304 Australian Veterinary Journal Volume 82, No 5, May 2004

ScientificScientificScientificScientific

Comparison of the analgesic effects of xylazine in sheepvia three different administration routes

C GRANT and RN UPTONa

Department of Anaesthesia and Intensive Care, University of Adelaide, South Australia 5005

Objective To examine the influence of administration routeon the analgesic effects of identical doses of xylazine in sheep.

Design A prospective, linear, randomised laboratory study.

Procedure The analgesic response to the administration of2.5 mg of the α2 agonist xylazine either intravenously, intra-muscularly or subcutaneously was assessed using an anal-gesia testing method based upon a learned response to apainful electrical stimulus.

Results Intravenous administration achieved the most rapidonset and highest peak analgesic values of all administrationmethods, but was characterised by a shorter duration of action(25 min). Intramuscular and subcutaneous administrationresulted in a longer duration of action (40 min) and a greatertotal analgesic response.

Conclusion For the routine management of acute pain,intramuscular administration provided the best combination ofonset, duration and total analgesic response of the routesexamined. The absence of adverse side effects, such as seda-tion, normally associated with the administration of α2 agonistsshould also encourage the use of this method as a simple andeffective means of providing significant analgesia in the sheep. Aust Vet J 2004;82:304-307

The use of analgesics to reduce pain and limit suffering inanimals is a cornerstone of veterinary practice. Forresearchers, ethical and legislative imperatives require suit-

able pain management procedures be in place for the treatment ofpain following surgical procedures.1 The validity of any dataacquired from animals suffering pain resulting from prior surgicalpreparation that is unrelated to the experimental procedure, mustalso be questioned.2 There are comprehensive recommendationson choosing suitable analgesics for pain relief.3 However, formany animals there is a lack of species specific data and recom-mendations are generally based upon anecdotal evidence or effi-cacy in other species.

The ideal analgesic should have a proven efficacy particular tothat species. In most circumstances it should be simple to admin-ister and provide a predictable magnitude and duration of anal-gesia with a minimum of adverse side effects, such as sedation,which may affect the recovery of the animal. Very little analgesictesting has been performed specifically in the sheep, but it appearsthat many traditional analgesics, such as opioids, fail to produceanalgesic effects in sheep.4,5 However, low doses of the α2 adreno-ceptor agonist xylazine produce consistent analgesic effectsfollowing intravenous6-8 and intramuscular5,9 administration.Unfortunately, these studies offer incomplete descriptions of the

analgesic action, with either no statistical examination of the dataor no determination of duration of effect.

There have been no cross over studies comparing different admin-istration routes in the same sheep under the same conditions.Intramuscular and subcutaneous injection offer simple methodsof administering analgesics, but the choice of administrationroutes can affect the efficacy of many drugs. While a dose givenintravenously is 100% available to the systemic circulation,extravascular injection can reduce both the rate and extent of drugabsorption. This can be due to factors such as the vascularity ofthe injection site and the degree of ionisation and lipid solubilityof the drug.10 This effect can be either a reduction in the magni-tude of peak analgesic effects, or a delay in the time-course ofanalgesic effects. Indeed, the selection of administration routemay provide a mechanism by which the analgesic profile can bebetter matched to the expected time-course of the pain experi-ence.

Before recommendations for pain control in sheep can be made,variations in the analgesic profile caused by different administra-tion routes need to be assessed. The aim of this study was toexamine differences in the duration and magnitude of analgesiceffects following low dose intravenous, intramuscular and subcu-taneous xylazine administration routes in sheep, with respect totheir potential for the management of pain.

MethodsStudies were approved by the Animal Ethics Committee of theUniversity of Adelaide and performed in seven adult Merinosheep from a single blood line, aged between 1.5 and 3 years ofage and weighing approximately 50 kg. They were housed in theanimal house facility of the Faculty of Health Sciences at theUniversity of Adelaide, and cared for according to the guidelinesfor animal care of the National Health and Medical ResearchCouncil of Australia.1 Sheep were kept within mobile metaboliccrates with free access to feed and water, and always housed atleast in pairs to avoid isolation stress. Sheep were housed in thisenvironment for at least 5 days before surgery commenced toallow them to adapt to their surroundings.

