Effect of sub-anesthetic xylazine and ketamine ( ketamine stun ) administered to calves immediately prior to castration

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1 Veterinary Anaesthesia and Analgesia, 2010, 37, doi: /j x RESEARCH PAPER Effect of sub-anesthetic xylazine and ketamine ( ketamine stun ) administered to calves immediately prior to castration Johann F Coetzee*, Ronette Gehring*, Jepkoech Tarus-Sang & David E Anderson* *Agricultural Practices Section, Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA PharmCATS Bioanalytical Laboratory, Department of Clinical Sciences, Kansas State University, Manhattan, KS, USA Correspondence: Johann Coetzee, Agricultural Practices Section, Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS , USA. jcoetzee@vet.ksu.edu Abstract Objective To describe the pharmacokinetics, cortisol response and behavioral changes associated with administration of sub-anesthetic xylazine and ketamine prior to castration. Study design Prospective, randomized experiment. Animals Twenty-two male beef calves ( kg). Methods Calves were randomly assigned to receive the following treatment immediately prior to surgical or simulated castration; 1) uncastrated, placebotreated control (CONT) (n = 4), 2) Castrated, placebo treated control (CAST) (n = 6), 3) castrated with intravenous xylazine (X) (0.05 mg kg )1 ) (n = 6), and 4) castrated with IV xylazine (X) (0.05 mg kg )1 ) combined with ketamine (K) (0.1 mg kg )1 )(n = 6). Blood samples collected over 10 hours post-castration were analyzed by LC-MS- MS for drug concentrations and chemiluminescent immunoassay for cortisol determination. Results Drug concentrations during the first 60 minutes post-castration fit a one-compartment open model with first-order elimination. The harmonic mean elimination half-lives (± pseudo SD) for X, X with K and K were 12.9 ± 1.2, 11.2 ± 3.1 and 10.6 ± 2.8 minutes, respectively. The proportion of the total area under the effect curve (AUEC) for cortisol during this period was significantly lower in the X group (13 ± 3%; p = 0.006) and the X+K group (14 ± 2%; p = 0.016) compared with the CAST calves (21 ± 2%). However, after 300 minutes the AUEC in the X group was higher than CAST. Significantly more calves demonstrated attitude that was unchanged from pre-manipulation behavior in the CONT (p = 0.021) and X+K treated calves (p = ) compared with the CAST calves. Conclusions Behavioral changes and lower serum cortisol concentrations during the first 60 minutes post-castration were associated with quantifiable xylazine and ketamine concentrations. Clinical relevance Low doses of xylazine and ketamine administered immediately prior to castration may offer a safe, efficacious and cost-effective systemically administered alternative or adjunct to local anesthesia. Keywords anesthesia, animal welfare, calves, castration, ketamine, xylazine. Introduction Castration of bull calves is one of the most common livestock management procedures worldwide and is considered one of the most stressful 566

2 experiences for livestock (AVMA 2009). Under the United Kingdom Protection of Animals (Anesthetics) Act, 1964 (c.39) it is an offence to castrate calves that have reached 2 months of age without the use of an anesthetic (OPSI, 2009). A recent survey of US veterinarians found that risk of injury to the operator is the most important consideration in the selection of a castration method (Coetzee et al. 2010). Injection of local anesthetics into the testicles and scrotum can be hazardous to the operator and may increase the time taken to process cattle. Furthermore, the onset of maximal local anesthesia may be variable resulting in sub-optimal pain relief at the time of castration in some practice situations (Hewson et al. 2007). A safe and efficacious systemic sedative-analgesic protocol for use alone or in combination with local anesthesia would benefit practitioners and enhance animal well-being. Xylazine is an a-2 adrenergic agonist that has sedative-analgesic effects when administered to cattle at mg kg )1 bodyweight (Garcia- Villar et al. 1981). Antinociceptive effects following IM administration at 0.05 mg kg )1 of xylazine in lambs have been described (Grant & Upton 2001). Ketamine is an NMDA-receptor antagonist that produces analgesia and dissociative anesthetic effects when administered at a dose of 2 4 mg kg )1 IV to calves (Posner & Burns 2009). Ketamine and its active metabolite, norketamine also bind mu and kappa opioid receptors producing analgesia (Annetta et al. 2005). Sub-anesthetic ketamine administered at mg kg )1 as an IV bolus is effective in managing acute postoperative pain in human medicine (Schmid et al. 1999). A sub-anesthetic combination of xylazine, administered at mg kg )1 and ketamine at mg kg )1 given IV or IM ( Ketamine Stun ) is reported to provide mild sedation without recumbency in cattle (Abrahamsen 2009). However, the pharmacokinetics and associated effects of this drug combination