Effect of a topical anaesthetic formulation on the cortisol response to surgical castration of unweaned beef calves

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Animal, page 1 of 7 © The Animal Consortium 2015 doi:10.1017/S1751731115001421 animal Effect of a topical anaesthetic formulation on the cortisol response to surgical castration of unweaned beef calves D. McCarthy†, S. Lomax, P. A. Windsor and P. J. White Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia (Received 21 October 2014; Accepted 1 July 2015) Impracticality and cost of existing pain management strategies during surgical castration of beef cattle have limited their widespread implementation on-farm. A farmer-applied topical anaesthetic formulation, originally developed and used commercially to mitigate the pain of mulesing in lambs, was investigated for its potential use for managing pain in surgically castrated calves. This formulation contained lidocaine, bupivacaine, adrenalin and cetrimide. In this study, 24 Angus bull calves were randomly allocated to (1) surgical castration (C, n = 8), (2) surgical castration with the post-operative application of topical anaesthetic (CTA, n = 8) and (3) sham castration/control (CON, n = 8). The experiment was conducted over 2 days, with treatment groups evenly represented across each day. Calves were habituated to handling before the experiment and blood samples were collected for plasma cortisol measurement at defined time periods before, at and post treatment, (at −0.5, 0 h, then +0.5, 1, 1.5, 2, 4 and 6 h). There was a significant effect of time on cortisol concentrations across all treatment groups ( P < 0.01), with lowest concentrations at −0.5 and 6 h and peak concentration at 0.5 h being significantly higher than the cortisol response at 0 h. The effect of treatment was not significant ( P = 0.077), however, there was a trend for CON calves to display lower cortisol

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cortisol response at 0 h. The effect of treatment was not significant ( P = 0.077), however, there was a trend for CON calves to display lower cortisol concentrations than C and CTA calves and CTA calves to display lower cortisol concentrations than C calves. The mean area under the curve (AUC) of CON calves was significantly lower than those of C and CTA calves ( P = 0.04), however, there was no significant difference between the AUCs of CTA and C calves. Immediate application of topical anaesthetic after surgical castration did not significantly reduce plasma cortisol concentrations. However, the trend for CTA calves to display lower cortisol concentrations than C calves warrants further investigation into the use of TA for pain relief of surgically castrated beef calves. Keywords: castration, cattle, cortisol, topical anaesthetic, pain management Implications Crowe, 2002; Coetzee, 2011) to prevent unwanted breeding, facilitate fattening (Puig et al., 2011) and improve meat This study investigated the use of a topical anaesthetic (TA) quality (Coetzee, 2013). Castration also reduces aggression formulation for post-operative pain relief of castrated calves, and mounting behaviours that cause injury and stress to which offers a practical option for producers to provide pain other cattle (Earley and Crowe, 2002). relief on-farm. In this study, TA had no significant effect on Pain and suffering in animals used in agriculture is of the cortisol response to surgical castration of unweaned increasing concern to consumers of livestock products (Earley Angus calves. However, there was a trend for calves treated and Crowe, 2002). Although the pain of castration in cattle with TA to have lower cortisol concentrations than untreated has been well documented (Fisher et al.,

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2002). Although the pain of castration in cattle with TA to have lower cortisol concentrations than untreated has been well documented (Fisher et al., 1996; Coetzee, castrated calves at some time points after castration. No 2011), the procedure is commonly performed without conclusions can be drawn from the current study regarding analgesic or anaesthetic intervention. Considerable research the effectiveness of TA to ameliorate pain during castration on pain alleviation in castrated calves has been published and further research is required. (Fisher et al., 1996; Earley and Crowe, 2002). However, the practicality and cost-effectiveness of these pain management strategies are a major limitation to their implementation Introduction (Petherick, 2005). To address these issues, a farmer-applied Castration of male calves is an important management ‘spray-on’ topical anaesthetic (TA) was recently studied in practice routinely performed in beef cattle (Earley and calves undergoing castration (Lomax and Windsor, 2013). This TA was shown to reduce pain-related behaviours and † E-mail: dominique.mccarthy@sydney.edu.au sensitivity of wounds and surrounding tissue for at least 24 h 1 McCarthy, Lomax, Windsor and White post-procedure (Lomax and Windsor, 2013). This followed Rural Products, QLD, Australia) for 2 min before exiting the previous studies demonstrating successful pain management race. Restraint involved manually catching the calf in the during mulesing and castration of lambs (Lomax et al., 2008; head bale in a standing position, and applying the squeeze 2010 and 2013). The TA (Tri-Solfen®; Bayer Animal Health, on the chute to reduce movement. This emulated how the Pymble, NSW, Australia) used in these studies and the calves would be handled during the trial for

