one 5-s interval), 2 (dominant behaviour in two 5-s

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red as 0 (not observed during 15 s), 1 (dominant behaviour in Effect of mulesing on lamb behaviour one 5-s interval), 2 (dominant behaviour in two 5-s intervals) On average, lambs that were mulesed and not administered or 3 (dominant behaviour in all three 5-s intervals) at each pain relief exhibited significantly more pain-related beha- measurement period. One observer from each plot on day 0 viours during the first 6 h post-mulesing than lambs that was used for the observations on days 1 to 4 and each were not mulesed (1.22 v. 0.22; P < 0.05; Table 2). The observer assessed the same lambs each day. On day 1, proportion of pain-related behaviours for mulesed lambs behaviours were recorded every 15 min for 2 h and on days 2 without pain relief was relatively constant over the first 6 h to 4 behaviours were recorded every 15 min for 1.25 h. On (P > 0.05; Table 3). Furthermore, over the first 6 h lambs days 1 to 4, observations of lamb behaviour on the first plot that were mulesed and not administered pain relief exhibited commenced at 9 am and once completed the observers significantly fewer normal behaviours and lying than lambs moved through the plots in numerical order. that were not mulesed (P < 0.05; Table 2). 2588 Behavioural effects of pain relief for mulesing 0.267 0.490 Control = lambs were not mulesed; Placebo = lambs were mulesed and administered no pain relief; MC-15 = lambs were mulesed and administered Metacam® 20 15 min before mulesing; TS = lambs were mulesed and administered 0.297 0.479 0.454 LSD* There were also significant differences in pain-related behaviours (0.50 v. 1.11; P < 0.05; Table 2) and normal < 0.001 < 0.001 0.265 0.741 0.631 P-Value behaviours (1.94 v. 1.31; P < 0.05; Table 2) on day 1 Tri-Solfen®; MC-15 + TS = lambs were

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ble 2) and normal < 0.001 < 0.001 0.265 0.741 0.631 P-Value behaviours (1.94 v. 1.31; P < 0.05; Table 2) on day 1 Tri-Solfen®; MC-15 + TS = lambs were mulesed and administered a combination of Metacam® 20 15 min before mulesing and Tri-Solfen®; MC0 = lambs were mulesed and administered Metacam® 20 in the cradle. between lambs that were mulesed without pain relief and those that were not mulesed; however, these differences 0.51A MC0 1.47 0.42 1.32 1.40 were not evident on days 2 to 4. There were no significant differences in the frequency of lying between mulesed and MC-15 + TS 0.88A non-mulesed lambs from days 1 to 4. There were no sex or 1.27 0.42 1.09 1.46 rear type effects between lambs in each treatment through- Lying3 out the experiment. 1.60AA 0.55A TS 0.48 1.18 1.74 There was a significant difference between treatments in live weight change of the lambs between days −7 and 10. On Control Placebo MC-15 0.47A 1.62A average, lambs that were not mulesed gained 3.9 kg com- 0.53 1.28 1.70 pared to 1.9 kg for lambs mulesed without pain relief (P < 0.001). 0.46A 1.68A 0.57 0.93 1.51 Effect of pain relief on lamb behaviour 0.83B 0.54 1.04 1.13 1.53 On average, lambs that were administered a combination of Tri-Solfen® and meloxicam 15 min before mulesing exhibited 0.322 0.458 0.485 0.516 0.444 LSD* significantly fewer pain-related behaviours throughout day 0 than those mulesed with no pain relief (0.85 v. 1.22; 0.018 < 0.001 0.042 0.255 0.292 P-Value P < 0.05; Table 2). Furthermore, lambs that were adminis- tered the combination of pain relief displayed significantly fewer pain-related behaviours than lambs administered Tri- 1.36A 1.47A MC0 1.30 1.20 1.50 Solfen® only on day 0 (0.85 v. 1.16; P < 0.001; Table 2). The MC-15 + TS magnitude of pain-related

