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Pharmacodynamics of alfaxalone after single‐dose intramuscular administration in red‐eared sliders (Trachemys scripta elegans): a comparison of two different doses at two different ambient temperatures

      Abstract

      Objective

      This study compares the pharmacodynamics of two different doses of alfaxalone administered intramuscularly (IM) to red‐eared sliders at two ambient temperatures.

      Study design

      Prospective blinded crossover experimental study.

      Animals

      Nine adult female sliders (Trachemys scripta elegans).

      Methods

      Following a 2‐week acclimation at 22–25 °C, nine sliders were randomly assigned to receive alfaxalone, 10 mg kg−1 (W10), or 20 mg kg−1 (W20) IM. Each turtle received each dose, with a minimum 7‐day washout period. A blinded observer evaluated heart rate (HR), palpebral and corneal reflexes, muscle relaxation, handling, and response to toe pinch at the following points: pre‐injection, and 5, 12, 20, 30, 45, 60, and 120 minutes post‐injection. Turtles then acclimated to 18–20 °C for 63 days, and the experiment was repeated in this lower‐temperature environment, with treatment groups C10 (alfaxalone 10 mg kg−1) and C20 (alfaxalone 20 mg kg−1) subjected to the same crossover design.

      Results

      C10 and C20 groups had significantly lower intraanesthetic HR than W10 or W20, respectively. C10 and W20 were significantly more relaxed and easier to handle than W10. No significant differences were observed in palpebral reflex, nor responsiveness to the toe pinch stimulus. None of the turtles lost corneal reflex. W20 and C20 had prolonged recoveries, compared to low‐dose groups within the same temperature environment. Recovery was also longer at C20 and C10 compared to W10.

      Conclusions

      Turtles given 10 mg kg−1 were more relaxed and easier to handle in cold than warm conditions. Warm turtles were more relaxed and easier to handle when given 20 mg kg−1 than those given 10 mg kg−1. Cold conditions correlated with lower HR and longer recovery time for each dose category.

      Clinical relevance

      The turtles had dose‐dependent and inconsistent responses to alfaxalone. Lower ambient temperature augmented the behavioral effects of this drug.