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the chute to reduce movement. This emulated how the Pymble, NSW, Australia) used in these studies and the calves would be handled during the trial for treatment and current study consists of lidocaine (40.6 g/l), bupivacaine blood collection. Cows and calves were released into the (4.2 g/l), adrenalin (24.8 mg/l) and cetrimide (5.0 g/l). This 4 ha paddock between habituation periods. product is currently only registered for use in lambs under- going mulesing. Experimental use of the product in cattle is Experimental design and treatments conducted under a research permit issued by the Australian The experiment was conducted over 2 days, with treatment pesticides and veterinary medicines authority. groups evenly represented across each day. Maximum daily Assessment of cortisol concentration has been widely used temperatures for these days were 26.4 and 31.0°C. On each as a measure of acute distress in animals. Cortisol con- day, cows and calves were moved from the paddock into centration as a measure of pain-induced distress is used the holding yard adjacent to the cattle race. Calves were extensively because the response magnitude and duration, separated from their mothers into a separate holding pen, as measured by peak height and integrated cortisol response, and the cows were released back into the paddock. Calves usually accord with the predicted noxiousness of certain were moved through the race, restrained in the head bale procedures (Mellor et al., 2000). Measurement of cortisol has and released after treatment and blood sampling for every been used in cattle to quantify the effects of different painful time point. Calves generally moved through the race well. procedures such as dehorning (Sylvester et al., 1998), If required, calves were gently touched on the

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t. Calves generally moved through the race well. procedures such as dehorning (Sylvester et al., 1998), If required, calves were gently touched on the back to branding (Lay et al., 1992) and castration (Fisher et al., encourage movement. Incorporated within the race were 1996). manual slide gates, which were used to separate calves. The aim of this study was to investigate the effect of TA on This avoided over-filling of races and facilitated with ordering the cortisol response to surgical castration of beef calves and of animals. Calves were randomly assigned to one of evaluate the effectiveness of TA as an option for pain relief. It three treatment groups: (1) sham castration/control (CON, was hypothesised that provision of TA would reduce the n = 8); (2) surgical castration (C, n = 8); and (3) surgical post-operative cortisol response of calves following surgical castration with post-operative application of TA (CTA, castration. n = 8). Four calves from each treatment group were treated each day. The random order of treatments was pre- determined before calves entering the race using the animals’ identification numbers. Material and methods All calves were treated within a 0.5 h time period, Animals and housing between 1000 and 1030 h on each of the 2 experimental A total of 24 unweaned, Angus bull calves (3 months old) days. For castration, the side gate of the crush was opened were sourced from a commercial herd at the University of after the calves were restrained in the head bale. A single, Sydney property ‘Arthursleigh’ (Marulan, NSW, Australia) in experienced operator manually restrained the calves in a November 2013. The experimental protocol was approved by standing position while performing the procedure. Calves the institutional animal ethics