All surgical procedures involving catheterisation of sheep wereperformed under general anaesthesia with strict asepsis. Sheepwere induced by rapid injection of 1000 mg sodium thiopentone(Abbott Australasia, Kernell, NSW) into the left internal jugularvein and then intubated. Anaesthesia was maintained using 1.5%halothane (Zeneca, Cheshire, England) in oxygen delivered via avaporiser and circle breathing system. Normocapnia was main-tained by artificial ventilation using a gas powered ventilator andmonitoring of end-tidal carbon dioxide tensions. The neck wasshorn of wool and the right jugular vein exposed via a longitu-dinal skin incision and a 7F polyethylene catheter (CookAustralia, Eight Mile Plains, Queensland) was inserted approxi-mately 10 cm in the jugular vein using Seldinger technique.

aDepartment of Anaesthesia and Intensive Care, Royal Adelaide Hospital,Adelaide, South Australia 5000

305Australian Veterinary Journal Volume 82, No 5, May 2004

ScientificScientificScientificScientific

Extension lines terminated with a three way stopcock (AbbottLaboratories, North Chicago, IL) were connected to the cathetersand lines kept patent by filling the volume of line and catheterwith 50 IU/mL sodium heparin (David Bull Laboratories,Melbourne) and capping. All sheep were able to stand and wereeating within 3 to 4 h of surgery.

Analgesia was assessed using a voluntary leg withdrawal responseto a subcutaneous electrical stimulus. The analgesic profile wasdetermined from the increase in current required to induce alearned leg withdrawal response, this method has been fullydescribed previously.11 In brief, two 13 mm 26G needles wereplaced subcutaneously, 5 mm apart, in the anterior aspect of thelower third of the sheep’s hind limb to act as electrodes betweenwhich the current could pass. The device delivered pulsed DCcurrent (50 Hz) to the needles increasing between 0 and amaximum of 50 mA over a period of approximately 5 seconds.The device automatically compensated for changes in skin resis-tance between the electrodes. This analgesia testing methodprovides a highly sensitive and repeatable index of analgesia, thestimulus is brief and causes no damage to the testing area.Examination of baseline stability has shown that the thresholdcurrent required to produce a leg withdrawal response does notchange over time.

The operator of the device was positioned alongside the sheepwhere any movement of the hindlimb was clearly visible. Deliveryof current was initiated by the operator depressing the hand-heldcutout button. The end-point was taken as a deliberate with-drawal of the leg at which point the current was terminated byreleasing the cutout button and the highest current level recorded.Readings were taken every 60 seconds. When end-point currentthresholds appeared stable for a period of at least 5 minutes, thereadings were considered to be at a baseline and xylazine wasadministered. During the course of each study sheep wereobserved for signs of sedation such as increased salivation, thedegree of ptosis and changes in alertness and response to externalstimuli such as noise. At times of maximal drug effect (whenthreshold currents were at their highest values), sheep were givenan innocuous non-painful stimulus such as gently touching theleg. If the sheep failed to withdraw its leg from this novel stimulusit was considered to be indicative of a depression of the centralreflexes. From this, it could be determined if changes in end-pointcurrent thresholds were due to ‘real’ analgesic effects or due to thesheep’s inability to respond as a result of inhibited locomotoractivity.

Dosing in adult sheepClassically in veterinary medicine dosing is expressed in terms ofdose per kg, this is done to provide an easy ‘rule of thumb’ forcalculating dosing requirements across a broad range of bodyweights. We have found that in adult sheep of the same blood linethe variation in body weight is small and dependent mostly uponfleece length and rumen contents. Normalising doses for massunder such circumstances can contribute to variability of effectrather than reduce it. The determinants of kinetic processes actu-ally scale better to lean body mass rather than total body weight,but measurement of lean body mass is difficult. With this knowl-edge, drug doses in adult sheep were not scaled for body weight.A xylazine dose of 2.5 mg was used throughout based upon thenominal weight of the adult sheep of 50 kg and a dose of 50µg/kg. This dose was chosen to provide effective analgesia with aminimum of sedation.5,8

Study designEach sheep received xylazine by three different administrationroutes on different experimental days in random order. On theexperimental day sheep were transported to an experimental roomand placed in a sling within their metabolic crate, whichprevented them from lying down, but otherwise appeared tocause no discomfort.