      Keywords

      Introduction

      Alfaxalone (3a‐hydroxy‐5a‐pregnane‐11, 20‐dione) is a synthetic neuroactive steroid that enhances binding of the gamma‐aminobutyric acid – A (GABAa) receptor to its endogenous ligand within the central nervous system, resulting in muscle relaxation and hypnosis (Rupprecht & Holsboer
      • Rupprecht R
      • Holsboer F
      Neuropsychopharmaco-logical properties of neuroactive steroids.
      ). The current formulation includes a 2‐hydroxypropyl‐β‐cyclodextrin excipient (Alfaxan‐CD RTU; Jurox, Australia), and is currently labeled for use in dogs and cats in Australia, New Zealand, South Africa and the UK (Ferre et al
      • Ferre PJ
      • Pasloske K
      • Whittem T
      • et al.
      Plasma pharmacokinetics of alfaxalone in dogs after intravenous bolus of Alfaxan-CD RTU.
      ; Muir et al.
      • Muir W
      • Lerche P
      • Wiese A
      • et al.
      Cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in dogs.
      ). This drug is widely utilized off‐label in other species, due to its reportedly wide therapeutic index, smooth induction and rapid recovery characteristics (Johnson
      • Johnson R
      ; Maddern et al.
      • Maddern K
      • Adams VJ
      • Hill NAT
      • et al.
      Alfaxalone induction dose following administration of medetomidine and butorphanol in the dog.
      ; Ambros et al.
      • Ambros B
      • Duke-Novakovski T
      • Pasloske KS
      Comparison of the anesthetic efficacy and cardiopulmonary effects of continuous rate infusions of alfaxalone-2-hydroxypropyl-β-cyclodextrin and propofol in dogs.
      ; Leece et al.
      • Leece EA
      • Girard NM
      • Maddern K
      Alfaxalone in cyclodextrin for induction and maintenance of anaesthesia in ponies undergoing field castration.
      ; Zaki et al.
      • Zaki S
      • Ticehurst KE
      • Miyaki Y
      Clinical evaluation of Alfaxan-CD® as an intravenous anaesthetic in young cats.
      ; Pasloske et al.
      • Pasloske K
      • Sauer B
      • Perkins N
      • et al.
      Plasma pharmacokinetics of alfaxalone in both premedicated and unpremedicated Greyhound dogs after single, intravenous administration of Alfaxan at a clinical dose.
      ). In contrast to propofol, it can be given by the intramuscular (IM) route. Propofol administered IM is ineffective for producing sedation or anesthesia and has been shown to cause significant inflammation and necrosis in rats. The IM propofol injection site lesions in these animals were characterized by neutrophil infiltration following the fascial planes, myocyte swelling, loss of cross‐striations, sarcoplasmic fragmentation, and formation of shrunken angular myocytes with pyknotic nuclei (McKune et al.
      • McKune CM
      • Brosnan RJ
      • Dark MJ
      • et al.
      Safety and efficacy of intramuscular propofol administration in rats.
      ). Alfaxalone has a pH (6.5–7.0) that is closer to that of physiologic fluid compared to ketamine, pH (3.5–5.5), so alfaxalone may be less painful on injection. There is no need to pair alfaxalone with additional drugs to achieve adequate muscle relaxation during induction of anesthesia in mammals (Muir et al.
      • Muir W
      • Lerche P
      • Wiese A
      • et al.
      Cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in dogs.
      ,
      • Muir W
      • Lerche P
      • Wiese A
      • et al.
      The cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in cats.
      ). While both alfaxalone and propofol lack an antimicrobial preservative, alfaxalone less readily supports bacterial growth and this feature represents a practical advantage with regard to storage and risk of iatrogenic infection (Strachan et al.
      • Strachan FA
      • Mansel JC
      • Clutton RE
      A comparison of microbial growth in alfaxalone, propofol and thiopental.
      ), especially when working in field conditions.
      The only published prospective study on alfaxalone in a reptile species demonstrated that as a single agent given IM at 20 mg kg−1 and 30 mg kg−1, this drug provided adequate immobilization and muscle relaxation for minor diagnostic or surgical procedures in green iguanas (Iguana iguana) (Bertelsen & Sauer
      • Bertelsen MF
      • Sauer CD
      Alfaxalone anaesthesia in the green iguana (Iguana iguana).
      ). In that study, IM alfaxalone provided variable loss of responsiveness to a toe pinch stimulus and brief hypnosis ranging from light sedation to surgical anesthesia, in a dose‐dependent manner.
      The onset of anesthesia from inhalation anesthetics delivered by mask can be prolonged, due to the dive reflex and breath‐holding behavior characteristic of many aquatic turtle species. Intravenous (IV) catheter placement can be difficult in small, noncompliant or unsedated reptiles. Consequently, clinical demand for more viable IM anesthetics in this order (Chelonia) is high.
      Hypothermia and prolonged recovery are common post‐anesthetic complications cited by veterinarians practicing reptile medicine (Read
      • Read MR
      Evaluation of the use of anesthesia and analgesia in reptiles.
      ). Prolonged recovery is likely associated with hypothermia because metabolic processes such as the hepatic enzyme system cytochrome P450, are reduced during hypothermia (Tortorici et al.
      • Tortorici MA
      • Kochanek PM
      • Polovac SM
      Effects of hypothermia on drug disposition, metabolism, and response: a focus of hypothermia-mediated alterations on the cytochrome P450 enzyme system.
      ). Ambient temperature has a dramatic effect on body temperature and reptile metabolism (White & Somero
      • White FN
      • Somero G
      Acid-base regulation and phospholipid adaptations to temperature. Time courses and physiological significance of modifying the milieu for protein function.
      ; Branco et al.
      • Branco LGS
      • Portner HO
      • Wood SC
      Interaction between temperature and hypoxia in the alligator.
      ). Therefore, it is expected that ambient temperature affects drug metabolism and hence recovery from anesthesia. An abundance of literature describes the effects of reduced oxygen availability and ambient temperature on reptile metabolism (Bickler & Buck
      • Bickler PE
      • Buck LT
      Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability.
      ). In vivo studies establishing the effects of ambient temperature on the pharmacodynamic properties of anesthetic drugs in reptiles are limited. Carregaro et al. (
      • Carregaro AB
      • Cruz ML
      • Cherubini AL
      • et al.
      Influence of body temperature on rattlesnakes (Crotalus durissus) anesthetized with ketamine.
      ) demonstrated that hypothermic rattlesnakes (Crotalus durissus) anesthetized with ketamine had lower heart rates and slower recoveries than those kept in normothermic environments.
      The aim of this study was to compare the hypnotic and muscle relaxant effects of two IM dose rates of alfaxalone in red‐eared sliders, and to examine the effect of ambient temperature on these clinical parameters. Our hypotheses were that both selected alfaxalone dose rates would provide muscle relaxation and adequate hypnosis for immobilization during a toe pinch stimulus, that the duration and intensity of muscle relaxation and hypnotic effects would be dose‐dependent, and that lower ambient temperatures would prolong anesthetic recovery.