when administered to calves prior to castration have not been described. If this regimen produces mild sedation and analgesia without recumbency, it would provide veterinarians with a practical and cost-effective systemic sedative-analgesic protocol to reduce pain and distress associated with castration. The objective of the study reported here was to describe the pharmacokinetics, cortisol response and behavioral changes associated with administration of sub-anesthetic doses of xylazine and ketamine to beef calves prior to castration. Materials and methods This protocol was approved by the Institutional Animal Care and Use Committee at Kansas State University. Given that an untreated, castrated control group was enrolled in the study, calves were assessed hourly for behavioral signs of excessive pain over a period of 10 hours following surgery. This was followed by twice daily monitoring for 7 days. Calves demonstrating postural changes, prolonged recumbency, anorexia and depression were scheduled to receive rescue analgesia with flunixin meglumine at 2.2 mg kg )1 IV, BID. No calves were determined to require rescue analgesia during the course of the study. Experimental animals Twenty-four Angus crossbred bull calves aged approximately 4 6 months and weighing between 260 and 310 kg were acquired from a livestock commission company. Two animals were removed prior to study commencement because they had been castrated previously. Scrotal circumference for each calf was measured using a commercially available scrotal tape measure (SireMaster; ICE Corp, KS, USA) and ranged from 25 to 30 cm. Upon arrival, the calves received an eight-way clostridial vaccine (Covexin 8; Schering Plough, NJ, USA), a single injection of tulathromycin (Draxxin; Pfizer, NY, USA) at 2.5 mg kg )1 bodyweight, and doramectin (Dectomax Pour-on; Pfizer) administered topically at 500 lg kg )1 bodyweight. Housing and husbandry Study animals were acclimated for approximately 6 weeks in dry lot confinement facilities. Cattle were fed a typical receiving diet composed of whole corn, wheat middlings, dry distiller s grain, soybean hull pellets, cottonseed hulls, molasses and a protein/vitamin/mineral supplement throughout the experiment. Feed and water were offered ad libitum. Bulls were moved to individual m 2 indoor stalls in the Veterinary Teaching Hospital 24 hours prior to study commencement. The hospital is a climate controlled facility designed to maintain ambient temperature at approximately 22 C and relative humidity approximately 40%. Each stall was fitted with a head gate to allow individual animal restraint. During the individual housing period, concentrate Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

3 feed was withheld but bulls had free access to water and grass hay. Group assignment and randomization procedure Study calves were blocked by bodyweight prior to random assignment to one of the following treatment groups; 1) uncastrated, placebo-treated control (CONT) (n = 4), 2) castrated, placebo treated control (CAST) (n = 6), 3) castrated with intravenous xylazine (X) (0.05 mg kg )1 ) (n = 6), and 4) castrated with IV xylazine (X) (0.05 mg kg )1 ) combined with ketamine (K) (0.1 mg kg )1 )(n = 6). Body weights were determined within 48 hours prior to study commencement. Group assignment within blocks was based on a corresponding list of computer-generated random numbers (Microsoft Excel 2007, Microsoft Corporation, WA, USA). Jugular catheterization Approximately 12 hours prior to study commencement, calves were restrained for intravenous catheter placement. Following restraint, the area over both jugular veins was clipped and disinfected using 70% isopropyl alcohol and povidone iodine scrub swabs. The catheter sites were infiltrated with 2% lidocaine injection, 2 ml intradermally, (Hospira Inc, IL, USA) prior to placement of two 14 gauge 130 mm extended use catheters (MILACATH; MILA International, KY, USA) which were sutured to the skin using #3 nylon suture. One catheter was designated for drug administration and the other for blood sample collection. Catheter patency was maintained using heparin saline flush containing 3 USP units heparin sodium per ml saline (Heparin Sodium Injection; Baxter Healthcare, IL, USA). Xylazine, ketamine and placebo administration Xylazine hydrochloride (20 mg ml )1 ) (Anased 20 Injection; Lloyd Laboratories, IA, USA) was administered intravenously at a dose of 0.05 mg xylazine kg )1 bodyweight. Ketamine hydrochloride ( mg ml )1 ) (Vetaket Injection; Lloyd Laboratories) was administered intravenously at a dose of 0.1 mg kg )1 ketamine. Both xylazine and ketamine were co-administered in the same syringe approximately 30 seconds prior to castration using the designated jugular catheter. A volume of 0.9% sterile sodium chloride solution (Baxter Health Care Corporation) equivalent to the volume of xylazine and ketamine was administered to calves in the placebo treated groups. Designated drug catheters were