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in a November 2013. The experimental protocol was approved by standing position while performing the procedure. Calves the institutional animal ethics committee (Approval No. were castrated standing up, instead of employing the use of 5832). Calves had not previously undergone any husbandry a calf cradle, to eliminate any potential stress associated with procedures. Calves were held with their mothers for 5 days lateral recumbency (Tagawa et al., 1994; Pesenhofer et al., before the experiment in a 4 ha paddock, adjacent to the 2006). Castration was performed using a technique that cattle handling facilities. During this time, cows and calves required initial transverse excision of the distal third of the had ad libitum access to water and pasture. Cows and calves scrotal skin with a sterilised knife. Each testis was manually were supplemented with lucerne hay daily due to low exteriorised by pulling from the tunica vaginalis, and the pasture levels in the holding paddock and to encourage a spermatic cord cut ~12 cm proximal to the head of the positive association with the experimental environment. epididymis. This method ensured that all tissue that had been Calves were ear-tagged 2 days before experimentation and handled or contaminated was exteriorised and removed weighed using cattle scales (Weigh scale and data recorder from the calf, reducing the chance of infection of retracted W810; Gallagher Group Ltd, Hamilton, New Zealand). Calf material. For CTA calves, before removal of each testis, the BW ranged from 77 to 102 kg. Before ear-tagging, calves had exposed testicular tissue was coated with Tri-Solfen® by not been separated from their mothers and had minimal inserting the applicator nozzle along the spermatic cord exposure to humans. Calves were habituated to

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en separated from their mothers and had minimal inserting the applicator nozzle along the spermatic cord exposure to humans. Calves were habituated to movement inside the tunica vaginalis, into the inguinal cavity and through handling facilities twice daily (at 0930 and 1600 h) applying 3 ml of TA. A quantity (2 to 3 ml) of TA was also for 4 days before experimentation. Cows and calves were applied to the cut skin edge of the scrotum. This application mustered into a holding yard and quietly moved through the of TA aimed to provide maximum coverage of exposed cut race with their mothers; 1 day before experimentation, calves tissue and ensured the retracted spermatic cord was covered were restrained in the cattle crush (‘Ultimate’ Crush; RPM in a pool of anaesthetic within the inguinal canal. 2 Topical anaesthetic for castration of cattle Blood sample collection Table 1 Mean cortisol concentration (nmol/l ± s.e.m.) of calves in each Calves were numbered (1 to 24) on each flank with white treatment group over time road marking spray paint at the first blood sample collection Cortisol concentration (nmol/l) to facilitate ordering of the calves for each sampling time point. Calves were always sampled in the same order. CON C CTA Blood samples (~4 to 9 ml) were collected into 9 ml EDTA vacutainers (Vacuette®, West Heidelberg, VIC, Australia) via Time (h) Mean s.e.m. Mean s.e.m. Mean s.e.m. jugular venipuncture within 2 min of securing the calves in the head bale and manually restraining their heads. Samples − 0.5 28.5 7.15 29.13 7.73 32.27 7.56 0 63.67 12.24 63.43 5.99 61.22 7.0 for baseline cortisol were drawn 0.5 h and immediately (0 h) 0.5 63.69 11.57 84.89 8.01 85.34 8.92 before treatment. Thereafter, samples were drawn at 0.5, 1, 1 58 9.17 79.7 7.20 78.33

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.5 h and immediately (0 h) 0.5 63.69 11.57 84.89 8.01 85.34 8.92 before treatment. Thereafter, samples were drawn at 0.5, 1, 1 58 9.17 79.7 7.20 78.33 10.12 1.5, 2, 4 and 6 h post-treatment. The first and last blood 1.5 50.07 6.17 80.2 10.13 70.11 10.42 samples were collected at 0930 and 1600 h, respectively, on 2 48.76 6.87 79.69 11.89 70.02 8.70 each day. Calves were kept as a group in the holding yard 4 28.4 6.17 59.22 7.78 44.43 8.13 adjacent to the cattle race between sampling time points where 6 11.76 1.50 27.92 11.18 30.49 7.22 they had ad libitum access to water and lucerne hay and where CON = sham castrated; C = castrated; CTA = castrated + topical anaesthetic. they could see and hear their mothers through a fence. Blood Descriptive statistics are based on predicted means (±s.e.m.). No significant samples were placed on ice immediately after collection and interaction was found (P = 0.463). stored until centrifugation. Blood samples were centrifuged within 4 h of collection at 3000 r.p.m. for 15 min. The plasma Table 2 Mean cortisol concentration (nmol/l ± s.e.m.) of all calves component of the samples was separated using 1 ml sterile over time pipettes and stored in 2 ml collection vials at −20°C. Time (h) Cortisol concentration (nmol/l) s.e.m. Plasma cortisol determination − 0.5 29.96a 7.17 Plasma cortisol concentrations were determined using a 0 62.77b 8.47 commercially available radio-immunoassay kit (Coat-A-Count 0.5 77.98c 9.91 Cortisol RIA; Siemens Pty Ltd, Los Angeles, CA, USA). The 1 72.01bc 9.27 inter-assay and intra-assay coefficients of variation were 1.5 66.79bc 9.87 2 66.16b 10.42 5.05% and 5.15%, respectively. 4 44.02d 8.49 6 23.39a 7.99 Statistical analysis a,b,c,d The program GenStat® (VSN International Ltd, Hemel Values within a