      Materials and methods

      This study was approved by the Institutional Animal Care and Use Committee (A2010 03‐043‐Y1‐A0) and animals were housed in an accredited facility. Nine adult female red‐eared sliders (Trachemys scripta elegans) were used for this study. An a priori power analysis was performed based on literature describing the use of other anesthetics and sedatives in chelonians, and revealed that a minimum of seven turtles was required to achieve an α of 0.05, β of 0.80 and to detect a 15 minute difference in anesthetic duration between treatment groups.
      The animals were obtained from a wholesale source, and for the first phase of the experiment, were housed individually in multiple aquatic tanks, with air temperatures maintained at approximately 26 °C, water temperatures between 25 and 28 °C, and basking areas provided using a broad spectrum mercury‐halide lamp. Animals were fed a commercial pelleted diet daily (Mazuri Freshwater Turtle Diet #5E08; Land O’Lakes, Inc., IN, USA). The turtles were allowed to acclimate to this environment for 2 weeks prior to the beginning of the experiment. For the second phase of the experiment, the turtles were housed together in one large tank (4 × 2 × 0.5 m), within a covered outdoor enclosure, that allowed direct sunlight for several hours per day between September and December, 2010. The water temperature in this tank was kept at approximately 19 °C, and the air temperature was approximately 10–21 °C. The turtles were allowed to acclimate to this colder environment for 63 days before the second phase of the experiment. The temperature of the room for the first (warm temperature) phase of the experiment was 22–25 °C, and room temperature during the second (cold temperature) phase was 18–20 °C.
      All turtles were screened for systemic illness based on physical examination and plasma biochemistry (Abaxis VetScan Classic Analyzer; Abaxis, CA, USA) before the beginning of the experiment and again before the last data collection day. Blood for these tests was drawn from the occipital venous sinus. Turtles were marked for identification by etching a number into one of their marginal scutes, then randomized by lottery to groups for the first (warm temperature) phase of the study: W10 (warm temperature treatment group receiving alfaxalone, 10 mg kg−1, IM) or W20 (warm temperature treatment group receiving alfaxalone, 20 mg kg−1, IM). In the second (cold temperature) phase of this study, turtles were again randomized by lottery to groups: C10 (cold temperature treatment group receiving alfaxalone, 10 mg kg−1, IM) or C20 (cold temperature treatment group receiving alfaxalone, 20 mg kg−1, IM). Within each phase, a crossover design was utilized, so each individual received each treatment, with a minimum 7‐day washout period.
      The commercial formulation of alfaxalone used in this study (Alfaxan‐CD RTU; Jurox, NSW, Australia) was available in a concentration of 10 mg mL−1 with a 2‐hydroxypropyl‐beta‐cyclodextrin excipient. On the morning of the experiment, each slider was removed from its tank, weighed and allowed to sit for a minimum of 5 minutes in a quiet environment, balanced on pedestals approximately 5 cm above the tabletop, such that neck and leg relaxation could be fully assessed without stimulation, and also to prevent ambulation.
      Alfaxalone was then administered by deep IM injection into the pectoralis major muscle of a thoracic limb with randomization of the chosen side, dosed according to each turtle's assigned treatment group. After injection, turtles were placed back on their pedestals for observation.
      The following measurements were taken by an individual (MS) blinded to drug dose at baseline (pre‐injection), 5, 12, 20, 30, 45, 60, and 120 minutes post injection: heart rate (HR), palpebral reflex, corneal reflex, muscle relaxation, ease of handling, and sensitivity to a toe pinch stimulus. Respiratory rate was not measured because some animals were observed to breathe less than once per minute. HR was obtained by counting Doppler sound (Parks Medical Electronics, OR, USA), with the piezoelectric crystal placed at the thoracic inlet, directed toward the heart. Palpebral and corneal reflexes were tested by gently touching a cotton‐tipped applicator to the lateral and medial canthus or cornea, respectively. Presence of a palpebral or corneal reflex was each assigned a score of “0” while absence of a response was assigned a score of “1”. Muscle relaxation and ease of handling were scored using a modification of a previously‐described, three‐point scale (Santos et al.
      • Santos ALQ
      • Bosso ACS
      • Alves Jr, JRF
      • et al.
      Pharmacological restraint of captivity giant Amazonian turtle Podocnemis expansa (Testudines, Podocnemididae) with xylazine and propofol.
      ; Appendix 1). Sensitivity to a noxious stimulus was determined by withdrawal of the limb in response to a toe pinch stimulus applied to the thoracic limb opposite that used for injection by applying a 16‐cm curved Kelly hemostat at the second joint of the phalanges and tightening to the first ratchet. A 4 × 4 gauze sponge was placed between the jaws of the hemostat and toe for each test, so as to reduce the likelihood of injury. A withdrawal response accompanied by simultaneous movement of the head or another limb received a score of 0. The absence of a response to the toe pinch or responses suggestive of a spinal reflex (limb withdrawal unaccompanied by other limb or head movement) received a score of 1. When a spinal reflex or no response occurred on the thoracic limb, the same toe pinch stimulus was applied to the contralateral pelvic foot. Gross movement of the head or other limbs at that time received a score of 0, while the absence of a response confirmed a score of 1. Induction of anesthesia was defined by the time point at which turtles achieved both maximal muscle relaxation and ease of handling.
      Beyond the 120‐minute time point, animals were observed approximately every 10 minutes for full return of palpebral and corneal reflex as well as the return of baseline scores for muscle relaxation and ease of handling. The time at which these parameters returned to baseline was recorded as each turtle's “recovery time”, and animals were then returned to their tanks. Three days after the conclusion of this study, these animals were included in an endoscopy (coeleoscopy) laboratory exercise, then euthanized under anesthesia at the end of the laboratory exercise and necropsied.