flushed with heparin-saline solution and removed following administration of the drug. Castration procedure The study commenced at 07:00. Castration or simulated castration was performed at 3-minute intervals. All castrations were performed by a single experienced veterinarian to minimize variation. Prior to castration, calves were restrained in a head gate and a pre-study blood sample was collected. Thereafter, the scrotum was washed with dilute chlorhexidine disinfectant and incised using a sharp, disinfected Newberry knife. The testes and spermatic cords were then exteriorized by blunt dissection and the scrotal fascia was stripped from each exteriorized testicle. A Henderson calf castration tool (Stone Manufacturing & Supply Company, MO, USA) attached to a 6-V cordless variable-speed hand drill with a 3/8 in chuck was clamped across the entire spermatic cord just proximal to the testicle. The drill was then powered to rotate the clamped spermatic cord at a slow to moderate speed until the testicle twisted off after approximately 10 turns of the tool which took <10 seconds. The tightly coiled sealed segment of the cord then retracted into the inguinal canal. The same procedure was used to remove the second testicle. Following castration, blood samples were collected as detailed in the next section. For each calf in the control group, the effect of manipulation associated with castration was simulated. The testes within the scrotum were firmly grasped, and ventral traction was applied for approximately 20 seconds. Behavioral scoring Behavioral changes in response to castration or simulated castration were recorded as described elsewhere (Coetzee et al. 2008). Behavior was assessed by assigning a score for vocalization and changes in attitude or temperament at the time of the castration procedure. Vocalization was scored on a scale of 0 3 (0 = no vocalization; 1 = snorting or grunting; 2 = momentary vocalization; and 3 = continuous vocalization during and immediately after testicular manipulation). Attitude was also scored on a scale of 0 3 (0 = unchanged from pre-manipulation behavior; 1 = head shaking, kicking, or tail flicking; 2 = momentary escape 568 Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

4 behavior [e.g. lunging against the head gate, head shaking, and kicking]; and 3 = violent escape behavior [e.g. repeated lunging against the head gate, head shaking and kicking throughout testicular manipulation]. Blood sample collection Blood samples were collected in syringes using a designated jugular catheter immediately prior to castration or simulated castration (designated as time 0) and at 3, 10, 20, 30, 40, 50 minutes and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8 and 10 hours thereafter. Blood was immediately transferred to 6 ml serum and lithium heparin vacutainer tubes (BD Diagnostics, NJ, USA) for cortisol and drug determination respectively. Samples were stored on ice prior to centrifugation for 15 minutes at 1500 g within 30 minutes of collection. Plasma and serum was then pipetted to cryovials and frozen at )70 C until analysis. Cortisol analysis Serum cortisol concentrations were determined using a solid-phase competitive chemiluminescent enzyme immunoassay and an automated analyzer system (Immulite 0 Cortisol; Siemens Medical Solutions Diagnostics, CA, USA). This method had been previously validated in bovine serum as described elsewhere (Coetzee et al. 2008). The reported calibration range for the assay was nmol L )1, and sensitivity was 5.5 nmol L )1. Laboratory technicians who analyzed the samples were masked with respect to treatment group. Determination of plasma xylazine and ketamine concentrations Xylazine and ketamine concentrations were determined as described by Gehring et al Briefly, ketamine and a deuterated internal standard (Cerilliant, TX, USA) were purchased as solutions in methanol (MeOH). Xylazine and chlorpromazine as an internal standard were purchased from Sigma- Aldrich (St. Louis, MO, USA). A calibration curve consisting of nine points (0.5, 4, 10, 40, 80,, 200, 400, and 800 ng ml )1 ) was constructed of which at least seven points were within 15% deviation of the nominal concentration for the analysis to be accepted. Following sample extraction a 50 ll aliquot of the supernatant was diluted with 450 ll of 0.1% formic acid in water and injected into the UPLC-MS/MS (Acquity; Waters, MA, USA) for analysis. A 2-minute gradient of mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water was used according to the following gradient flow: 0 (95:5), 0.1 (95:5), 0.7 (55:45), 1 (25:75), 1.6 (5:95), 1.9 (95:5), 2 minutes (95:5). The flow rate for the gradient used was 0.4 ml minute )1 on an Acquity UPLC BEH C lm column. Multiple selected reaction-monitoring transitions of initial product ions for ketamine (mass to charge ratio [m/z], fi ); xylazine (m/z, 221 fi 90); and chlorpromazine (m/z, 319 fi 86) were used for detection and quantitation. The quantitation