Effects of a topical anaesthetic formulation and systemic carprofen, given singly or in combination, on the cortisol and behavioural responses of Merino lambs to castration

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PRODUCTION ANIMALS Effects of a topical anaesthetic formulation and systemic carprofen, given singly or in combination, on the cortisol and behavioural responses of Merino lambs to castration DR Pauli, C Lee, iG Colditz? and AD Fisher’* ( vee and tail docking of lambs are standard husbandry Objective To determine the effectiveness of a topical anaes- procedures in many sheep production systems. Castration thetic formulation (Tri-Solfen) with or without the administration permanently prevents the development of behavioural of a non-steroidal anti-inflammatory drug (carprofen) on the pain characteristics of male rams such as aggression towards other rams and distress response associated with ring or surgical castration of and unwanted matings, Tail docking significantly reduces the risk ram lambs, of flystrike.’ Counterbalancing the life-long outcomes of these Procedures Merino ram lambs (n = 78) were allocated to 10 husbandry practices is the associated short-term pain. Considerable treatment groups: 4 groups of knife-castrated lambs and 4 groups research has therefore examined the welfare consequences of vari- of ring-castrated lambs received carprofen (4 mg/kg SC) and ous castration and tail docking methods, and the effectiveness of Tri-Solfen; 2 control groups (sham) received carprofen at 0 or4 mg/kg analgesic strategies,” SC, Measurements included plasma cortisol and haptoglobi n Dinniss et al found that infiltrating local anaesthetic into the scrotal concentrations, haematology, and behaviour, including posture, neck was an effective method of reducing the cortisol response of Results Knife-castrated jambs had higher peak cortisol and lambs to ring castration.’ Similarly, injection of local anaesthetic into integrated cortisol responses for the first 6h

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had higher peak cortisol and lambs to ring castration.’ Similarly, injection of local anaesthetic into integrated cortisol responses for the first 6h after treatment and the spermatic cords and scrotal neck reduced animal responses to greater concentration s of circulating acute phase proteins burdizzo and ring castration, with bupivacaine producing a longer than ring-castrated lambs, both of which were significantly different lasting benefit than lignocaine.’ Typically, surgical castration of from the sham controls. Tri-Solfen applied to the knife castration lambs produces a greater physiological stress response than ring wound significantly reduced both the castration,’ although the types of behaviours induced by surgical and peak plasma cortisol concentration and the integrated cortisol response for the ring castration are different, making it difficult to compare these first 6 h and improved lying behaviour in the first 12 h. Carprofen reduced methods using behaviour alone.”* the cortisol response to knife castration at 30 min, but elevated the For tail docking, both behavioural and cortisol data indicate that cortisol responses at 24 and 48h. Carprofen nearly halved the performing the procedure with a heated cautery docking iron is less number of acute pain behaviours associated with ring castration. stressful than tail docking with a knife, with ring application being There were no significant additive or synergistic effects from intermediate.’ The behavioural responses to hot iron docking have combining the analgesic treatments. Tri-Solfen applied to the tail been shown to be ameliorated by subcutaneous or epidural adminis- wound provided no detectible benefits during ring castration + tail tration of local anaesthetic.’ The intramuscular

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subcutaneous or epidural adminis- wound provided no detectible benefits during ring castration + tail tration of local anaesthetic.’ The intramuscular administration of docking. diclofenac 20 min before treatment reduced the cortisol responses to Conclusions The physiological and behavioural responses rubber-ring castration in lambs, but increased behavioural changes,’ suggest that ring castration has less impact on the lamb than In a separate study, carprofen administration did not alleviate knife castration. The specific analgesic treatments can provide modest discomfort-associated behaviours in lambs castrated and tail-docked amelioration of the pain and discomfort associated with castra- with rubber rings.’ A topical analgesic spray was of only slight benefit tion. Alternative doses or application methods may enhance their in reducing the behavioural responses of lambs to tail docking with efficacy. rubber rings,’ Despite extensive research, there has been little uptake of anaesthetic Keywords anaigesia; castration; cortisol; lambs; pain; tail docking approaches to reduce the pain and stress responses of lambs to castration and tail docking, which may be related to problems of Abbreviations ADG, average daily gain; AUC, area under curve; practicality in achieving effective local anaesthetic infiltration of the CV, coefficient of variation; DM, dry matter; NSAID, non-steroidal scrotum and tail of young lambs, particularly given the numbers of anti-inflammatory drug animals that are typically treated on farms. Regulatory issues also Aust VetJ2009;87:230-237 dok: 10.1111/.1751-0813.2009.00429.x limit the use of analgesic treatments in sheep, We recently found that a combination of a topical anaesthetic formu- lation and the non-steroidal anti-inflammatory drug