      Data analysis

      Body weight and baseline HR of each turtle over the course of the experiment were analyzed using a repeated measures anova. For categorical data, an area under the curve (AUC) was calculated, using the time points as the y axis and each individual turtle's value at that time point for the x axis. This provided an individual AUC for each turtle for each domain (muscle relaxation, handling, sensitivity to a toe pinch stimulus). A one‐way repeated measures anova was then used to compare these AUC data, using each treatment condition as a group. Anesthetic duration for each group was compared using a one‐way anova with Tukey's multiple comparison post test. Significance was set at p < 0.05. HR were compared over time using a Friedman's test, with significance set at p < 0.01. Statistical tests were performed using GraphPad Prism v 5 (GraphPad Software Inc., CA, USA).

      Results

      Initial body weight of the animals in this study ranged from 896–1692 g, and there were no significant differences in body weights during the study weeks. Two of nine turtles were mildly to moderately anemic prior to the beginning of the experiment, with packed cell volumes (PCV) of 15% and 15.5% (normal 28.7%, range 16–47%) (ISIS
      • International Species Information System (ISIS)
      ). Other laboratory abnormalities prior to beginning the experiment included mild hypoproteinemia (3/9 turtles), hypoalbuminemia (3/9 turtles), mild hyperglobulinemia (1/9), hypophosphatemia (2/9), and hypouricacidemia (3/9). At the end of the experimental period, 7/9 turtles were hypophosphatemic, ranging from 1.5 to 2.3 mg dL−1 (normal 4.8 mg dL−1, range 2.7–7.7 mg dL−1) (ISIS
      • International Species Information System (ISIS)
      ). There was no significant difference in recovery time between turtles that were hypoproteinemic (192 ± 62 minutes) and those that were not (204 ± 59 minutes) (p = 0.60).
      Although baseline HR was not significantly different between treatment groups (Table 1), t‐test comparing the AUC generated from each group's HR over time showed significant differences between warm and cold conditions. HR AUC was found to be significantly lower in the C10 than the W10 group, and also was significantly lower in the C20 group, compared to the W20 group (p < 0.05). C10 and W20 turtles had significantly higher scores for muscle relaxation (p < 0.01, p = 0.01, respectively) and handling (p < 0.01, p = 0.04, respectively) than W10 turtles. No significant differences were observed in palpebral reflex or responsiveness to a toe pinch stimulus for the duration of the experiment. None of the turtles lost corneal reflex throughout the experiment.
      Table 1Heart rates (beats minute−1) of turtles before (baseline) and after administration of alfaxalone 10 mg kg−1 (C10 and W10) or 20 mg kg−1 (C20 and W20) IM (mean ± SD). C = cold, W = warm. No significant differences in baseline HR were found between treatment groups. HR AUC was significantly lower in C10 turtles compared to W10 turtles (*), and also in C20 turtles compared to W20 turtles (†) p < 0.05
      Time (minutes)Group
      C10*C20 †W10*W20 †
      Baseline18 ± 925 ± 1025 ± 1025 ± 13
      525 ± 929 ± 335 ± 1233 ± 12
      1227 ± 327 ± 331 ± 1133 ± 4
      2025 ± 225 ± 334 ± 1334 ± 7
      3024 ± 326 ± −332 ± 1234 ± 8
      4525 ± 325 ± 333 ± 1335 ± 6
      6026 ± 327 ± 336 ± 836 ± 6
      12023 ± 425 ± 426 ± 1032 ± 9
      Maximal muscle relaxation was achieved in all nine turtles of the C20 group by 12 minutes post‐injection, while the C10, W10 and W20 groups only showed maximal relaxation in a fraction of individuals within their respective groups (Table 2). Maximal ease of handling was achieved in only five of nine turtles in the C20 group, three of nine turtles in the W20 group and two of nine turtles in the C10 group (Table 2). Induction time was variable between treatment groups, and induction was not achieved in the W10 group. By 12 minutes post injection, induction was achieved in five of nine turtles in the C20 group, three of nine turtles in the W20 group, one of nine turtles in the C10 group, and none of the turtles in the W10 group (Table 2).
      Table 2Number of turtles per group (n = 9) that achieved maximal muscle relaxation, maximal ease of handling, and anesthetic induction (maximal muscle relaxation and ease of handling) at 5, 12, and 20 minutes after receiving alfaxalone 10 mg kg−1 (C10 and W10) or 20 mg kg−1 (C20 and W20) IM. C = cold, W = warm
      GroupMuscle relaxationEase of HandlingInduction
      Minutes after injectionMinutes after injectionMinutes after injection
      512205122051220
      C10039212012
      C20399255155
      W10002000000
      W20345233233
      Mean recovery time for W10, W20, C10 and C20 groups were 126 ± 15, 216 ± 34, 206 ± 46 and 257 ± 25 minutes, respectively. High dose turtles had significantly longer recoveries than low dose turtles within each temperature category. Recovery was also significantly longer in C10 and C20 groups compared to the W10 group (Fig. 1).
      Figure thumbnail gr1
      Figure 1Time (mean ± SD) elapsed between injection of alfaxalone and recovery from anesthesia (defined as the return to baseline scores for palpebral and corneal reflex, muscle relaxation and ease of handling) for each treatment group. Significant differences were between C10 and W10 (*), W10 and W20 (†), W10 and C20 (‡), and C10 and C20 (§), p ≤ 0.05.
      All turtles survived and no abnormalities, such as injection site reaction, were found at necropsy.