of ketamine and xylazine was performed by injection of blank samples, standard solution, and spiked samples. The response factor consisting of the ratio between the area of the analyte peak and the area of the internal standard was established. A commercially available software program (MASSLYNX 4.1; Waters, MA. USA) was used to generate calibration curves for quantitation of the analytes. The lower limit of quantitation (LLOQ) of the assay was 0.5 ng ml )1 for all analytes. Precision and accuracy were within the required 15% deviation (20% for the LLOQ). The intra- and inter-run relative standard deviation for ketamine was <6.5% with recovery >89%. The intra- and inter-run relative standard deviations for xylazine were <4.7% and recovery was >92%. Pharmacokinetic analysis An open compartmental model with first-order elimination was used to describe the time concentration data for xylazine and ketamine over the first 60 minutes post-administration using commercially available analytical software (WINNONLIN 5.2; Pharsight Corporation, NC, USA). The complete model for ketamine has been described (Gehring et al. 2009). A one-compartment model fit the ketamine and xylazine data best, based on comparisons of the value of Aikake s Information Criterion and inspection of plots of predicted versus observed data and residuals. Pharmacokinetic parameters needed for calculating doses were determined using standard equations (Gibaldi & Perrier 1982). These parameters were area under the concentration versus time curve (AUC), the elimination half-life (T 1/2 ), the back-extrapolated drug concentration at time zero (C0), plasma drug clearance (CL) and volume of distribution at steady state (Vdss). Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

5 Pharmacodynamic analysis The area under the time effect curve (AUEC) for cortisol was calculated with the linear trapezoidal rule (Leon-Reyes et al. 2008). The maximum serum cortisol concentration (C cortmax ) and the time at which this occurred (T cortmax ) were also determined using non-compartmental analysis. Both these analyses were implemented using commercially available software (WINNONLIN 5.2; Pharsight Corporation, NC, USA). Statistical analysis Data were analyzed using a commercial statistical software program (JMP 7.0.2; SAS Institute Inc, NC, USA). Xylazine concentrations and pharmacodynamic data from each treatment group were compared using analysis of variance (ANOVA) and Kruskal Wallis one-way analysis of variance on ranks for the non-normally distributed T cortmax data. Planned comparisons (based on previous studies) of the AUEC for cortisol in the control, castrated, xylazine and xylazine/ketamine treated calves were conducted using two-sided Student t-tests (Ramsey & Schafer 2002). Individual confidence levels were controlled for all planned comparisons. Statistical significance was designated a priori as a p-value Behavioral scores from calves in each treatment group were compared using an ordinal logistic fit model. Likelihood-ratio Chi-square tests were used to determine differences in attitude and vocalization scores between treatment groups. Statistical significance was designated as p Results Pharmacokinetic analysis Mean compartmental pharmacokinetic parameters for xylazine, xylazine combined with ketamine and ketamine following intravenous administration are presented in Tables 1 3. Plasma drug concentration curves for xylazine, xylazine with ketamine and ketamine are simulated in Figs 1 3. Co-administration of ketamine appeared to reduce the elimination half-life, and the volume of distribution and produced a higher extrapolated C0 of xylazine compared with parameters for xylazine alone. However, these differences were not statistically significant. Pharmacodynamic analysis Mean non-compartmental analysis parameters for cortisol are summarized in Table 4. Mean serum cortisol concentrations over time for the four treatment groups are presented in Fig. 4a d. Mean peak serum cortisol concentrations (C cortmax ) tended to be higher in the CAST and CONT group compared with the sedative-analgesic treated groups but this difference was not statistically significant (p = 0.396). Furthermore, the ID Vd (ml kg )1 ) AUC (minutes ng ml )1 ) K10 half-life (minutes) C0 (ng ml )1 ) Mean SD CV Harmonic 12.9 mean Pseudo-SD 1.2 CL (ml minute )1 kg )1 ) Table 1 One-compartment modelpharmacokinetic parameters for xylazine administered at 0.05 mg kg )1 IV prior to castration Vd, volume of distribution; AUC, area under the plasma concentration time curve; K10, elimination rate constant; C0, the back-extrapolated drug concentration at time zero; CL, plasma drug clearance; CV, coefficient of variation. 570 Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