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nalgesic treatments in sheep, We recently found that a combination of a topical anaesthetic formu- lation and the non-steroidal anti-inflammatory drug (NSAID), car- profen, significantly reduced the stress and pain associated with *Correspondence author. CSIRO FD McMaster Laboratory, Locked Bag 1, Armidale, NSE 2350 surgical mulesing.’ Previous research demonstrated the efficacy of "Faculty of Veterinary Science, University of Melbourne, VIC 3010; carprofen in attenuating the development of hyperalgesia in an adfisher@unimelb.edu.au ischaemic pain model in sheep,'° and carprofen has a relatively long 230 © 2009 The Authors Australian Veterinary Journal Volume 87, No 6, June 2009 Journal compilation © 2009 Australian Veterinary Association PRODUCTION ANIMALS elimination half-life in this species,'' Ring and knife castration are be effective in controlling pain associated with mulesing of lambs.’ likely to cause different pain responses to those accompan ying The remainder of the experimental animals received 1 mL pyrogen mulesing, so in the present study we examines the potential for these free saline SC at this time. A 3-mL dose of topical anaesthetic (40.6 g/L drugs to provide analgesia following castration and tail docking. Our lignocaine, 4.5 g/L bupivacaine, 24.8 mg/L adrenaline, 5.0 g/L cetrimide: hypothesis was that the application of either carprofen or the topical Tri-Solfen, Animal Ethics, VIC, Australia) was sprayed onto the anaesthetic formulation would reduce the pain and stress responses wound resulting from knife castration and/or onto the butt of the tail to castration and tail docking, and that the effect would be enhanced following hot knife tail docking. by a combination of the two classes of drug for surgical castration. To facilitate the

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the effect would be enhanced following hot knife tail docking. by a combination of the two classes of drug for surgical castration. To facilitate the collection of biological samples and behavioural observations, the experiment was performed in eight replicates, Materials and methods blocked across treatments. Thus one lamb from each of the 10 groups Animals and treatments was allocated to each block. One block per day was subjected to The experiment was undertaken at CSIRO’s FD experimentation over a 2-week period. The day before castration McMaster and Laboratory, Armidale, NSW and was approved by the institutio tail docking, a block of lambs and ewes was placed in a study pen nal Animal Ethics Committee. Seventy-eight fine adjoining the main group. The study pen was divided in half such wool Merino ram lambs at a mean age of 5 weeks were randomised on body that each section (4.5 x 3.0 m) housed five lambs and ewes. At this weight into treatment groups as shown in Table 1. Half the time, lambs were weighed and individually identified with coloured lambs in the castration treatments were castrated by elastrator rubber spray to assist behavioural observations. Ewes and lambs were rings and the held in other half were surgically castrated by knife. For the knife-cast the study pen for 24 h after castration, during which time blood samples rated animals, the lower half of the scrotum was excised and were collected via venepuncture and behaviours were recorded the testes on removed by the pull method. video. The ewes and lambs were then returned to the larger group until the completion of blood sampling and then put back on pasture. The ewes and lambs were accommodated in group pens in an animal Lambs were again weighed 21 days post castration. All lambs had house for a

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n pasture. The ewes and lambs were accommodated in group pens in an animal Lambs were again weighed 21 days post castration. All lambs had house for a 2-week period before the commencement of the study, to EMLA topical cream (2.5% lidocaine, 2.5% prilocaine; AstraZenec a, acclimatise them to the feed rations and housing. Sheep were fed a North Ryde, NSW, Australia) applied to the site of venepuncture on ration of sheep pellets (Ridley Agriproducts, Tamworth, NSW, Australia; the neck at the time of analgesic or saline administration and were 22% crude protein DM; 11.1 Mj/kg DM) at a rate of 0.8 kg/dry sheep given a clostridial 6-in-1 vaccination (Glanvac, CSL, Parkville, VIC, equivalent/day. Lambs were quietly handled for a 5-min period on Australia) and an intramuscular injection of oxytetracycline antibiotic two occasions during the week before treatment with the aim of at a dose of 10 mg/kg (Pharm Tech, West Pymble, NSW, Australia) reducing sampling stress during the experiment. During handling, just prior to castration to minimise the risk of post-surgical infection wool on the neck adjacent to the jugular vein was removed with while in the animal house. Blood samples were taken from the jugular clippers to facilitate blood sampling, vein via venepuncture into 5-mL EDTA vacutainers (Becton Dickenson, Carprofen was administered subcutaneously 90 min before Plymouth, UK) at six time points over the course of the experiment castration , and tail docking at a dose of 4 mg/kg, which was previousl commencing prior to analgesic treatment and again at y shown to 0.5, 6, 12, 24 and 48 h post castration, At each time point, blood samples were taken from lambs in the same sequence. Table 1. Experimental design At the time of castration, lambs were tail docked with a gas-fired

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od samples were taken from lambs in the same sequence. Table 1. Experimental design At the time of castration, lambs were tail docked with a gas-fired hot knife. Procedures were performed on lambs restrained in lamb- Castration n Tail dock Systemic Topical anaesthetic’ marking cradles. Sham-treatment lambs were placed in cradles for method analgesia? approximately 15 to 20 s, being the typical time taken to complete the Castration Tail husbandry procedure, but were not tail docked or castrated. Lambs wound wound were then placed in the study pens with their dams. Knife 8 + - + Cortisol determination Knife 8 + + + Plasma cortisol concentrations were determined using a commercia l Knife 8 + - + radioimmunoassay (Spectria Cortisol RIA, + Orion Diagnostica, Knife 8 + Espoo, Finland), adapted and validated for ovine plasma as described - - + previously,’ The intra-assay coefficient of variation (CV) for samples Ring 7 + + - - containing 35.4, 79.8 and 167.7 nmol/L cortisol, respectivel y, was 7.9, Ring 7 + - + 8.9, and 8.9%. The inter-assay CV for the same samples was 5.1, 2.8 Ring 8 + - - and 3.8%, respectively, Ring 8 + - - - Haptoglobin Sham 8 - + NA NA Haptoglobin concentration was assessed using a modified technique Sham 8 ~ - of Jones and Mould,” based on the principle that the haptoglobi NA NA n/ haemoglobin complex initiates a peroxidase reaction, which releases *Carpofen 4 mg/kg administered SC 90 min before treatment . oxygen from introduced hydrogen peroxide, oxidising colourless *Tri-Solfen 3 mL applied topically to wound. guaiacol to brown-coloured tetraguaiacol. Plasma samples and NA, not applicable. standards (50 WL) were added in triplicate to wells of a microtitre © 2009 The Authors Journal compilation © 2009 Australian Veterinary Association