      Discussion

      This study showed that alfaxalone given at 10 mg kg−1 or 20 mg kg−1 IM may be used in red‐eared sliders to produce variable degrees of muscle relaxation and improve compliance to handling for diagnostic or medical procedures such as radiography, physical exam and venipuncture. While the higher dose (20 mg kg−1) of alfaxalone and the colder temperature environment intensified relaxation and compliance to handling in these animals, neither dose nor ambient temperature significantly changed palpebral reflex, corneal reflex or response to toe pinch stimulus – parameters used by 85.8%, 45.0% and 36.0% of practitioners, respectively, to determine anesthetic depth in anesthetized reptiles (Read
      • Read MR
      Evaluation of the use of anesthesia and analgesia in reptiles.
      ). In contrast to our results, one study demonstrated that 20 mg kg−1 of alfaxalone IM was sufficient for induction of anesthesia in iguanas undergoing minor surgical procedures or for intubation prior to maintenance with inhalation anesthetics, based on loss of righting reflex and subjective evaluations of spontaneous movement, muscle tone, palpebral and corneal reflex and responsiveness to a “hard toe pinch” (Bertelsen & Sauer
      • Bertelsen MF
      • Sauer CD
      Alfaxalone anaesthesia in the green iguana (Iguana iguana).
      ). The investigators were able to intubate all iguanas given 20 mg kg−1 in that study. It is unknown whether intubation would have been feasible in the turtles in this study, although compliance to opening of their mouths was assessed as part of the “handling” score. The decision was made not to attempt intubation based on the concern that repeated intubation attempts may traumatize the glottis and compromise the welfare of animals in this study.
      Recovery from the effects of alfaxalone was significantly longer in groups given the higher dose of alfaxalone, as well as groups kept in colder ambient temperature environments. The turtles with longest recovery time (257 ± 25 minutes) were those administered 20 mg kg−1 and kept in the cold condition, while turtles with the shortest recovery time (126 ± 15 minutes) were those administered 10 mg kg−1 in the warm condition. The authors recognize that recoveries in excess of four hours may realistically limit the clinical utility of this drug in turtles, particularly those living in low‐ambient temperature environments immediately prior to anesthesia. Compared to green iguanas, this species exhibits more prolonged recovery from alfaxalone. Bertelsen & Sauer (
      • Bertelsen MF
      • Sauer CD
      Alfaxalone anaesthesia in the green iguana (Iguana iguana).
      ) described full recovery of reflexes and muscle relaxation in iguanas administered 20 mg kg−1 alfaxalone IM by 37 ± 9 minutes post‐injection (Bertelsen & Sauer
      • Bertelsen MF
      • Sauer CD
      Alfaxalone anaesthesia in the green iguana (Iguana iguana).
      ). Recovery from the same dose in our study population was nearly seven times longer than that observed in iguanas. Recovery from alfaxalone anesthesia is also comparatively shorter in dogs and cats. Whittem et al. (
      • Whittem T
      • Pasloske KS
      • Heit MC
      • et al.
      The pharmacokinetics and pharmocodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan at clinical and supraclinical doses.
      ) described anesthetic duration (defined as time to end of recumbency) of approximately 45 minutes in cats that received a single 5 mg kg−1 IV dose of alfaxalone. Cats receiving the same dose that were subsequently exposed to a noxious stimulus recovered in approximately 7 minutes (Whittem et al.
      • Whittem T
      • Pasloske KS
      • Heit MC
      • et al.
      The pharmacokinetics and pharmocodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan at clinical and supraclinical doses.
      ). One study of dogs administered 6 mg kg−1 IV injection of alfaxalone recorded recovery times of approximately 30 minutes, when recovery was defined as the time from intubation to extubation or duration of nonresponsiveness to a noxious stimulus (Muir et al.
      • Muir W
      • Lerche P
      • Wiese A
      • et al.
      Cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in dogs.
      ). Shorter anesthetic duration in mammals intuitively may be attributed to their endothermic physiology and a metabolism less dependent on ambient temperature. Reasons for the difference in recovery between the turtles in this study and previously reported recovery of green iguanas are unclear.
      The role of colder ambient temperatures in prolonging drug elimination in an ectothermic species is not an unexpected finding. The relationship between metabolic rate and environmental temperature is well established in reptiles (Branco et al.
      • Branco LGS
      • Portner HO
      • Wood SC
      Interaction between temperature and hypoxia in the alligator.