6 Table 2 One-compartment modelpharmacokinetic parameters for xylazine (0.05 mg kg )1 IV) co-administered with 0.1 mg kg )1 ketamine IV prior to castration Calf Vd (ml kg )1 ) AUC (minutes ng ml )1 ) K10 half-life (minutes) C0 (ng ml )1 ) Mean SD CV Harmonic 11.2 mean Pseudo-SD 3.1 CL (ml minute )1 kg )1 ) Vd, volume of distribution; AUC, area under the plasma concentration time curve; K10, elimination rate constant; C0, the back-extrapolated drug concentration at time zero; CL, plasma drug clearance; CV, coefficient of variation. Table 3 One-compartment model pharmacokinetic parameters for ketamine (0.1 mg kg )1 IV) co-administered with 0.05 mg kg )1 xylazine IV prior to castration Animal Vd (ml kg )1 ) AUC (minutes ng ml )1 ) K10 half-life (minutes) C0 (ng ml )1 ) Mean SD CV Harmonic 10.6 mean Pseudo-SD 2.8 CL (ml minute )1 kg )1 ) Vd, volume of distribution; AUC, area under the plasma concentration time curve; K10, elimination rate constant; C0, the back-extrapolated drug concentration at time zero; CL, plasma drug clearance; CV, coefficient of variation. mean time to maximum cortisol concentration tended to be longer in the X+K treated calves compared with the X and CONT groups, but this difference was also not statistically significant (p = 0.639). There was also no statistically significant difference between any of the groups in the total area under the effect curve (AUEC) for serum cortisol (p = 0.559). To associate changes in plasma xylazine and ketamine concentrations to serum cortisol response, the total AUEC for cortisol was subdivided into four periods based on the predicted pharmacokinetic profile of xylazine and ketamine and the blood sampling schedule (Figs 4 & 5). The proportion of the total AUEC for cortisol that occurred during the first 60 minutes following drug administration and castration was significantly higher in the CAST calves (21 ± 2%) compared with the X (13 ± 3%; p = ) and X+K (14 ± 2%; p = 0.016) treated calves. There was no statistically significant difference in the AUEC for cortisol between the four groups during the period and minutes post-castration. However, the proportion of the total AUEC for cortisol during the period minutes post-castration was significantly higher in the CONT (p = 0.036) and X (p = 0.024) treated calves compared with the CAST calves. Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

7 Log concentration (ng ml 1 ) 0 10 Predicted Observed Time (minutes) Figure 1 Log transformed mean plasma xylazine concentrations (ng ml )1 ) following IV administration of 0.05 mg kg )1 xylazine to calves prior to castration. Log concentration (ng ml 1 ) Log concentration (ng ml 1 ) Predicted Time (minutes) Predicted Time (minutes) Observed Observed Figure 2 Log transformed mean plasma xylazine concentrations (ng ml )1 ) following IV co-administration of 0.05 mg kg )1 xylazine and 0.1 mg kg )1 ketamine to calves prior to castration Figure 3 Log transformed mean plasma ketamine concentrations (ng ml )1 ) following IV co-administration of 0.05 mg kg )1 xylazine and 0.1 mg kg )1 ketamine to calves prior to castration. Behavioral scoring Vocalization and attitude scores in calves following IV administration of a placebo or 0.05 mg kg )1 xylazine alone or co-administered with 0.1 mg kg )1 ketamine prior to castration are presented in Fig. 6. All calves remained standing during and immediately following the castration procedure. However, two calves in the X+K treated group became recumbent during the intensive blood sampling period but were easily aroused with mild tactile and auditory stimulation. There was a significant difference in attitude scores between the four treatment groups (p = 0.023). Significantly more calves demonstrated attitude that was unchanged from pre-manipulation behavior in the CONT (p = 0.021) and X+K treated calves (p = ) compared with the CAST group. There was no statistically significant difference in vocalization scores between the four groups (p = 0.12). Discussion The study reported here was conducted to test the hypothesis that administration of sub-anesthetic xylazine alone or in combination with ketamine reduces distress associated with castration, without causing recumbency. None of these calves became recumbent during or immediately after castration although two calves became recumbent during the post-operative period. Although both treatments significantly reduced serum cortisol response over 0 60 minutes post-castration, co-administration of ketamine with xylazine resulted in more significant behavioral effects compared with the castrated controls. Cortisol response during the period minutes post-castration was significantly higher in the uncastrated and xylazine treated calves compared with the castrated calves but this did not occur to the same extent in calves that also received ketamine. This finding suggests that the addition of low doses of ketamine to low-dose xylazine may have enhanced the sedative-analgesic effects of the drug combination beyond the period of quantifiable plasma concentrations. To our knowledge, this is the first report evaluating the effects of low-dose xylazine and ketamine administered prior to castration and associating these effects with plasma drug concentrations. This is relevant because these drugs, when administered at these doses, are not associated with clinically apparent untoward physiological effects such as recumbency, 572 Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