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e. standards (50 WL) were added in triplicate to wells of a microtitre © 2009 The Authors Journal compilation © 2009 Australian Veterinary Association Australian Veterinary Journal Volume 87, No 6, June 2009 231 PRODUCTION ANIMALS plate (Sarstedt), appropriately diluted in phosphate-buffered saline ring-castrated lambs than in control lambs, and significantly higher for the linear range of the standard curve. An equal volume of in knife-castrated lambs than in ring-castrated lambs (Table 3). The methaemoglobin solution (0.12 mg/mL in 0.15 mol/L NaCl solution) area under the curve (AUC) from 0 to 6h also showed the same was added to all wells and lightly tapped to mix. Following pattern of significant differences, whereas in the interval from 6 to incubation for 10 min at room temperature, 100 HL of guaiacol 48h there were no significant differences and between 0 and 48h (Sigma Aldrich Pty Ltd, Castle Hill, NSW) solution was added (6.82 g ring and knife castration were both significantly greater than the guaiacol/930 mL, 0.24 mol/L acetic acid; pH 4.0; mixed 3 parts control lambs (Table 3). Topical anaesthetic applied to the castration guaiacol with 1 part H,O,). The absorbance was read at 450 nm ona wound reduced the peak cortisol concentration and the cortisol AUC plate reader after 3 min. The inter-plate CV for quality control for 0 to 6 h (Table 3). There were no other significant main effects or samples containing 0,013, 0.038 and 0.091 mg/mL was 11.0, 6.6 and interactions between analgesic treatments for cortisol response. 6.2%, respectively, Haptoglobin Haematology Haptoglobin concentrations were significantly elevated (P < 0.001) Haematology to determine total leukocyte, neutrophil, lymphocyte 24 and 48h after knife castration (0.403 and 0.98 mg/mL, and

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tly elevated (P < 0.001) Haematology to determine total leukocyte, neutrophil, lymphocyte 24 and 48h after knife castration (0.403 and 0.98 mg/mL, and erythrocyte counts was performed using a Cell Dyn 3500 respectively), but not after ring castration (0.123 and 0.179 mg/mL, haematology unit (Abbott Diagnostics, USA) and blood samples respectively) in comparison with control lambs (0.09 and 0.091 mg/ collected at —1.5, 6, 24, and 48 h after treatment. mL, respectively), Carprofen tended to decrease the haptoglobi n concentrations 48h after knife castration (0.652 mg/mL) as Body weight and growth compared with nil carprofen (1.056 mg/mL), but the difference did Sheep were weighed at —1, and 21 days relative to the time of not reach significance (P = 0.124). There were no other significant castration and their growth rate calculated over the experimental main effects or interactions between analgesic treatments on haptoglobin period, ; response, Behaviour Haematology The behaviour of lambs in the study pen was captured using four The greatest effect of castration on the haematological parameters digital video cameras for a total of 12h after treatment . Active was on the neutrophil count at 6h after surgery, which was still avoidance behaviours were observed for a 1-min period at 5-min significantly elevated 24h post treatment (P < 0.01), but declined intervals for a total of 1 h after castration. Postural behaviour s were from this point to 48 h, Total red blood cell count decreased over the observed at 10-min intervals for 12 h, consisting of three periods of course of sampling (P <0.001). Total leukocyte count increased 4h duration. The behaviours were categorised as shown in Table 2 by significantly over the 48h sampling period (P < 0.001). Although one observer,

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sed 4h duration. The behaviours were categorised as shown in Table 2 by significantly over the 48h sampling period (P < 0.001). Although one observer, who was unaware of the treatment codes. castrated lambs had higher leukocyte counts, there was no significant difference between the various analgesic treatment groups. The Statistical analysis neutrophil-lymphocyte ratio was elevated at 6, 24 and 48 h after both Analyses, performed in ASReml," examined the effects of castration knife (1.35, 1.01 and 0.80, respectively) and ring (1.34, 1.00 and 0.79, method on response variables in an incomplete factorial design respectively) castration in comparison with controls (0.88, 0.66 and encompassing all treatment groups. The software did not permit 0.52, respectively), although these values failed to reach significance interactions to be fitted for the unbalanced experimental design, The for method of castration. Carprofen tended to decrease the ratio fitted effects were castration (3 levels), carprofen on castration following both knife (1.22, 0.91, 0.74 vs 1.58, 1.18, 0.95 respectively, wound (2 levels), Tri-Solfen on the castration wound (2 levels) and P = 0.057) and ring (1.266, 0,84, 0.68 vs 1.53, 1.01, 0.83 respectively, Tri-Solfen on the tail wound (2 levels), Separate analyses were P=0.18) castration. conducted for ring and knife castration to examine the effect of analgesic treatment (2 levels of carprofen by 2 levels of Tri-Solfen ) Growth rate nested as a 2 x 2 factorial design within each castration method. Knife castration significantly reduced average daily gain (ADG) over Where appropriate, data underwent logarithmic transform ation the 21 days following castration (94+ 21.4 g/day) in comparison with (cortisol, neutrophil count, neutrophil-lymphocyte ratio,