After a period of approximately 30 min to acclimatise to theirsurroundings, baseline threshold current values were recorded andsheep received 2.5 mg of xylazine by one of the routes describedbelow.

Intravenous administration — A 30 mL solution of xylazinewas prepared in 0.9% saline (0.1 mg/mL). Saline was chosen asthe vehicle because intravenous infusions of hypotonic solutions,such as when some drugs are prepared in water, have beenshown to produce possible cardiovascular perturbations insheep.12 After baseline values were recorded, a programmablepump (Model 33, Harvard Apparatus Ltd, Kent, England) wasthen used to deliver a 2 min infusion of the xylazine solution at12.5 mL/min. Readings were continued until end-point currentthresholds had returned to baseline for at least 10 min.

Intramuscular administration — After baseline values had beenrecorded, 2.5 mg of xylazine prepared in 1 mL of 0.9% salinewas injected into the rump of the contra-lateral leg to which theelectrodes for analgesia assessment were attached. Readings werecontinued until end-point current thresholds had returned tobaseline for at least 10 min.

Subcutaneous administration — After baseline values had beenrecorded, 2.5 mg of xylazine prepared in 1 mL of 0.9% salinewas injected under the wool free area of skin between the insidefront leg and sternum. The skin was raised and great care takento ensure correct needle placement at the time of injection.Readings were continued until end-point current thresholds hadreturned to baseline for at least 10 min.

Data analysisThreshold current values were expressed as a percentage changefrom each sheep’s own baseline values. This was done to accountfor differences in the point at which sheep terminate the currentby eliciting a leg lifting response. These differences can be due tothe variation between each sheep in their ability to tolerate thepainful stimuli and differences in the electrode needle placementon the foreleg. Although great care was taken to ensure that theposition, depth and distance between the needles was consistentbetween sheep, small variations can cause differences in thecurrent density (mA x cm2 ).

The total antinociceptive response was determined by measuringthe area under the current curve (AUC) using the trapezoidal rule forthe duration of possible analgesic effects, from the time of injectionup to 60 minutes. The maximal analgesic response was calculatedby averaging the maximum threshold current value of each sheepfor the experimental period for each administration method.

Statistical analysisTo determine any statistically significant change in the antinoci-ceptive response from baseline values, one-way ANOVA wasperformed with post-hoc multiple comparisons using theNewman-Keuls method. Paired t-tests were used to assess anydifference in the baseline, peak and total (AUC) antinociceptiveresponses between different administration methods. A P value of

306 Australian Veterinary Journal Volume 82, No 5, May 2004

ScientificScientificScientificScientific

measured by AUC, due to the achievement of relatively rapidpeak analgesia followed by slow drug wash out. Intramuscularadministration also produces fewer negative side effects thanintravenous administration. In the present study there was littleevidence of sedation and the negative respiratory and cardiovas-cular effects which are seen following intravenous administrationof α2 agonists19-21 can also be greatly reduced with intramuscular

less than 0.05 was considered significant in all tests. Statisticalanalysis was performed using the data analysis language ‘R’.13

ResultsIntravenous administration of xylazine demonstrated the fastestonset of all administration routes with the shortest time to peakthreshold current values (Table 1). However, this was also associ-ated with the shortest period where threshold current values weresignificantly above baseline (Figure 1) and three sheep showedsigns of sedative effects with increases in the degree of ptosis andsalivation. These changes were seen during the 30 min periodimmediately following administration, but did not prevent thesheep from responding to non-painful stimuli such as touchingthe forelimb. No overt signs of sedation were observed during theintramuscular or subcutaneous studies.

Intramuscular administration of xylazine showed the greatest totalantinociceptive response (AUC) and subcutaneous the leastresponse (Figure 2). However, these differences were not statisti-cally significant. The period for which threshold current valueswere significantly above baseline was similar for both the intra-muscular and subcutaneous administration methods (Figure 1).