8 Table 4 Non-compartmental analysis of serum cortisol concentrations following IV administration of 0.05 mg kg )1 xylazine alone or co-administered with 0.1 mg kg )1 ketamine or a placebo prior to castration Group Mean (SEM) C cortmax (nmol L )1 ) Mean (SEM) T cortmax (minutes) Total mean (SEM) AUEC (minute nmol L )1 ) Uncastrated (44.03) (10.80) 69, (14336) control Castrated (14.77) (2.24) 53, (5694) control Xylazine (28.23) (8.41) 68, (11405) Xylazine and (7.86) (57.26) 65, (4456) ketamine p-value (a) 350 (b) 350 Plasma concentration (nmol L 1 ) Plasma concentration (nmol L 1 ) (c) Time after castration (minutes) (d) Time after castration (minutes) Plasma concentration (nmol L 1 ) Plasma concentration (nmol L 1 ) Time after castration (minutes) Time after castration (minutes) Figure 4 Mean plasma cortisol concentrations (nmol L )1 ) in calves following IV administration of (a) 0.9% sterile sodium chloride placebo prior to surgical castration (CAST); (b) 0.9% sterile sodium chloride placebo prior to sham castration (CONT); (c) 0.05 mg kg )1 xylazine prior to surgical castration (X); and (d) 0.05 mg kg )1 xylazine co-administered with 0.1 mg kg )1 ketamine prior to surgical castration (X+K). rumen tympany, bradycardia and hypotension often associated with administration of xylazine alone. Castration is one of the most common cattle management practices in the United Kingdom with over 1 million procedures performed annually (DEFRA, 2009). Although it is considered an offense to castrate calves older than 2 months of age without an anesthetic in the UK, similar legislation does not exist in other territories (AVMA 2009). For example, a recent survey of Canadian veterinarians revealed that only 20% of beef calves and 33% of dairy calves >6 months old received an analgesic at the time of castration (Hewson et al. 2007). Respondents that did use an analgesic at the time of castration used xylazine (>50% of respondents) more frequently than lidocaine (<30% of respondents). Administration of local anesthetics into the testicles is considered by some to be dangerous and time consuming with unpredictable efficacy, especially when circumstances do not allow sufficient time for maximal anesthesia to take effect (Hewson Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

9 Xylazine + Ketamine a a a ab Treatment group Xylazine Control Castration a a a a ab a a a b a a b Figure 5 Mean proportion of the total area under the effect curve (AUEC) for cortisol represented by successive periods (in minutes) after castration. Treatment groups not connected by the same letter are significantly different (p < 0.05) Percent of total AUEC AUEC 0-60 AUEC AUEC AUEC Attitude Vocalization p = Number of calves 4 p = Control Castrate Xylazine Xylazine + ketamine Treatment group Figure 6 Behavioral scores in calves following IV administration of a placebo or 0.05 mg kg )1 xylazine alone or coadministered with 0.1 mg kg )1 ketamine prior to castration. Vocalization scores are not significantly different between groups (p = 0.12). Significant differences in attitude scores between groups are indicated. Vocalization scores: 0 = no vocalization; 1 = snorting or grunting; 2 = momentary vocalization; and 3 = continuous vocalization during and immediately after testicular manipulation. Attitude scores: 0 = unchanged from pre-manipulation behavior; 1 = head shaking, kicking, or tail flicking; 2 = momentary escape behavior [e.g. lunging against the head gate, head shaking, and kicking]; and 3 = violent escape behavior [e.g. repeated lunging against the head gate, head shaking and kicking throughout testicular manipulation]. et al. 2007). The results reported herein suggest that intravenous administration of sub-anesthetic xylazine and ketamine is an efficacious sedativeanalgesic protocol. This regimen may also replace the need for intra-testicular anesthetic injection and thus enhance animal well-being and operator safety. Cortisol is widely used to quantify distress associated with nociception in calves because the response magnitude (C cortmax ), duration and/or integrated response (AUEC) is reported to correspond with the predicted noxiousness of different animal husbandry procedures (Chase et al. 1995; Fisher et al. 1997; Mellor et al. 2000; Fisher et al. 2001; Earley & Crowe 2002; Stafford et al. 2002; Ting et al. 2003). However, cortisol concentrations may also be increased in response to the stress of handling alone (Coetzee et al. 2008). This was observed in the uncastrated control group in the present study, although these results may be skewed by the unbalanced group size with fewer animals in this group. The results presented here are consistent with studies reviewed by Stafford & Mellor (2005) that report peak cortisol concentrations within 574 Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