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rithmic transform ation the 21 days following castration (94+ 21.4 g/day) in comparison with (cortisol, neutrophil count, neutrophil-lymphocyte ratio, haptoglob in controls (223 + 29,4 g/day, P = 0.013), while ADG in ring-castrated and active pain behaviours) before analysis. Linear contrasts were lambs (160 + 20.9 g/day) was intermediate and did not differ from performed between least squares means using the Ryan-Hol m the control or knife-castration groups. Analgesic treatments had no step-down procedure for multiple comparisons." significant main effects or interactions on ADG. Results Behaviour Cortisol Acute pain behaviours in the first hour. Restlessness, kicking and Cortisol levels were significantly elevated 30 min, 6h and 24h foot stamping and the total abnormal behaviour were significantly following knife castration and 30 min following ring castration increased in the first hour following ring castration, whereas only (Figure 1). Carprofen significantly reduced the cortisol concen- rolling and licking were increased following knife castration (Table 4). tration in blood 6h after knife castration and was followed by a Carprofen tended to reduce restlessness (P = 0,09) and decreased rebound at 24 and 48 h when cortisol was higher in carprofen- treated total pain-associated behaviours following ring castration (P = 0.05). knife-castrated lambs than in untreated animals (Figure 1). The peak There were no other significant main effects or interactions between cortisol value detected within each animal was significantly higher in analgesic treatments on pain avoidance behaviours in the first hour. 232 Australian Veterinary Journal Volume 87, No 6, June 2009 © 2009 The Authors Journal compilation © 2009 Australian Veterinary Association PRODUCTION ANIMALS Table

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Veterinary Journal Volume 87, No 6, June 2009 © 2009 The Authors Journal compilation © 2009 Australian Veterinary Association PRODUCTION ANIMALS Table 2, Description of behaviours Behaviour Abbreviation Description Active pain avoidance Restlessness RST No. of times lamb stood up and laid down, Instances of lamb tising as far as its knees included in the one count Foot stamping/kicking FSK Either a front or hindlimb (usually hindlimb) was lifted and forcefully placed on the ground while standing or was used to kick while standing or lying Rolling RL Lamb rolled from lying on one side to the other without getting up; half roils, in which the lamb roiled on its back and then returned to lying on the same side, included Jumping JMP Lamb moved forward using bunny hops with its hindlimbs Licking/biting wound site LBW Movement of the head beyond the shoulder, including both looking and touching at the source of pain and grooming Teat seeking TS No differentiation of with or without sucking Pain behaviours RST+FSK+RL+JMP+LBW Ail pain avoidance behaviours pooled Postural indicators of pain Normal ventral lying v1 Lay on sternum with legs tucked in and head up or down Abnormal ventral lying V2 Ventral lying with hindlimbs partially or fully extended or keeping scrotal region off the ground (dog sitting) Ventral lying other vu Lamb lying ventrally, but unable to clearly categoris e the posture Lateral lying L Lateral (on side) with one shoulder on ground, extension of hindlimbs with head up or down Abnormal lying L+V2 Abnormal lying categories pooled Total lying V14V24+VU4L All lying categories pooled Normal standing S1 Standing with no apparent abnormalities Statue standing SS Immobile standing with an obvious withdrawal from interaction with other pen members and outside

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ing with no apparent abnormalities Statue standing SS Immobile standing with an obvious withdrawal from interaction with other pen members and outside stimuli; legs positioned further back than normal; may show arched back Abnormal standing $2 Standing hunched or unsteadily, often associated with foot stamping, kicking and tail wagging Standing other SU Lamb standing, but unable to clearly categorise the posture Normal walking W1 Walking with no apparent abnormalities Abnormal walking W2 Walking unsteadily or stiffly, includes walking backward s, on knees, moving forward with bunny hops, circling, leaning or falling Walking other Wu Lamb walking, but unable to clearly categorise the type Total standing $14+52+SS+SU-+W1+W2+WU All standing and walking categories pooled Total abnormal behaviours V2+SS+524+W2 All abnormal! posture categories pooled Postural behaviours in the 12 h following castrati on. The pro- (P< 0.05). There were no other significant main effects or portion of time spent in each postural behaviou r divided into three interactions between analgesic treatments on postural periods over the 12 h of video surveillance is presente behaviours, d in Table 5. From 0 to 4h, ring castration resulted in an increase in lying behaviours and total abnormal behaviours and a decrease Discussion in standing behaviours, whereas knife castration altered the time spent standing The physiological and behavioural findings of the present study (P < 0.05). From 4 to 8h and 8 to 12 h, knife castration significantly conform with previous studies of the pain and discomfor reduced the amount of time spent lying and increase t associated d the time with castration of lambs.”* Behavioural and cortisol responses spent standing. Topical anaesthetic applied to the persist castration