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bs administered Tri- 1.36A 1.47A MC0 1.30 1.20 1.50 Solfen® only on day 0 (0.85 v. 1.16; P < 0.001; Table 2). The MC-15 + TS magnitude of pain-related behaviours exhibited by lambs 1.10A 1.13A 1.72B 1.21 1.40 from all other pain relief treatments did not differ post- Behaviours Normal2 mulesing. Overall meloxicam administered 15 min before mulesing 1.08A 1.19A 1.28A 1.43 1.10 TS did not significantly affect pain-related behaviour on day 0. Table 2 Effects of pain relief on average daily behaviour 5 days post-mulesing in Merino lambs However, at 4 and 5 h post-mulesing, the lambs exhibited Control Placebo MC-15 1.37AA 1.11A 1.32 1.33 1.19 significantly fewer pain-related behaviours in comparison to lambs mulesed with no pain relief (P < 0.05). This was con- 1.31A 1.23A 1.13 1.72 1.38 sistent with the response at 4 and 5 h for lambs treated with the combination of meloxicam and Tri-Solfen® (Table 3). There were no significant differences in pain-related 1.62B 1.94B 1.97B 1.66 1.48 Means within a row are significantly different to the Placebo treatment (P < 0.05). Means within a row are significantly different to the Control treatment (P < 0.05). behaviour between mulesed lambs that received pain relief and those that did not on days 2, 3 or 4. In addition, there 0.304 0.428 0.336 0.161 0.109 LSD* was no significant difference between lambs that were mulesed and administered pain relief and those that were < 0.001 < 0.001 0.099 0.513 0.278 P-Value not when pain-related behavioural scores were averaged Total pain = hunched and abnormal standing and stiff walking. across days 0 and 1 (1.17 v. 1.11; P = 0.12). There were no Values are respresented as observations scored out of three. 0.94A 1.18A MC0 0.47 0.10 0.06 sex or rear type effects on pain-related behaviour

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were no Values are respresented as observations scored out of three. 0.94A 1.18A MC0 0.47 0.10 0.06 sex or rear type effects on pain-related behaviour between lambs that were provided pain relief and those that were not. Total lying = ventral, lateral and ventral/lateral lying. MC-15 + TS Furthermore, live weight had no significant impact on pain- 0.85AB 1.40A 0.64A 0.15 0.03 related behaviour between lambs that received pain relief Total normal = normal standing and walking. Pain1 and those that did not (P = 0.264). 1.16A 1.33A 0.13A Normal behaviours exhibited by lambs on day 0, on 0.36 0.16 TS average, were not significantly different between treat- Day Control Placebo MC-15 1.03A 1.31A ments regardless of whether pain relief was administered or 0.42 0.11 0.02 not. The number of observations of normal behaviour increased significantly in the 6 h following mulesing 1.22A 1.11A 0.30 0.12 0.09 (P < 0.05; Table 3). Female lambs also displayed more nor- *LSD (P = 0.05). mal behaviour than males (1.38 v. 1.16; P = 0.01). From days 0.22B 0.50B 0.13 0.01 0.01 1 to 4, there were no significant differences in normal behaviour between pain relief treatments, sex or rear type of the lambs. 0 1 2 3 4 A B 1 2 3 2589 2590 Table 3 Effects of pain relief on average hourly behaviour 6 h post-mulesing in Merino lambs Average hourly behaviours on day 0 Inglis, Hancock, Laurence and Thompson Pain1 Normal2 Lying3 Hour Control Placebo MC-15 TS MC-15 + TS MC0 P-value LSD* Control Placebo MC-15 TS MC-15 + TS MC0 P-value LSD* Control Placebo MC-15 TS MC-15 + TS MC0 P-value LSD* 1 0.39B 1.21A 1.09A 1.08A 0.84 0.99A 0.015 0.478 1.60B 0.92A 0.80A 0.84A 1.09 1.01A 0.021 0.513 0.33 0.48 0.83A 0.72 0.71 0.75 0.332 0.470 2 0.19B 0.88A 0.82A 0.99A 0.88A 0.86A 0.009 0.470 1.38 1.33 1.16 0.71AB