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% and 5.15%, respectively. 4 44.02d 8.49 6 23.39a 7.99 Statistical analysis a,b,c,d The program GenStat® (VSN International Ltd, Hemel Values within a column with different superscripts differ significantly at P < 0.05. Descriptive statistics are based on predicted means (±s.e.m.). Hempstead, UK) was used to conduct all statistical analyses A significant effect was found (P < 0.001). and generate LSD values. Data on cortisol concentrations were subjected to residual maximum likelihood for repeated measures. The fixed effects of the model were treatment P < 0.001, Table 2). Cortisol concentrations increased (CON, C, CTA), time (−0.5, 0, 0.5, 1, 1.5, 2, 4, 6 h), day (1, 2) between −0.5 and 0.5 h relative to castration, and decreased and BW (covariate). The random effect of the model was calf. between 1 and 6 h after castration. Lowest concentrations The integrated cortisol response, or area under the curve were at −0.5 h (29.96 nmol/l) and 6 h (23.39 nmol/l) and (AUC − 0.5 to 6), was calculated for each calf and then peak concentration at 0.5 h (77.98 nmol/l) was significantly analysed using a one-way ANOVA. The suitability of the AUC higher than the cortisol response at 0 h (62.77 nmol/l). data for parametric ANOVA was tested using a probability The cortisol response at 0 h was significantly higher than plot of the residuals to determine the normality of the the cortisol response at −0.5 h. There was a statistical data, and a plot of residuals against fitted values to tendency for treatment to be significant (F = 2.95; determine the homogeneity of variance. For all statistical d.f. = 2, 19; P = 0.077). CON, C and CTA calves had mean calculations, P ⩽ 0.05 was considered statistically significant cortisol concentrations of 44.11 ± 10.05, 63.02 ±

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.077). CON, C and CTA calves had mean calculations, P ⩽ 0.05 was considered statistically significant cortisol concentrations of 44.11 ± 10.05, 63.02 ± 11.5 and and P ⩽ 0.05 ⩽ 0.1 were considered statistical tendencies. 59.03 ± 10.68 nmol/l, respectively. There was a significant Differences between means were considered statistically effect of treatment on integrated cortisol response significant if they were greater than the generated LSDs. (F = 3.78; d.f. = 2, 21; P = 0.04). The mean AUC for CON calves (253 ± 40.49 nmol/l per h) was significantly lower than the mean AUCs of C (394 ± 38.22 nmol/l per h) and CTA (372 ± 31.39 nmol/l per h) calves. Results There was no significant interaction between time and Discussion treatment (F = 0.99; d.f. = 14, 147; P = 0.463, Table 1). There was a significant effect of time on cortisol response The results of this study did not support our hypothesis that across all treatment groups (F = 25.49; d.f. = 7, 161; provision of TA would reduce the post-operative cortisol 3 McCarthy, Lomax, Windsor and White response of calves following surgical castration. The main This study is not the first to find a non-significant effect of finding was that TA had no significant effect on cortisol locally administered anaesthetic on the cortisol response of concentrations of surgically castrated calves. This was castrated calves (Fisher et al., 1996; Webster et al., 2013). the first time that the cortisol response of castrated calves The effect of lidocaine HCl, a component of TA, has been treated with TA had been studied. widely investigated for its effects on the cortisol response to In this study we elected to use cortisol as an indirect castration of calves (Coetzee, 2011). One study found that measure of pain associated