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ciated d the time with castration of lambs.”* Behavioural and cortisol responses spent standing. Topical anaesthetic applied to the persist castration wound for 6 to 12h, with peak cortisol concentrations occurring marginally increased abnormal lying postures in each within time interval 30 min of castration and being significantly higher than for the sham following knife castration, and carprofen improve d the time spent treatments. Of the analgesic treatments used in this study, topical standing normally in each time interval following ring castration anaesthetic applied to the castration wound reduced both peak © 2009 The Authors Journal compilation © 2009 Australian Veterinary Association Australian Veterinary Journal Volume 87, No 6, June 2009 233 PRODUCTION ANIMALS 120 Cc 2 v = 100 + \ z ——®— Sham I\ —-O— Ring Ss | \ ——%— Knife = 80 - 1 \ oO i \ B i \ ce) oc B a. 60 4 p \g as \ \ 40 4 —, _ ; 20 4 -~@ 2 Oo 0 T T T qT T § 0 10 20 30 40 50 3 Knife Ring = 140 © 4204 ——6-— No top anaes ~—@—_No top anaes 100 | “--O-- Top anaes (e) Top anaes 80 + 60 | 40 4 20 | fe) 0 0 120 4 Q ——@— No carprofen ——6—— No carprofen 100 J ~~ OQ Carprofen ~~ O-- Carprofen ao! | 6o{ | 40; J 20 ° o\ P00 0 A A : : : : : 0 10 20 30 40 50 0 10 20 30 40 50 Time (hours) Figure 1. Effect of castration method and analgesic treatment on plasma cortisol responses (nmol/L) to castration and tail docking in Merino lambs. Data are back-transformed least squares means. 48Significant differences. Contrasts are between castration method (sham, ring, knife) and between level of analgesic treatment (- and 4) within each analgesic treatment nested within each castration method. plasma cortisol concentration and the integrated cortisol response knife-castrated

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thin each analgesic treatment nested within each castration method. plasma cortisol concentration and the integrated cortisol response knife-castrated or sham-treated controls. This result is similar to the for the first 6h after castration, Carprofen reduced the cortisol findings of others.*° What was somewhat surprising was the relative response to knife castration at 30 min, but elevated the cortisol lack of effect of carprofen at reducing the cortisol response to ring responses at 24 and 48h. In addition, carprofen nearly halved the castration, especially given the reduction in behavioural changes number of acute pain behaviours and improved standing behaviours produced by prior carprofen administration in ring-castrated lambs. associated with ring castration. There were no additive or synergistic It is possible that restriction of blood supply to the area immediately effects from combining analgesic treatments. Topical anaesthetic after elastrator ring placement could limit the concentrations of NSAID formulation applied to the tail wound provided no detectable reaching the major site of inflammation. Conversely, lignocaine benefits for ring castration + tail docking. injected into the testes prior to ring castration may have been more beneficial, through maintaining the drug at the site of action for a Ring castration produced a lesser integrated cortisol response over longer period than would be normally expected? Another explanation the first 6h than knife castration, although in the experimental could be that the relatively modest increase in cortisol produced by period from 6 to 48 h, ring-treated animals were not different to the ring castration offered little scope for detecting the effects of carprofen. © 2009 The Authors 234 Australian Veterinary

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ls were not different to the ring castration offered little scope for detecting the effects of carprofen. © 2009 The Authors 234 Australian Veterinary Journal Volume 87, No 6, June 2009 Journal compilation © 2009 Australian Veterinary Association PRODUCTION ANIMALS Table 3. Peak plasma cortisol concentrations and area under the cortisol curve at intervals following castration Variable Castration method Knife Ring Sham Knife Ring Carprofen TAon Carprofen TAon castration wound tail wound - - + - + - + Peak cortisol (nmol/L) 29.84 111.6¢ 60.48 112 108 125.48 96.44 74.5 63.5 67.8 69.8 AUC (nmolh/L) 0-6h 924 515° 795° 473 432 505° 405° 238 199 191 248 6-48 h 843 1337 1239 1238 1501 1353 1374 1226 1131 1153 1202 0-48 h 9374 1833° 14608 1749 1954 1878 1819 1503 1379 1410 1469 Data are back-transformed least squares means. ‘8<Significant differences. Contrasts are between castration method (sham, ring, knife) and between level of analgesic treatment (— and +) within each analgesic treatment nested within castration method, AUC, area under curve; TA, topical anaesthetic. Table 4. Effect of treatment on pain avoidance behaviours of lambs for 7h following castration Variable Castration method Knife Ring Sham Knife Ring Carprofen TAon Carprofen TAon castration wound tall wound - + - + - + ~ + Restlessness of of 3.48 0,3 fC) 2.3 2.0 49 27 3.5 39 Kicking/foot stamping o* of 1.88 0 0 ) 0 2.0 17 18 1.9 Rolling of 0,348 0.58 ) ) 0 0 0.1 0.7 0.3 0.9 Jumping 0 0 0.2 0 0 0 0 0.1 0.2 01 0,2 Licking/biting wound site 0 0.2 0.3 0 0 0 0 0.2 0.3 0.6 0.1 Teat seeking 0.3 0.1 0.7 0.1 0.1 2.0 2.2 0.4 0.8 0.6 0.6 Sum of pain behaviours of 0.14 5,9° 0.3 0 0.3 0 7.8° 4.6 57 64 Data are back-transformed least squares means of behaviours observed in lambs. A8Significant differences.