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0.80A 0.84A 1.09 1.01A 0.021 0.513 0.33 0.48 0.83A 0.72 0.71 0.75 0.332 0.470 2 0.19B 0.88A 0.82A 0.99A 0.88A 0.86A 0.009 0.470 1.38 1.33 1.16 0.71AB 0.93 1.06 0.234 0.554 1.03 0.60 0.70 0.95 0.99 0.77 0.593 0.547 3 0.30B 1.21A 1.36A 1.32A 0.86A 0.99A < 0.001 0.511 1.37B 0.75 1.06A 0.81 1.07 1.10 0.323 0.538 1.17 0.76 0.43A 0.60A 0.94 0.64 0.171 0.547 4 0.16B 1.47A 0.93AB 1.18A 0.72AB 1.08A < 0.001 0.509 1.58 1.08 1.57 1.29 0.89A 1.57 0.182 0.619 0.89B 0.28A 0.43 0.39A 1.27B 0.24A < 0.001 0.469 5 0.11B 1.37A 0.83AB 1.55A 0.71AB 0.94A < 0.001 0.481 1.84 1.35 1.66 1.32 1.38 1.49 0.534 0.576 0.87B 0.26A 0.44 0.44 0.83B 0.41A 0.069 0.455 6 0.05B 0.95A 1.01A 1.07A 0.87A 0.60A < 0.001 0.492 2.00 1.54 1.77 1.51 1.42A 1.89 0.304 0.549 0.78 0.42 0.07A 0.33 0.57 0.25A 0.074 0.458 Values are respresented as observations scored out of three. Control = lambs were not mulesed; Placebo = lambs were mulesed and administered no pain relief; MC-15 = lambs were mulesed and administered Metacam® 20 15 min before mulesing; TS = lambs were mulesed and administered Tri-Solfen®; MC-15 + TS = lambs were mulesed and administered a combination of Metacam® 20 15 min before mulesing and Tri-Solfen®; MC0 = lambs were mulesed and administered Metacam® 20 in the cradle. A Means within a row are significantly different to the Control treatment (P < 0.05). B Means within a row are significantly different to the Placebo treatment (P < 0.05). 1 Total pain = hunched and abnormal standing and stiff walking. 2 Total normal = normal standing and walking. 3 Total lying = ventral, lateral and ventral/lateral lying. *LSD (P = 0.05). Behavioural effects of pain relief for mulesing Lambs that were administered the combination of observed in response to mulesing in the current study are Tri-Solfen® and

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of pain relief for mulesing Lambs that were administered the combination of observed in response to mulesing in the current study are Tri-Solfen® and meloxicam 15 min before mulesing spent unknown. more time lying than lambs administered no pain relief on Lambs that were only treated with Tri-Solfen® had sig- day 0 (P < 0.05; Table 2). This again reflected treatment nificantly more pain-related behaviours on day 0 in compar- differences 4 h post-mulesing (Table 3). There were no sig- ison to lambs treated with the combination of meloxicam and nificant differences in lying behaviour between sexes or rear Tri-Solfen®, and there was no difference in pain related types from days 0 to 4. There was also no significant differ- behaviours when compared to lambs mulesed and adminis- ence in live weight change from days −7 to 10 between tered no pain relief. The absence of an effect of Tri-Solfen® lambs administered pain relief and those that were not alone on lamb behaviour in response to mulesing, is contrary (P >0.05). to previous studies (Paull et al., 2007; Lomax et al., 2008 and 2013). Paull et al. (2007) found Tri-Solfen® to have moderate analgesic effects 4 h post-mulesing as evidenced by less hunched standing compared to lambs mulesed without pain Discussion relief. However, Lomax et al. (2013) found wound sensitivity Mulesing significantly increased the pain-related behaviours and pain-related behaviours were reduced for 24 h. The as evidenced by hunched standing and stiff walking in shorter term of pain relief observed by Paull et al. (2007) was comparison to lambs that were not mulesed. The 20% dif- suggested to be caused by the collection of blood samples to ference in pain-related behaviours across days 0 and 1 was measure cortisol concentrations, which