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ponse to In this study we elected to use cortisol as an indirect castration of calves (Coetzee, 2011). One study found that measure of pain associated with castration as the cortisol 2% lidocaine HCl, injected into the testes and scrotum response to castration of cattle, and the amelioration of this 15 min before castration, did not reduce the integrated response by use of anaesthetics and analgesics, has been cortisol response to surgical or burdizzo castration of Friesian well documented (Coetzee, 2011). However, despite this, it is calves (Fisher et al., 1996), despite significantly reducing still widely accepted that cortisol secretion occurs in response cortisol concentrations from 0.25 to 1 h. Fisher et al. (1996) to a variety of stressors other than pain (Molony and Kent, suggested that this was likely attributable to the short 1997). These stressors include weaning, social isolation, duration of action (~1 h) (Reichl and Quinton, 1987) of transport, social mixing, novelty, restraint and handling lidocaine HCl. This suggestion is not suitable to explain the (Stilwell et al., 2010). In addition, there are other variables, lack of difference between the integrated cortisol response of such as diurnal changes and individual variation that influ- CTA and C calves in the current study. The TA in the current ence cortisol concentration. This can implicate interpretation study consists of the anaesthetic agent bupivacaine HCl in of experimental results (Molony and Kent, 1997). The results addition to lidocaine HCl. Bupivacaine is a long acting local of the current study highlight the responsiveness of cortisol anaesthetic with a duration of action of ~5 to 8 h (Coetzee, to factors other than pain. While it is ideal to combine 2011). In addition, the adrenaline

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aesthetic with a duration of action of ~5 to 8 h (Coetzee, to factors other than pain. While it is ideal to combine 2011). In addition, the adrenaline component of TA has been multiple physiological, neuroendocrine and behavioural suggested to slow the rate of systemic absorption of lido- measures to reduce the impact of non-painful factors on caine and bupivacaine, thereby prolonging their duration of results, this usually requires using separate groups of animals action (Lomax et al., 2013). Another study that measured for each measure. As we only utilised a single group of calves cortisol concentrations of surgically castrated dairy calves for this study, our options for obtaining other data in addition found that 20 ml of 2% lidocaine HCl administered in a to cortisol concentrations were limited due to the constant subcutaneous ring block at the neck of the scrotum, just movement and repeated handling of the calves for blood above the testes, did not reduce the cortisol response to sampling. In addition, other measurements could have caused castration (Webster et al., 2013). It is likely that administra- distress which would have affected our cortisol findings. tion of lidocaine alone as a subcutaneous ring block was However, a previous study conducted by the same research ineffective at mitigating the pain of castration. It was sug- group, on the same property, provides information on the gested that the relatively high dose rate and the injection behavioural response and wound sensitivity of calves sub- into the testes rather than the spermatic cords may have jected to the same treatments as those in the current study caused tissue irritation or inflammation. It was also proposed (Lomax and Windsor, 2013). The study found that calves that the twisting and