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ours of 0.14 5,9° 0.3 0 0.3 0 7.8° 4.6 57 64 Data are back-transformed least squares means of behaviours observed in lambs. A8Significant differences. Contrasts are between castration method (control, ring, knife) and between level of analgesic treatment (- and +) within each analgesic treatment nested within each castration method. There were no significant interactions between analgesic treatments. TA, topical anaesthetic. Interestingly, the use of a combined regimen of analgesics (topical immediately to the surgical site. Infiltrating the affected tissues with anaesthetic and carprofen) that has been demonstrated to be successful lignocaine before castration has been successfully demonstrated in a mulesing model’ was of little benefit in surgical castration, previously in sheep’ and in cattle.'*"” Applying topical anaesthetic to all Although the extent of tissue damage at mulesing is perceived to be the exposed tissues after excision of the lower portion of the scrotum greater, it is generally superficial and effective coverage of the entire and before severing the testes may provide better analgesic cover of surgical site with topical analgesic is achievable. In the case of surgical the affected tissues than the application method used in this study castration, the genitofemoral, superior spermatic, scrotal branch of (M. Sheil, personal communication). The physiological responses the pudenal and distal cutaneous branch of the sacral plexus nerves seen here suggest that post-surgical treatment with topical anaesthetic are all severed and activated during the procedure. Innervation of the formulation provides a modest decrease in the discomfort and pain latter two groups of scrotal nerves would be blocked quickly and associated with knife castration.

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ovides a modest decrease in the discomfort and pain latter two groups of scrotal nerves would be blocked quickly and associated with knife castration. effectively by topical anaesthetic application to the site, but the former neural groups and associated soft tissue would be stretched Haptoglobin concentrations were not significantly elevated follow- during the castration procedure and would then retract up into the ing ring castration or tail docking compared with sham-treated inguinal canal and thereby not be exposed to the analgesic applied lambs. Clearly, the tissue damage produced in tail docking and ring © 2009 The Authors Journal compilation © 2009 Australian Veterinary Association Australian Veterinary Journal Volume 87, No 6, June 2009 235 PRODUCTION ANIMALS Table 5. Effect of treatment on postural behaviours of lambs for 12 h following castration » Variable Castration method Knife Ring <x = Sham Knife Ring Carprofen TAon : = Carprofen TAon castration wound tail wound 9 3 - + - + - + - + S a. Period 1 (0-4 h) Abnormal ventrallying 6,341.24 62+0.9% 1264088 58 £07 56407 4.7+07% 6740.78 127+07 105407 124£0.7 10.6407 Abnormal lying 6.5412 6140.9 1354098 58+ 07 56407 4740.74 674078 134407 114407 132+ 08 11.6408 Normal standing 12.7+1.0° 94407 684074 83+08 93£08 87+08 8.9408 584074 7.64075 73407 61407 Total standing 20.141.49 1664108 12.541.0° 1984+1,2 19.34£1.2 20.7412 18541.2 125+09*% 149+0.98 14.141. 13.3412 Total abnormal 614118 8740.8 1414088 88+ 10 7841.0 7.6409 9.0409 143406 124406 13.9+40.7 12.9407 Period 2 (4-8 h) Abnormal ventrallying 8.6 + 1.2 5340.9 9140.8 48407 4740.7 3840.74 574078 90+07 7.1407 8940.7 7,240.7 Abnormal lying 88+ 1.2 5.3409 9240.9 5.0407 47407 3.84074 584078 91407 7.2£0,7 90408 7.3+08 Normal standing 8841.0 9.04 0.7 7.7407

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1407 8940.7 7,240.7 Abnormal lying 88+ 1.2 5.3409 9240.9 5.0407 47407 3.84074 584078 91407 7.2£0,7 90408 7.3+08 Normal standing 8841.0 9.04 0.7 7.7407 79408 90408 84+408 8540.8 68407 864078 82407 7.1407 Total standing 16.941.4% 10341.0* 11341.0% 16.7412 6241.2 175412 154412 11.340.9* 1374098 129412 12.7£1.2 Total abnormal 8441.1 7040.8 9440.8 7I1£10 61410 59409 7.3409 96+£0.6 7.7406 9240.7 82+0,7 Period 3 (8-12 h) Abnormal ventral lying 9.0 + 1.2 6140.9 8440.8 37407 55407 464074 664072 82+07 6.3 40.7 8.2407 6440.7 Abnormal lying 9541.2 61409 85+09 58407 55407 4.74074 674078 84407 6440.7 82+08 66+08 Normal standing 44+1.0 7.30.7 5.3 £0.7 62408 72408 66408 6.7£0.8 4440.7* 614078 5840.7 47407 Total standing 13,741.48 5041.04 72£10*° 135412 13.0412 143412 12.2£1.2 7.2£0.9% 964099 8841.2 8041.2 Total abnormal 88411 75£0,8 8.8+0.8 76£1.0 6641.0 6440.9 7.8409 8.9+0.6 7040.6 844207 742407 Data are untransformed least squares means (+ SEM). ‘®Significant differences. Contrasts are between castration method (control, ring, knife) and between level of analgesic treatment (- and +) within each analgesic treatment nested within each castration method. TA, topical anaesthetic, castration has minimal effect on the acute phase protein response, The administration of analgesics did not reduce the effect of castra- Surgical castration resulted in significantly elevated plasma hap- tion on the growth of lambs for a 3-week period after the procedure. toglobin concentrations 24 and 48 h after the procedure, but less than However, there were significant differences in the growth rates of lambs levels observed at similar time points following mulesing.”* Although castrated by knife, while the ring castrated lambs were intermediat e the extent of excised scrotal tissue is