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d to be caused by the collection of blood samples to ference in pain-related behaviours across days 0 and 1 was measure cortisol concentrations, which likely exacerbated pain similar in magnitude to that reported by Paull et al. (2007 responses. The present study suggests this was not the case as and 2008). Lambs that were not mulesed displayed sig- the experimental design mimicked an on-farm scenario with nificantly fewer pain-related behaviours in the 30 h following no physical disturbances and the effectiveness of Tri-Solfen® marking compared to lambs that were mulesed. This result, was not reflected in behaviour. The previous studies discussed and the results referred to from previous research, provides had relatively low animal numbers in comparison to this study, evidence in support of the initial hypothesis that behavioural and the animals used were either housed indoors or on observations can be used to assess the effect of pain relief in pasture-covered pens less than one-tenth the size of what was mulesed lambs. used in this experiment. This may account for the differences The combination of meloxicam and Tri-Solfen® sig- observed between experiments in the effectiveness of Tri- nificantly reduced pain-related behaviours in the 6 h post- Solfen® in reducing pain-related behaviours. mulesing compared to lambs mulesed and administered no The average incidence of pain related behaviours on day 0 pain relief, supporting our second hypothesis. Independently, was similar for lambs administered the combination of Tri-Solfen®, meloxicam administered 15 min before mulesing meloxicam and Tri-Solfen® and individual administration of and meloxicam administered in the cradle did not affect the meloxicam 15 min before mulesing. Furthermore, adminis- average

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dividual administration of and meloxicam administered in the cradle did not affect the meloxicam 15 min before mulesing. Furthermore, adminis- average incidence of pain-related behaviour during the day tration of meloxicam 15 min before mulesing significantly after mulesing compared to lambs mulesed and given no decreased pain-related behaviours at 4 and 5 h post- pain relief. As there were no differences in pain-related mulesing when compared to lambs that received no pain behaviours between the administration times of meloxicam relief. Therefore, the present study suggests the main our third hypothesis was rejected. Overall, our findings sug- analgesia responsible for the effectiveness of the combina- gest that under the conditions of this study a combination of tion treatment was meloxicam. This is consistent with Small a topical analgesia and meloxicam was most effective at et al. (2014) who reported that buccal meloxicam decreased decreasing pain-related behaviours associated with mulesing the combined abnormal behaviours (hunched standing, in Merino lambs. The effectiveness of combining meloxicam standing with stretched posture and walking stiffly) for 8 h and Tri-Solfen® to decrease pain-related behaviours is con- following knife castration and tail docking compared to sistent with Paull et al. (2007). They found the combination lambs offered no pain relief. of carprofen and a topical anaesthetic reduced the acute rise The relative effectiveness of meloxicam in reducing pain- in cortisol for 24 h and peak plasma cortisol concentrations related behaviours may be related to dose and time of were not significantly different to non-mulesed animals. administration. Paull et al. (2008) found meloxicam admi- Twelve hours of behavioural observations also indicated

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ly different to non-mulesed animals. administration. Paull et al. (2008) found meloxicam admi- Twelve hours of behavioural observations also indicated that nistered at time of mulesing at 0.5 mg/kg had no effect on animals administered the combination of analgesics spent behaviour. Colditz et al. (2011) administered meloxicam at less time standing and in a hunched posture and were not 1 mg/kg, which relieved sheep from lameness at 6 to 8 h different from non-mulesed animals. Pain alleviation is likely over a 24-h period. Mulesing causes soft tissue damage that due to two mechanisms; the anaesthetic properties of the results in rapid inflammation. Given NSAIDs preferentially topical analgesia working directly on nerve fibres to block accumulate in inflamed tissue, administration of a NSAID at pain signals (Sheil, 2015), and the NSAID inhibiting inflam- the time of mulesing will allow for quick absorption (Paull mation and associated pain by reducing pressure on nerve et al., 2008; Schweitzer et al., 2009). However, the analgesic endings (Davies et al., 1984). The contribution of these effect will depend largely on the dose administered. Phar- mechanisms to the expression of the pain related behaviours macokinetics of NSAIDs cannot be transferred from one 2591 Inglis, Hancock, Laurence and Thompson species to another (Welsh et al., 1993), and to the authors’ References knowledge only two studies have investigated the pharma- Chapman R, Fell L and Shutt D 1994. A comparison of stress in surgically cokinetics of meloxicam in sheep and showed that plasma and non-surgically mulesed sheep. Australian Veterinary Journal 71, concentrations of meloxicam were detectable for 24 to 72 h 243–247. (Shukla et al., 2007; Stock et al., 2013). Dose rates and Coetzee JF, Mosher R