10 30 40 minutes of surgical castration. However, the magnitude of the peak cortisol response in both castrated and uncastrated control calves was considerably higher in the present study than the peak concentration of 129 ng ml )1 reported in this review. Another study reported peak cortisol responses ranging from ± nmol L )1 to ± nmol L )1 following a 24-hour acclimatization period and castration with a Henderson castration tool (Coetzee et al. 2007). The difference between the cortisol results reported could thus be associated with the use of the Henderson castration tool as opposed to manual twisting and pulling of the testicles. Although the Henderson tool is not widely used in the UK, a recent survey of 189 veterinarians in the USA suggests that approximately 15% respondents use this technique in older calves especially where postsurgical hemorrhage is a concern (Coetzee et al. 2010). Faulkner et al. (1992) investigated the health and performance effects of intravenous butorphanol (0.07 mg kg )1 ) and xylazine (0.02 mg kg )1 ) coadministration to weanling bulls at the time of castration. Co-administration of xylazine and butorphanol resulted in reduced chute activity and clinical sedation characterized by muscle relaxation and occasional (<15 20%) difficulty in exiting the chute. It is noteworthy that cortisol concentrations immediately post-castration were not evaluated in this study. However, treated calves were found to have significantly higher cortisol concentrations at 3 days post-castration compared with castrated controls. The authors conclude that butorphanol and xylazine did not reduce stress or improve performance. This finding is contradicted by the results presented here that demonstrate significant cortisol suppression for 60 minutes post-castration following xylazine and ketamine administration. This suggests that ketamine, and its active metabolite norketamine, may be superior analgesics than butorphanol when co-administered with xylazine. Despite the lack of specificity of increased cortisol to measure pain, Fisher et al. (1996) demonstrated that a decrease in acute cortisol response coincides with the analgesic effect of lidocaine local anesthesia administered prior to surgical or burdizzo castration in calves. Cortisol suppression lasted minutes which coincides with the reported duration of local anesthesia (Lemke & Dawson 2000; Spoormakers et al. 2004). It has also been shown that non-steroidal anti-inflammatory drugs, ketoprofen and meloxicam, reduce acute plasma cortisol response in cattle when administered at the time of castration or dehorning (Earley & Crowe 2002; Stafford et al. 2002; Ting et al. 2003; Heinrich et al. 2009). In one study, ketoprofen was found to more effectively attenuate plasma cortisol response following burdizzo castration than a local anesthetic or a xylazine epidural (Ting et al. 2003). These data support the use of plasma cortisol as a pharmacodynamic indicator of distress associated with pain following castration (Stafford & Mellor 2005). A deficiency of the present study is that cortisol determination is unlikely to accurately differentiate between sedative and analgesic drug effects since both can attenuate plasma cortisol response. Furthermore, a centrally acting a-2 adrenergic inhibition of corticotrophin releasing factor (CRF) synthesis/release by xylazine has been demonstrated in goats (Sanhouri et al. 1992). This suggests that cortisol suppression may also be independent of direct sedative or analgesic effects of xylazine. Sub-anesthetic ketamine exhibits analgesic properties that are related to plasma drug concentration in humans (Annetta et al. 2005). In humans plasma ketamine concentrations above 4 5 lmol L )1 (0 ng ml )1 ) are required to produce anesthetic effects while analgesic effects are associated with plasma concentrations below 1 lmol L )1 (275 ng ml )1 ) or 1/10th 1/5th of the anesthetic dose (Eide 1999). Previously Grant & Upton (2004) reported that 0.05 mg kg )1 xylazine IV in sheep produced analgesia lasting 25 minutes with only 3/7 animals showing signs of mild sedation. In the study reported here the same dose of xylazine combined with 1/20th the recommended anesthetic dose of ketamine in calves diminished behavioral responses without recumbency. Attenuation of serum cortisol response was also associated with ketamine concentrations below the anesthetic dose in humans. In the absence of a robust, specific measure of pain in animals, the mitigation of plasma cortisol response observed in the present study was likely due to a combination of mild sedative and analgesic drug effects. The pharmacokinetics of ketamine (5 mg kg )1 )in anesthetized calves, administered alone or 10 minutes after xylazine premedication (0.2 mg kg )1 ) has been previously described (Waterman 1984). Garcia-Villar et al. (1981) reported the pharmacokinetics of xylazine administered at 0.2 mg kg )1 IV to four calves. A two-compartment open model was fit Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