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me points following mulesing.”* Although castrated by knife, while the ring castrated lambs were intermediat e the extent of excised scrotal tissue is generally minimal, the bruising between treatment groups and not significantly different from each associated with the stretching of tissues during this procedure may treatment. Knife-castrated lambs had reduced ADG of 63% and ring- well add to the cumulative haptoglobin response. The analgesic castrated lambs had ADG reduced by 33% compared with sham-treated treatments used in this study did not modify the rise in haptoglobin lambs. It is likely that castration either reduced feed intake and/or concentration following knife castration. reduced growth through altering the utilisation or partitioning of the available nutrients. Activation of the hypothalamic-pituitary- Castration resulted in elevated neutrophil counts, which peaked at adrenal axis and inflammation via the expression of cytokines are 6 h and then slowly returned to normal values. There was a lack of well-known mechanisms for nutrient partitioning in surgical models,” consistency between ring and knife castration in the haematolo gical Clearly, knife castration had a greater effect on the experimental responses. The greatest effect following castration was on the lambs than ring castration, Similar effects on the growth of lambs by neutrophil-lymphocyte ratio. Both methods of castration had a both ring and surgical castration have been demonstrated elsewhere,’ significantly higher ratio than sham-treated animals at 6h and the whereby part of the decrease in growth was attributed to reduced ratios declined from there on, but were still significantly higher at the feeding in the first 24h after surgical castration. Likewise in cattle, final sampling at

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ed from there on, but were still significantly higher at the feeding in the first 24h after surgical castration. Likewise in cattle, final sampling at 48 h, Analgesic treatments had no significant effects reduced feed intake in the 5 days following surgical castration on haematology, as previously observed over a similar time frame contributes to reduced growth rates in the subsequent week.” Nutrient following the administration of ketoprofen to cattle prior to band partitioning provides substrates for the inflammatory and healing and burdizzo castration.” response to surgical procedures at the expense of normal growth rates. 236 Australian Veterinary Journal Volume 87, No 6, June 2009 © 2009 The Authors Journal compilation © 2009 Australian Veterinary Association PRODUCTION ANIMALS The behaviours measured in the study were successful at differentia ting between castrated and non-castrated lambs, Lambs displayed References more pain avoidance behaviours in the 1 h period following ring castration 1, French NP, Wall R, Morgan KL. Lamb tail docking: a controlled field study of the than following knife castration. In a study examining different effects of tail amputation on health and productivity. Vet Rec 1994;134:463 tail -467. docking methods, the use of elastrator rings produced the greatest 2. Mellor DJ, Stafford KJ, Acute castration and/or tailing distress and its incidence of abnormal behaviours, which included both alleviation in lambs. NZ VetJ 2000;48:33-43, abnormal lying and abnormal standing postures,’ In the same study, subcuta- 3. Dinniss AS, Mellor DJ, Stafford KJ, Bruce RA, Ward RN. Acute cortisol responses of lambs to castration using a rubber ring and/or a castrating neous injection of bupivacaine was effective in overcomin clamp with or g

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sol responses of lambs to castration using a rubber ring and/or a castrating neous injection of bupivacaine was effective in overcomin clamp with or g abnormal without local anaesthetic, NZ VetJ 1997;45:1 14-121. lying behaviours to the ring method, but had only minimal effects on 4. Melches S, Mellema SC, Doherr MG, Wechsler B, Steiner A. Castration of lambs: abnormal standing postures. The current study supports the findings a welfare comparison of different castration techniques in lambs over 10 weeks of Dinniss et al, highlighting the major problems in comparing ofage. VetJ 2007;173:554-563, behavioural responses between differing pain models as experienc 5. Lester SJ, Mellor DJ, Ward RN, Holmes RJ. Cortisol responses of young ed lambs to by ring versus knife castration.” Clearly, the ischemic pain produced castration and tailing using different methods. NZ Vet / 1991;39:134 ~138, 6. Lester SJ, Mellor DJ, Holmes RJ. Ward RN, Stafford KJ, Behavioural by ring castration results in a more overt modification of behaviour and cortisol s responses of lambs to castration and tail docking using different for at least 1 h. Other pain models in this species are often characteri methods, NZ sed VetJ 1996;44:45-54, by the sheep’s stoicism, which contrasts to the heightened abnormal 7. Graham MJ, Kent JE, Molony V. Effects of four analgesic treatments on the behaviours expressed in response to ischemic pain. The difference behavioural and cortisol responses of 3-week-old lambs to tail docking. in VetJ abnormal behaviours was relatively short and did not extend beyond 1997;153:87-97, the first 4 h, The greater postural pain-related behaviours in response 8, Price J, Nolan AM. Analgesia of newborn lambs before castration and tail to ring castration, particularly in the first