      ; Mosley
      • Mosley CAE
      Anesthesia and analgesia in reptiles.
      ; Bickler & Buck
      • Bickler PE
      • Buck LT
      Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability.
      ). Despite the fact that hypothermia prolongs the anesthetic effects of alfaxalone in this species, the authors of this study recognize that intentional cooling is not an ethically appropriate method of intensifying anesthetic effect. Based on a wealth of physiological reptile studies, hypothermia is generally not regarded as an efficacious anesthetic strategy for this phylogenetic Class (Martin
      • Martin BJ
      Evaluation of hypothermia for anesthesia in reptiles and amphibians.
      ). While both mammalian and reptilian peripheral nerve conduction velocities decrease with decreasing temperature (DeJong et al.
      • DeJong RH
      • Hershey WN
      • Wagman IH
      Nerve conduction velocity during hypothermia in man.
      ; Rosenberg
      • Rosenberg ME
      Thermal relations of nervous conduction in the tortoise.
      ), neuronal function has been demonstrated in tortoises at temperatures below which mammals are expected to develop conduction blockade (Rosenberg
      • Rosenberg ME
      Temperature and the nervous conduction in the tortoise.
      ). In addition, anesthetic monitoring may be confounded by the paralysis caused by severe hypothermia. Muscular movement is made undetectable at temperatures higher than those associated with nerve blockade (Hunsaker & Lansing
      • Hunsaker D
      • Lansing RW
      Electroencephalographic studies of reptiles.
      ; DeJong et al.
      • DeJong RH
      • Hershey WN
      • Wagman IH
      Nerve conduction velocity during hypothermia in man.
      ; Rosenberg
      • Rosenberg ME
      Thermal relations of nervous conduction in the tortoise.
      ). Given the questionable effectiveness of hypothermia in causing unconsciousness or analgesia, the general difficulty of properly monitoring this state, and potential tissue damage from overzealous cooling, hypothermia's role in anesthesia should only be that of a preexisting condition likely to modify the animal's response to a proven, humane anesthetic protocol.
      The effects of ambient temperature on metabolism may be difficult to separate from those attributable to circadian and biannual changes known to occur in this species. Reyes & Milsom (
      • Reyes C
      • Milsom WK
      Circadian and circannual rhythms in the metabolism and ventilation of red-eared Sliders (Trachemys scripta elegans).
      ) demonstrated that metabolic rate (specifically oxygen consumption and total ventilation) in this species peaks during fall and spring (the reproductive seasons) and decreases during the winter and summer months. The cold‐temperature phase of this experiment occurred during the winter months while these turtles were housed outdoors, in a covered, screened‐in enclosure. Through the course of this study, sunlight hours as well as temperature steadily decreased, which undoubtedly influenced circadian rhythms within this population, and may have affected their responses to alfaxalone.
      Animals in this study were found to have decreases in serum albumin and phosphorus over the course of the experiment, which may have influenced their drug responses. Possible explanations for these changes include deficiency of dietary vitamin D3 or calcium, or inadequate ultraviolet light B (UVB) exposure (Mader
      • Mader DR
      Metabolic bone disease.
      ; Wilkinson
      • Wilkinson R
      Clinical pathology.
      ). The significance of these findings is unclear, as necropsies performed on all the animals revealed no gross abnormalities. Indeed, these abnormalities may be artifactual as reference blood ranges for this population have not been established. We relied upon published biochemical and hematological ranges for turtles that were likely kept under different management conditions, and may have represented a greater variety in sex and life stage. The extent of this drug's plasma protein binding also is not yet described in reptiles. Alfaxalone exhibits relatively low plasma protein binding in mammalian species (Whittem et al.
      • Whittem T
      • Pasloske KS
      • Heit MC
      • et al.
      The pharmacokinetics and pharmocodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan at clinical and supraclinical doses.
      ). If the protein binding properties of this drug in reptiles are similar to that of mammals, the effect of hypoproteinemia on our turtles’ response to alfaxalone would be minimal. This study showed no significant difference in recovery time between hypoproteinemic turtles and those with normal serum protein concentrations.