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rrent study caused tissue irritation or inflammation. It was also proposed (Lomax and Windsor, 2013). The study found that calves that the twisting and severing of spermatic cords by the treated with TA expressed significantly less pain-related Henderson tool may have stimulated nociceptors proximal to behaviour than untreated calves and withstood greater pres- the site of lidocaine injection (Webster et al., 2013). In the sure applied to the wound and surrounding skin as measured current study, the castration procedure and the mode of by an electric von Frey anaesthesiometer (0 to 1000 g; IITC Life anaesthetic application differs to the study by Webster et al. Sciences, Woodland Hills, CA, USA). There were no significant (2013). The spermatic cords were severed using a knife after treatment differences when wound sensitivity was measured the distal third of the scrotum was excised and the testes with a von Frey monofilament (300 g; Bailey Instruments Ltd, were exposed. TA was applied postoperatively, directly onto Manchester, UK) (Lomax and Windsor, 2013). exposed, injured tissue. Therefore, a more likely explanation In this study, an increase in plasma cortisol from −0.5 to for the lack of difference between C and CTA calves in the 0 h was apparent for all treatment groups (Tables 1 and 2). current study is that the castration procedure itself caused This may reflect the stress of separation from mothers tissue damage, inflammation, stimulation of nociceptors, (Loberg et al., 2008), handling through a race, and restraint and haemmorhage, all of which can induce a rise in cortisol in a head bale (Cooke et al., 2009) before treatment. Cortisol (Gann and Egdahl, 1965; Earley and Crowe, 2002). This concentration further increased from 0 to 0.5 h to reach a

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al., 2009) before treatment. Cortisol (Gann and Egdahl, 1965; Earley and Crowe, 2002). This concentration further increased from 0 to 0.5 h to reach a explanation is also applicable when comparing the results of peak, with the rise more apparent in C and CTA calves than the current study to contrasting results from previous litera- CON calves (Table 1). In addition, the integrated cortisol ture. In some studies, local administration of 2% lidocaine response of CON calves was significantly lower than those of HCl has been shown to significantly reduce the acute cortisol C and CTA calves. The significantly greater AUCs of C and response to castration of Friesian calves, though not com- CTA calves, along with the tendency for these calves to pletely eliminating it (Ting et al., 2003; Stewart et al., 2010). display higher peak cortisol concentrations, is indicative In these studies, lidocaine HCl was injected 10 (Stewart et al., of a response to castration which involves tissue damage, 2010) or 20 min (Ting et al., 2003) before castration. stimulation of nociceptors (Earley and Crowe, 2002) and Pre-operative administration of lidocaine HCl would haemorrhage (Gann and Egdahl, 1965). have ensured amelioration of both peri-operative and acute 4 Topical anaesthetic for castration of cattle post-operative pain. TA, being applied postoperatively, had head restraint of calves. Due to the design of the current no effect on peri-operative pain, which may induce a rise in study, calves needed to be moved through the race and into cortisol (Mellor et al., 2000). In addition, the study by Ting the crush and restrained in a head bale in order to collect et al. (2003) employed the burdizzo method for castration, blood samples, regardless of indwelling catheter or jugular which causes

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n order to collect et al. (2003) employed the burdizzo method for castration, blood samples, regardless of indwelling catheter or jugular which causes a restriction in blood flow to the testes before venipuncture. The risk of the catheters being damaged removal. This reduces haemorrhage (Stafford and Mellor, or pulled out by this form of restraint meant that jugular 2005), of which cortisol secretion is a concomitant (Gann and venipuncture was a more practical option. Furthermore, Egdahl, 1965). there are contradictory results in the literature on the effects It is important to note that in the previously mentioned of venipuncture on cortisol, with some suggestions that it studies (Stafford et al., 2002; Webster et al., 2013), cortisol has no effect (Alam and Dobson, 1986) and some sugges- concentrations of uncastrated calves were significantly lower tions that it causes an increase in cortisol (Veissier and Le than those of untreated castrated calves. In the current Neindre, 1988). Hopster et al. (1999) suggest that jugular study, although the integrated cortisol response of CON venipuncture may induce an increase in cortisol concentra- calves was significantly lower than that of C and CTA calves, tion, but it seemingly relates to the handling experience of there was no significant difference between the mean corti- cattle. In the current study, manual restraint for sampling, sol concentrations of any treatment group. These findings and jugular venipuncture, may have increased cortisol con- have been demonstrated previously in a study comparing centrations. Further, in the current study, calves had never plasma concentrations of substance P and cortisol in beef experienced separation from their mothers before the calves after castration or simulated castration