DiscussionAnalgesic testing using a painful electrical stim-ulus showed all three administration routesproduced significant analgesia. As expected fora route with no absorption delay, intravenousadministration of xylazine was characterised bya rapid but relatively brief increase in antinoci-ception. These findings are consistent withprevious studies using mechanical and thermalstimuli.7,8 Analgesic testing methods usingbrief painful mechanical, thermal or electricalstimuli obviously cannot encompass thetotality of all pain experiences, such as chronicor visceral pain states. However, the use ofchronic pain models to examine such issues isbeset with grave ethical and moral considera-tions.14 The electrical stimulus method used inthe present study provides a brief painful stim-ulus and because it is gradually ramped thesheep determines at which point the stimulusis stopped. A good correlation between anal-gesia values from such methods and painscores, blood concentrations or known effectshas been seen in both humans and sheep for arange of analgesics and anaesthetics.15-17

The 2.5 mg dose used throughout the studywas chosen to provide significant analgesiceffects with a minimum of sedative effects. Thesedative effects of xylazine become moreapparent with increasing dose18 and dosesabove 50 µg/kg IM in sheep have been shownto cause sedation.5 The slight sedationobserved in 3 sheep following intravenousadministration was probably due to the rapidmixing of the drug with blood and the conse-quent high initial blood concentrations ofxylazine anticipated for this route.

Intramuscular injection of xylazine resulted inthe greatest total analgesic response, as

Table 1. Baseline and peak end-point current values following three differ-ent administration routes of 2.5 mg xylazine in seven sheep.

Route of administration

Current threshold Intravenous Intramuscular Subcutaneous

Baseline (mA) 4.1 ±1.1 2.7 ± 0.6 4.5 ± 0.9

Peak(mA) 27.1 ± 8.7 16.6 ± 6.5 16.5 ± 2.4

Time to peak current (min) 13.8 ± 1.8AB 25.1 ± 3.9B 29.9 ± 2.2A

Data are presented as mean ± SEM. Superscript A indicates P < 0.01 betweentreatment groups, and superscript B indicates P < 0.05 between treatment groups.

Figure 1. Analgesic effects of 2.5 mg xylazine following intravenous, intramuscularand subcutaneous injection in seven sheep. Thick bar indicates periods wherevalues were significantly different from baseline (P < 0.05). Data are presented as amean of the percentage change from baseline SEM.

307Australian Veterinary Journal Volume 82, No 5, May 2004

ScientificScientificScientificScientific

administration.22 Differences in the baseline threshold currentvalues between intramuscular and the other two administrationgroups (Table 1) were likely due to differences in skin conduc-tivity between the electrodes, or slight differences in the electrodeneedle placement on the foreleg causing changes in the currentdensity across the electrodes. A variation in behavioural responsebetween sheep to the painful stimulus was unlikely to be a factorbecause each sheep participated in all three studies and werematched against themselves for statistical comparisons betweenadministration routes.

Subcutaneous administration routes are widely considered toprovide more variable drug effects than other routes due to theirdependence upon the rate of local skin perfusion, which can varydue to changes in temperature or physiological responses such asshock. In the current study, subcutaneous administration ofxylazine produced reasonably consistent antinociceptive effects,however, factors such as room temperature and animal handlingmethods remained constant among all sheep.

Of all the administration routes examined intramuscular injectionof xylazine fulfilled the greatest number of criteria for the routinemanagement of pain in sheep with peak analgesic effects compa-rable to intravenous administration, but with a longer duration ofsignificant analgesia and less risk of sedation. The difficulties asso-ciated with achieving consistent analgesic effects following subcu-taneous administration make this route less suitable for generaluse in less controlled environments. Unfortunately, the duration

of analgesic effect provided by a single intramuscular bolus wasonly 60 minutes and may not be sufficient for the treatment of allforms of pain. Further studies examining repetitive dosing, alter-native preparations and different α2 agonists may provide dosingstrategies that could provide longer term pain control.

AcknowledgmentsThe authors would like to thank Mrs. Allison Martinez forassisting in a number of the studies.