      Two of nine animals in our study population were anemic on initial evaluation. Possible explanations for this finding include parasitism, lymph contamination and natural seasonal changes in circulating red blood cell mass. Although no specific tests were performed to isolate specific parasites, no evidence of parasitism was found on initial physical exam, during any of the experimental sessions throughout the course of the study, nor on necropsy. Hematocrit has been documented to vary significantly throughout the year in captive, winter‐hibernating chelonians, possibly due to sequestration of red blood cells in the liver or spleen (Lawrence & Hawkey
      • Lawrence K
      • Hawkey C
      Seasonal variations in haematological data from Mediterranian tortoises (Testudo graeca and Testudo hermanni) in captivity.
      ). Normal hematology and biochemistry reference ranges for wild‐caught healthy chelonians also vary a great deal within each species, depending on each individual's reproductive phase, circadian, circannual and life stage (McArthur et al.
      • McArthur SD
      • Wilkinson RJ
      • Meyer J
      ).
      A toe pinch technique was used in this study to simulate a painful mechanical stimulus. Evaluation of pain in reptiles is difficult, and pain evaluation models are poorly substantiated. One survey‐based study showed that the majority of private practitioners recognize a reptile's ability to experience pain, but they are reluctant to administer analgesics (Read
      • Read MR
      Evaluation of the use of anesthesia and analgesia in reptiles.
      ). This reluctance to treat pain in reptiles may be due to the paucity of literature addressing reptile pain assessment and analgesia. To the authors’ knowledge, the only standardized, published pain model in reptiles is a thermal model, utilizing infrared heat stimuli and thermal withdrawal latencies (Sladky et al.
      • Sladky KK
      • Miletic V
      • Paul-Murphy J
      • et al.
      Analgesic efficacy and respiratory effects of butorphanol and morphine in turtles.
      ,
      • Sladky KK
      • Kinney ME
      • Johnson SM
      Analgesic efficacy of butorphanol and morphine in bearded dragons and corn snakes.
      ; Baker et al.
      • Baker BB
      • Sladky KK
      • Johnson SM
      Evaluation of the analgesic effects of oral and subcutaneous tramadol administration in red-eared slider turtles.
      ), which may or may not simulate the intensity of pain most commonly experienced by these animals undergoing surgery or other semi‐invasive procedures in a clinical environment. While no mechanical pain assessment model has been validated for chelonians, a toe pinch stimulus has been validated for assessment of pain in rodents (Chapman et al.
      • Chapman CR
      • Casey KL
      • Dubner R
      • et al.
      Pain measurement: an overview.
      ). Bertelsen & Sauer (
      • Bertelsen MF
      • Sauer CD
      Alfaxalone anaesthesia in the green iguana (Iguana iguana).
      ) used a toe pinch to assess pain in green iguanas, however, the administration was performed by a non‐blinded individual. The toe pinch technique in the present study was standardized using the same hemostat and ratchet‐tightening technique for each animal, and was administered by one individual blinded to drug dose. While animals varied in size, which might have had impact on the force of the toe pinch between individuals, the cross over design allowed valid intra‐individual comparison. Given the difficulty in establishing the analgesic potential of alfaxalone in this species, this drug should not be used as a single anesthetic agent in reptiles undergoing invasive or potentially painful procedures. A multimodal approach to anesthesia must be selected for these cases, including drugs such as morphine or tramadol, which have proven analgesic properties in chelonians (Sladky et al.
      • Sladky KK
      • Miletic V
      • Paul-Murphy J
      • et al.
      Analgesic efficacy and respiratory effects of butorphanol and morphine in turtles.
      ; Baker et al.
      • Baker BB
      • Sladky KK
      • Johnson SM
      Evaluation of the analgesic effects of oral and subcutaneous tramadol administration in red-eared slider turtles.
      ).
      This study demonstrates that IM administration of alfaxalone (10 and 20 mg kg−1) provides dose‐dependent sedation in red‐eared slider turtles. Lower ambient temperatures potentiated muscle relaxation, sedation (compliance to handling), and the duration of action of this drug in turtles. This information serves to alert practitioners of the likelihood of increased effects of this drug in wild chelonians living in environments below their preferred temperature range. Hypothermia should never be intentionally used to enhance the anesthetic effects of this drug. As the analgesic properties of alfaxalone in this species remain unclear we recommend that alfaxalone be paired with proven analgesic drugs in chelonians undergoing invasive or potentially painful procedures.