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71, concentrations of meloxicam were detectable for 24 to 72 h 243–247. (Shukla et al., 2007; Stock et al., 2013). Dose rates and Coetzee JF, Mosher R and Allen PS 2009. Phatmacokentics of intraveous and expected duration of action for meloxicam (and other oral Meloxicam in ruminant calves. Veterinary Therapeutics 10, 1–8. NSAIDs) in previous sheep research has been extrapolated Coetzee H and Smith R 2010. Recommendations for castration and dehorning of cattle. In Proceedings of the 43rd Annual Conference of the American Associa- from data from other species (Paull et al., 2008). This tion of Bovine Practitioners, Albuquerque, NM, USA, 19–21 August 2010, pp. includes osteoarthritis in cats and dogs and castration of 40–45. cattle and pigs (Lees et al., 1998; Mathews, 2002; Zöls et al., Colditz I, Paull D, Hervault G, Aubriot D and Lee C 2011. Development of a lameness model in sheep for assessing efficacy of analgesics. Australian Veter- 2005). Coetzee et al. (2009) documented a half-life of 27.5 h inary Journal 89, 297–304. in calves, supporting the notion that meloxicam is a good Davies P, Bailey PJ, Goldenberg MM and Ford-Hutchinson AW 1984. The role of choice for long-lasting analgesia in ruminants. With the arachidonic acid oxygenation products in pain and inflammation. Annual amount of soft tissue damage caused by mulesing and Review of Immunology 2, 335–357. minimal analgesia observed in this current experiment, Fell L and Shutt D 1989. Behavioural and hormonal responses to acute surgical increased dose rates of meloxicam administered in the cradle stress in sheep. Applied Animal Behaviour Science 22, 283–294. could be investigated in combination with Tri-Solfen®. To Grant C 2004. Behavioural responses of lambs to common painful husbandry

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cience 22, 283–294. could be investigated in combination with Tri-Solfen®. To Grant C 2004. Behavioural responses of lambs to common painful husbandry procedures. Applied Animal Behaviour Science 87, 255–273. enhance adoption of injectable meloxicam by commercial Keita A, Pagot E, Prunier A and Guidarini C 2010. Pre-emptive meloxicam for sheep producers ease of application is crucial, such as the opst-operative analgesia in piglets undergoing surgical castration. Veterinary ability to administer pain relief in the marking cradle. Anaesthesia and Analgesia 37, 367–374. We conclude that using a combination of meloxicam and Lane J, Jubb T, Shephard R, Webb-Ware J and Fordyce G 2015. Priority list of Tri-Solfen® can reduce pain-related behaviours in the first endemic diseases for the red meat industries. Meat & Livestock Australia, Sydney, NSW, Australia. 6 h post-mulesing. However, this study did not observe Lee C and Fisher AD 2007. Welfare consequences of mulesing of sheep. analgesic effects through behavioural measures 24 h post- Australian Veterinary Journal 85, 89–93. mulesing regardless of the type of pain relief administered. Lees P, McKellar Q, Foot R and Gettinby G 1998. Pharmacodynamics and This paper adds support to the premise that mulesing sheep pharmacokinetics of tolfenamicacid in ruminating calves: evaluation in models is painful, this pain is measurable using pain-related beha- of acute inflammation. The Veterinary Journal 155, 275–288. viour and that a combination of analgesics is most effective Lomax S, Sheil M and Windsor P 2008. Impact of topical anaesthesia on pain alleviation and wound healing in lambs after mulesing. Australian Veterinary in mitigating the pain. Journal 86, 159–168. Lomax S, Sheil M and Windsor P 2013. Duration of