11 in these data in contrast with a one-compartment model used in the present study. This difference was likely due to the lower plasma drug concentrations, associated with a sub-anesthetic dose, that decreased rapidly to concentrations below the LLOQ. This resulted in an abbreviated terminal phase of the slope that could not be adequately described by a two-compartment model. The extrapolated peak ketamine concentration (C0) associated with sub-anesthetic dosing (0.1 mg kg )1 ) was ± ng ml )1 compared with 9450 ± 1260 ng ml )1 reported previously (5 mg kg )1 ). However, the calculated CL total of ketamine following xylazine premedication was similar to the clearance reported here at ml kg )1 minute )1. The extrapolated peak xylazine concentration (C0) associated with sub-anesthetic dosing at 0.05 mg kg )1 IV was also significantly lower at ± ng ml )1 (without ketamine) and ± (with ketamine) compared with 1050 ng ml )1 previously reported (2 mg kg )1 IV). Further comparison between these reports is confounded by the substantial differences in the anesthetic regimen, pharmacokinetic model and analytical methodology. Waterman (1984) previously reported that premedication with xylazine significantly reduced the volume of distribution and clearance of ketamine without affecting the half-life. In the present study, co-administration of ketamine with xylazine appeared to reduce the elimination half-life, and the volume of distribution of xylazine and produced a higher extrapolated C0 compared with parameters for xylazine alone. Although these differences were not statistically significant, it is noteworthy that mean plasma clearance of xylazine in the two groups was similar, suggesting that the shorter halflife for xylazine combined with ketamine was likely due to a reduction in the apparent volume of distribution. This finding suggests that ketamine may alter xylazine pharmacokinetics in a similar manner to xylazine premedication altering ketamine pharmacokinetics, as was previously reported. A potential criticism of the present study is that only a limited number of calves were available for enrollment. Although our sample size was relatively small, these data were sufficient to demonstrate significant differences in AUEC for cortisol between treated and untreated groups during the first 60 minutes post castration (statistical power = 0.72). Previous reports examining plasma cortisol response typically used 8 10 animals per treatment group instead of the six animals used in the present study (Stafford & Mellor 2005). In the present study the statistical power ranged from 0.23 to 0.72 for AUEC calculations. However, this use of postexperiment power calculations should be interpreted with caution (Hoenig & Heisey 2001). In conclusion, a sub-anesthetic dose of xylazine at 0.05 mg kg )1 and ketamine at 0.1 mg kg )1 administered as an IV bolus significantly reduced distress behavior at the time of castration and attenuated serum cortisol response for 60 minutes thereafter. This is comparable with the duration of cortisol attenuation previously reported following intratesticular lidocaine administration prior to castration (Fisher et al. 1996). These findings suggest that low doses of xylazine and ketamine may offer an efficacious and cost-effective systemically administered alternative to local anesthesia prior to castration. However, this study also highlights deficiencies associated with the use of serum cortisol for pain assessment and emphasizes the need for more robust and specific pain biomarkers. Further assessment of both local and systemic analgesic techniques is required in order to formulate sciencebased analgesic recommendations to enhance animal wellbeing following castration. Acknowledgements This research was funded in part by a Career MAPS award administered under the KSU ADVANCE Institutional Transformation Program and by the KSU College of Veterinary Medicine. Dr Coetzee is supported by USDA-CSREES, Animal Protection (Animal Well-being), NRI Grant # The authors wish to acknowledge the invaluable contributions of the following KSU faculty members, staff and students who assisted with study design, sample collection, processing and care of the study animals: Dr Brian Lubbers, Dr Dan Thomson, Erin Evanson, Dan Linden, Paul Wagoner, Elliot Stevens, Keith DeDonder, Shauna England, Kellie Triplett, Bryan Kerling, Lindsay Waechter-Mead, Sarah Weber and Sara McReynolds. This study also benefited from technical laboratory assistance provided by Kara Smith and Rita Doyle from the KSU-Veterinary Clinical Sciences laboratory. References Abrahamsen EJ (2009) Chemical restraint in ruminants. In: Current Veterinary Therapy Food Animal Practice 576 Ó 2010 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 37,

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