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lated behaviours in response 8, Price J, Nolan AM. Analgesia of newborn lambs before castration and tail to ring castration, particularly in the first 4 h following docking with rubber rings. Vet Rec 2001;149:321~324, castration, is 9. Paull DR, Lee C, Colditz IG, Atkinson SJ, Fisher AD. The effect consistent with an integrated score based on pain behaviour of a topical s used by anaesthetic mixture, and systemic flunixin or carprofen, on modifying the Grant” and consistent with the same method of castration in calves,"® pain and stress responses of Merino lambs to mulesing. Aust VetJ 2007;85:98- The two previous studies examined behaviours over a much shorter 106. period after castration (1.5 and 4h, respectively); however, 10. Welsh EM, Nolan AM. Effects of non-steroidal anti-inflammatory drugs on in the the present study the chronic physiological responses exhibited hyperalgesia to noxious mechanical stimulation induced by the application of by the a lambs were not associated with abnormal behaviours over this tourniquet to a forelimb of sheep. Res Vet Sci 1994;57:285~-291, latter 11, Welsh EM, Baxter P, Nolan AM. Pharmacokinetics of carprofen administered period. intravenously to sheep. Res Vet Sci 1992:53:264-266, Despite the increase in abnormal behaviours exhibited 12. Jones GE, Mould DL. Adaptation of the quaiacol test for haptoglobins. by ring- Res Vet Sci 1984;37:87-92, castrated lambs, the administration of carprofen resulted in 13. Gilmour AR, Gogel BJ, Cullis BR, Welham SJ, Thompson R. ASReml user guide, amelioration of the postural changes seen over the first 4h and a Release 1.0. VSN International Ltd, Hemel Hempstead, UK, 2002. reduction in total pain avoidance behaviours in the first hour, These 14, Ludbrook J. Multiple comparison procedures

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l Ltd, Hemel Hempstead, UK, 2002. reduction in total pain avoidance behaviours in the first hour, These 14, Ludbrook J. Multiple comparison procedures updated. Clin Exp Pharmacol results suggest that the NSAID had some effect in counterin Physiol 1998;25:1032-1037. g the short-term ischaemic pain induced by ring application. It is 15. Stafford KL, Mellor DJ, Todd SE, Bruce RA, Ward RN. Effects difficult of local to fully explain the marginal increase in abnormal lying postures anaesthesia or local anaesthesia plus a non-steroidal anti-inflamm atory drug on recorded in knife-castrated lambs treated with topical anaesthetic the acute cortisol response of calves to five different methods of castration, Res , Vet Sci 2002;73:61-70, One explanation could be that these animals were comfortable 16. Earley B, Crowe MA. Effects of ketoprofen alone or in combination enough to be more likely to lie down, but that once they did with local so, they anesthesia during the castration of bull calves on plasma cortisol, immuno- were still inclined to lie in a manner that avoided pressure on logical and inflammatory responses. J Anim Sci 2002;80:1044-1052, the scrotal area. 17. Thuer S, Mellema S, Doherr MG, Wechsler B, Nuss K, Steiner A. Effect of local anaesthesia on short- and long-term pain induced by two bloodless castration Where some previous research has concluded that ring castration is methods in calves. Vet/ 2007;173:333-342, more stressful to the animal than other methods, most 18. Paull DR, Lee C, Atkinson SJ, Fisher AD. The effects of meloxicam of those and conclusions were made following behavioural assessmen tolfenamic acid administration on the pain and stress responses of Merino ts with minimal physiological data to support them. Comprehensive lambs to mulesing. Aust

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id administration on the pain and stress responses of Merino ts with minimal physiological data to support them. Comprehensive lambs to mulesing. Aust VetJ 2008;86:303-311. studies involving cortisol, haptoglobin, haematology, active behavioura 19, Pang WY, Earley B, Sweeney T, Crowe MA, Effect of carprofen administration l during banding or burdizzo castration of bulls on plasma cortisol, in vitro responses and growth parameters provide a clearer picture of the interferon-gamma production, acute-phase proteins, feed Intake and overall animal response. Taking all these factors into account, growth, ring J Anim Sci 2006;84:351-359, castration can be considered to have a lesser impact on 20. Johnson RW. Inhibition of growth by pro-inflammatory cytokines: the animal’s an homeostasis than knife castration, the other widely used method integrated view. J Anim Sci 1997;75:1244-1255, of castration for sheep in Australia. Modest pain relief for ring 21. Fisher AD, Crowe MA, Alonso de la Varga ME, Enright WJ. Effect of castration castration can be provided by single-shot carprofen injection while modest method and the provision of local anesthesia on plasma cortisol, pain scrotal circumference, growth and feed intake of bull calves, J Anim Sci 1996;74:2336 relief for knife castration can be provided by application of - topical 2343, anaesthetic formulation to the castration wound. 22, Dinniss AS, Stafford KJ, Mellor DJ, Bruce RA, Ward RN. The behaviour pattern of lambs after castration using a rubber ring and/or castrating clamp with or without local anaesthetic, NZ VetJ 1999:47:198-203, Acknowledgments 23. Grant C. Behavioural response of lambs to common painful husbandry The authors thank Andrew Eichorn, Sue Belson and Troy Kalinowsk procedures. Appl Anim Behav Sci

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avioural response of lambs to common painful husbandry The authors thank Andrew Eichorn, Sue Belson and Troy Kalinowsk procedures. Appl Anim Behav Sci 2004;87:255-273, i for their skilled technical contributions in the conduct of this study, (Accepted for publication 23 November 2008) © 2009 The Authors Journal compilation © 2009 Australian Veterinary Association Australian Veterinary Journal Volume 87, No 6, June 2009 237