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oncentrations of substance P and cortisol in beef experienced separation from their mothers before the calves after castration or simulated castration (Coetzee et al., experimental days, where they were separated for a period of 2008). In this study, mean cortisol concentrations of castra- 7 to 8 h. Studies investigating the stress of weaning have ted and uncastrated calves were not significantly different at found that separating calves from their mothers (Lay et al., any point up to 4 h following the procedure. In addition, the 1998; Loberg et al., 2008; O’Loughlin et al., 2014), and mean cortisol response of castrated and uncastrated calves additionally, altering normal milk intake (Lay et al., 1998), was similar regardless of whether castrated calves vocalised results in an elevated cortisol response (Lay et al., 1998; or displayed aversive behaviour during the procedure. Similar Loberg et al., 2008; O’Loughlin et al., 2014). The calves used to the current study, Coetzee et al. (2008) used Angus in the current study were unweaned beef calves that before crossbred calves and habituation for the experiment con- experimentation had minimal exposure to humans. Studies sisted of restraint in a head bale and a rope halter for 15 to reporting an effect of castration on cortisol (Ting et al., 2003; 30 min daily for 5 days. It was proposed that non-painful Stewart et al., 2010) used dairy calves, which under com- stressors, such as handling, had an effect on cortisol that mercial conditions are permanently separated from their was disproportional to that of the nociceptive stimulus of mothers and artificially reared by humans within hours of castration (Coetzee et al., 2008). Non-painful stressors their birth (Budzynska and Weary, 2008). Commercial beef experienced by

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umans within hours of castration (Coetzee et al., 2008). Non-painful stressors their birth (Budzynska and Weary, 2008). Commercial beef experienced by the calves in the current study include production systems typically wean calves at ~6 months of separation from their mothers, novel exposure to human age, hence the period of separation from mothers in handling, and restraint. Other studies have habituated calves the current study likely caused distress and hence a major to intensive handling and holding facilities for 3 weeks cortisol response. before experimentation commenced (Ting et al., 2003; The effect of TA on the cortisol response to painful Stewart et al., 2010). This extensive habituation reduced the husbandry procedures has been explored in other production effect of handling on treatment outcomes, resulting in a animal species. A study investigating a short acting TA, significant effect of castration on cortisol response (Ting and a long acting TA found that both formulations were et al., 2003; Stewart et al., 2010). The calves used in the unsuccessful at reducing the cortisol response to castration in current study underwent a less intensive, shorter habituation piglets. The short acting TA contained 14% Benzaine, 2% process. Hence, the intensity and duration of habituation Butamben and 2% Tetracaine hydrochloride and the long may not have been sufficient to eliminate the stress caused acting TA was the same product as that used in the current by handling and restraint in a head bale. Furthermore, pre- study (Sutherland et al., 2010). Sutherland et al. (2010) vious studies also inserted indwelling jugular catheters 1 day suggests that as TA is applied postoperatively, the pain before experimentation to facilitate intensive blood sampling of the

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jugular catheters 1 day suggests that as TA is applied postoperatively, the pain before experimentation to facilitate intensive blood sampling of the castration procedure itself may have overshadowed and minimise animal handling (Ting et al., 2003; Stewart any effect of TA on cortisol. It was also suggested that et al., 2010). Calves in one of these studies were held in the anaesthetic or application method was inadequate individual pens for the duration of the trial. For that reason, (Sutherland et al., 2010). These limitations can be applied to manual restraint for each blood sample was possible the current study. A study investigating the pain relieving (Ting et al., 2003). In the other study, blood samples effects of the same TA for mulesing and tail docking in lambs were only taken −20, −10, 15 and 20 min relative to found that TA significantly, yet only moderately, reduced the castration, which meant that each calf could be restrained peak cortisol response to the procedure and it had no effect one at a time in a squeeze chute for the duration of sample on the AUC (Paull et al., 2007). Paull et al. (2007) found that collection (Stewart et al., 2010). Therefore, in both of these combining this TA with the non-steroidal anti-inflammatory studies, access to the catheter did not require movement or drugs (NSAIDs) carprofen and flunixin, resulted in a greater 5 McCarthy, Lomax, Windsor and White decrease in peak cortisol than TA alone, as well as a Gann DS and Egdahl RH 1965. Responses of adrenal corticosteroid significant reduction in AUC. Therefore, the effect of TA in secretion to hypotension and hypovolemia. The Journal of Clinical Investigation 44, 1–7. combination with an NSAID on the cortisol response of Hopster H, van der Werf JTN, Erkens JHF and