References1. National Health and Medical Research Council. Australian code of practice forthe care and use of animals for scientific purposes. Australian GovernmentPublishing Service, Canberra, 1995.2. National Research Council. Recognition and alleviation of pain and distress inlaboratory animals. National Academy Press, Washington DC, 1992.3. Flecknell P. Laboratory Animal Anaesthesia. Academic Press, London, 1987.4. Livingston A, Acevedo M, Kiles A, Waterman A. The effects of droperidol onfentanyl induced dysphoria in sheep. Acta Vet Scand 1991;Suppl 87:170-172.5. Grant C, Upton R, Kuchel T. Efficacy of intra-muscular analgesics for acute painin sheep. Aust Vet J 1996;73:129-132.6. Nolan A, Livingston A, Waterman A. The use of a thermal device to assessanalgesics in the sheep. Br J Pharmacol 1985;86 Suppl:462.7. Ley S, Waterman A, Livingston A. Variation in the analgesic effects of xylazinein different breeds of sheep. Vet Rec 1990;126:508.8. Nolan A, Livingston A, Morris R, Waterman A. Techniques for comparison ofthermal and mechanical nociceptive stimuli in the sheep. J Pharm Meth1987;17:39-49.9. Grant C, Upton R. The anti-nociceptive efficacy of low dose intramuscularxylazine in lambs. Res Vet Sci 2001;70:47-50.10. Baggot JD. Pharmacokinetics: Disposition and fate of drugs in the body, In:Adams HR, editor. Veterinary Pharmacology and Therapeutics. 7th edn.IowaState University Press, Iowa, 1995:18-52.11. Ludbrook G, Grant C, Upton R, Penhall C. A method for frequent measure-ment of sedation and analgesia in sheep using the response to a ramped elec-trical stimulus. J Pharmacol Toxicol Methods 1995;33:17-22.12. Huang Y, Upton R, Rutten A. Adverse haemodynamic effects of the rapid intra-venous injection of hypotonic solutions in sheep. Res Vet Sci 1996;60:209-212.13. Ihaka R, Gentleman R. R: A language for data analysis and graphics. J CompGraph Stat 1996;5:299-314.14. Lineberry CG. Laboratory Animals in Pain Research. In: Gay WI, editor.Methods of Animal Experimentation, Academic Press, New York. 1981:237-311.15. Ludbrook G, Upton R, Grant C, Gray E. Brain and blood concentrations ofpropofol after rapid intravenous injection in sheep. Anaesth Intensive Care1996;24:445-452.16. Upton R, Ludbrook G, Grant C, Gray E. In vivo relationships between the cere-bral pharmacokinetics and pharmacodynamics of thiopentone in sheep aftershort-term administration. J Pharmacokinet Biopharm 1996;24:1-18.17. Doverty MA, Somogyi AM, White J et al. Methadone maintenance patientsare cross-tolerant to the antinociceptive effects of morphine. Pain 2001;93:155-63.18. Maze M, Tranquilli W. Alpha 2-adrenoceptor agonists: defining the role in clin-ical anesthesia. Anesthesiology 1991;74:581-605.19. Klide AM, Calderwood HW, Soma LR. Cardiopulmonary effects of xylazine indogs. Am J Vet Res 1975;36:931-935.20. Wagner AE, Muir WW, Hinchcliff KW. Cardiovascular effects of xylazine anddetomidine in horses. Am J Vet Res 1991;52:651-657.21. Waterman A, Nolan A, Livingston A. Influence of idazoxan on the respiratoryblood gas changes induced by alpha 2-adrenoceptor agonist drugs in conscioussheep. Vet Rec 1987;121:105-107.22. Grant C, Upton R. Cardiovascular and haemodynamic effects of intramusculardoses of xylazine in conscious sheep. Aust Vet J 2001;79:58-60.

(Accepted for publication 29 September 2003)

Figure 2. A comparison of the AUC of the analgesic responseversus time curves following either intravenous (IV), intramuscular(IM) or subcutaneous (SC) administration of 2.5 mg xylazine inseven sheep. Data are presented as mean SEM.