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d healing in lambs after mulesing. Australian Veterinary in mitigating the pain. Journal 86, 159–168. Lomax S, Sheil M and Windsor P 2013. Duration of action of a topical anaes- thetic formulation for pain management of mulesing in sheep. Australian Veterinary Journal 91, 160–167. Acknowledgements Mathews KA 2002. Non‐steroidal anti‐inflammatory analgesics: a review of The authors thank Boehringer Ingleheim (Sydney, Australia) for current practice. Journal of Veterinary Emergency and Critical Care 12, 89–97. funding. L.I. was the recipient of an Australian Wool Education Mellor D and Murray L 1989. Effects of tail docking and castration on behaviour Trust and Universities Federation for Animal Welfare (UK) and plasma cortisol concentrations in young lambs. Research of Veterinary scholarship and their support is sincerely thanked. Science 46, 387–391. Molony V and Kent J 1997. Assessment of acute pain in farm animals using S. Hancock, 0000-0002-4115-4642 beahvioural and physiological measurements. Journal of Animal Science 75, M. Laurence, 0000-0003-1215-2848 266–272. Molony V, Kent J and McKendrick I 2002. Validation of a method for assess- ment of an acute pain in lambs. Applied Animal Behaviour Science 76, 215–238. Paull D, Lee C, Atkinson S and Fisher A 2008. Effects of meloxicam or tolfenamic Declaration of Interest acid administration on the pain and stress responses of Merino lambs to None. mulesing. Australian Veterinary Journal 86, 303–311. Paull D, Lee C, Colditz I, Atkinson S and Fisher A 2007. The effect of a topical anaesthetic formulation, systemic flunixin and carprofen, singly or in combina- tion, on cortisol and behavioural responses of Merino lambs to mulesing. Aus- tralian Veterinary Journal 85, 98–106. Ethics Statement Phillips

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ombina- tion, on cortisol and behavioural responses of Merino lambs to mulesing. Aus- tralian Veterinary Journal 85, 98–106. Ethics Statement Phillips CJ 2009. A review of mulesing and other methods to control flystrike All procedures described were performed in accordance with (cutaneous myiasis) in sheep. Animal Welfare 18, 113–121. the Australian Code of Practice for the Use of Animals for Ricciotti E and FitzGerald GA 2011. Prostaglandins and inflammation. Arterio- Scientific Purposes 2013 and were approved by the Murdoch sclerosis, Thrombosis, and Vascular Biology 31, 986–1000. University Animal Ethics Committee (R2903/17). Sackett D, Holmes P, Abbott K, Jephcott S and Barber M 2006. Assessing the economic cost of endemic disease on the profitability of Australian beef cattle and sheep producers. Meat and Livestock Australia, Sydney, NSW, Australia. Schweitzer A, Hasler-Nguyen N and Zijlstra J 2009. Preferential uptake of the Software and data repository resources non steroid anti-inflammatory drug diclofenac into inflamed tissues after a There are no software and/or data repository resources. single oral dose in rats. BMC Pharmacology 9, 5. 2592 Behavioural effects of pain relief for mulesing Sheil ML 2015. Analgesia for surgical husbandry procedures in sheep and other Stock ML, Coetzee JF, KuKanich B and Smith BI 2013. Pharmacokinetics of livestock. Animal Ethics Pty Ltd, Associate Sydney University Faculty of Veter- intravenously and orally administered meloxicam in sheep. American Journal of inary Science, Sydney, NSW, Australia. Veterinary Research 74, 779–783. Shukla M, Singh G, Sindhura B, Telang A, Rao G and Malik J 2007. Comparative Thornton P and Waterman-Pearson A 1999. Quantification of the pain and plasma pharmacokinetics of meloxicam

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