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emia. The Journal of Clinical Investigation 44, 1–7. combination with an NSAID on the cortisol response of Hopster H, van der Werf JTN, Erkens JHF and Blokhuis HJ 1999. Effects of calves to castration is worth future investigation. repeated jugular puncture on plasma cortisol concentrations in loose-housed dairy cows. Journal of Animal Science 77, 708–714. Lay DC Jr, Friend TH, Bowers CL, Grissom KK and Jenkins OC 1992. A com- Conclusion parative physiological and behavioral study of freeze and hot-iron branding using dairy cows. Journal of Animal Science 70, 1121–1125. In this study there was no significant effect of treatment on Lay DC Jr, Friend TH, Randel RD, Bowers CL, Grissom KK, Neuendorff DA and the cortisol response of unweaned beef calves. It is likely that Jenkins OC 1998. Effects of restricted nursing on physiological and behavioral an insufficient habituation period, in addition to separation reactions of Brahman calves to subsequent restraint and weaning. Applied Animal Behaviour Science 56, 109–119. of calves from their mothers, may have caused an increase in Loberg JM, Hernandez CE, Thierfelder T, Jensen MB, Berg C and Lidfors L 2008. calf cortisol concentrations independent of pain. This may Weaning and separation in two steps – A way to decrease stress in dairy calves have masked any effects of TA on the pain of castration. The suckled by foster cows. Applied Animal Behaviour Science 111, 222–234. tendency for castrated calves treated with TA to have Lomax S, Dickson H, Sheil M and Windsor PA 2010. Topical anaesthesia alle- reduced cortisol concentrations at some time points after viates short-term pain of castration and tail docking in lambs. Australian Veterinary Journal 88, 67–74. castration and a reduced integrated cortisol

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viates short-term pain of castration and tail docking in lambs. Australian Veterinary Journal 88, 67–74. castration and a reduced integrated cortisol response com- Lomax S, Sheil M and Windsor PA 2008. Impact of topical anaesthesia on pain pared with untreated castrated calves warrants further alleviation and wound healing in lambs after mulesing. Australian Veterinary investigation. Future studies should incorporate more Journal 86, 159–168. extensive habituation of calves to reduce the impact of stress Lomax S, Sheil M and Windsor PA 2013. Duration of action of a topical anaes- on results. thetic formulation for pain management of mulesing of sheep. Australian Veterinary Journal 91, 160–167. Lomax S and Windsor PA 2013. Topical anesthesia mitigates the pain of cas- tration in beef calves. Journal of Animal Science 91, 4945–4952. Acknowledgements Mellor DJ, Cook CJ and Stafford KJ 2000. Quantifying some responses to pain as The authors gratefully acknowledge the financial support a stressor. In The biology of animal stress: basic principles and implications for of Meat and Livestock Australia and Bayer Animal Health animal welfare (ed. GP Moberg and JA Mench), pp 171–198. CABI Publishing, Wallingford, UK. Australia. The authors are grateful for the technical assistance Molony V and Kent JE 1997. Assessment of acute pain in farm animals of Steve Burgun and his staff at Arthursleigh, Marulan, NSW, using behavioral and physiological measurements. Journal of Animal Science 75, and honours students Charissa Harris and Samantha Faber from 266–272. the University of Sydney. The authors thank Kim Heasman O’Loughlin A, McGee M, Doyle S and Earley B 2014. Biomarker responses to from the University of Sydney for assistance with laboratory weaning stress in beef

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