      Acknowledgements

      Thanks to Dr. Shaun Boone for assistance in animal husbandry and Ms. Lynn Reece and Ms. Fran Cantrell for logistical support.

      Appendix 1

      Numerical rating scale describing the muscle relaxation, ease of handling and sensitivity to a toe pinch stimulus exhibited by red‐eared sliders following IM administration of 10 mg kg−1 or 20 mg kg−1 alfaxalone in two different temperature ranges. Adapted from Santos et al.
      • Santos ALQ
      • Bosso ACS
      • Alves Jr, JRF
      • et al.
      Pharmacological restraint of captivity giant Amazonian turtle Podocnemis expansa (Testudines, Podocnemididae) with xylazine and propofol.
      .
      Tabled 1
      Muscle relaxation(0) The animal keeps its head up and retracted
      (1) The head, legs and tail retain a mild degree of muscle tone
      (2) The head, legs and tail remain extended and relaxed
      Handling(0) Difficulty in flexing and extending the head, legs and tail and in opening the mouth manually
      (1) Mild resistance to manipulation of the head, legs and tail, or to opening the animal's mouth
      (2) No resistance to manipulation of the head, legs and tail, or to opening the animal's mouth
      Sensitivity to toe pinch stimulus(0) Withdrawal response accompanied by movement of the head or another limb
      (1) Absence of a response to the toe pinch or responses suggestive of a spinal reflex (limb withdrawal unaccompanied by limb or head movement)

      References

        • Ambros B
        • Duke-Novakovski T
        • Pasloske KS
        Comparison of the anesthetic efficacy and cardiopulmonary effects of continuous rate infusions of alfaxalone-2-hydroxypropyl-β-cyclodextrin and propofol in dogs.
        Am J Vet Res. 2008; 69: 1391-1398
        • Baker BB
        • Sladky KK
        • Johnson SM
        Evaluation of the analgesic effects of oral and subcutaneous tramadol administration in red-eared slider turtles.
        J Am Vet Med Assoc. 2011; 15: 220-227
        • Bertelsen MF
        • Sauer CD
        Alfaxalone anaesthesia in the green iguana (Iguana iguana).
        Vet Anaesth Analg. 2011; 38: 461-466
        • Bickler PE
        • Buck LT
        Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability.
        Annu Rev Physiol. 2007; 69: 145-170
        • Branco LGS
        • Portner HO
        • Wood SC
        Interaction between temperature and hypoxia in the alligator.
        Am J Physiol. 1993; 265: R1339-R1343
        • Carregaro AB
        • Cruz ML
        • Cherubini AL
        • et al.
        Influence of body temperature on rattlesnakes (Crotalus durissus) anesthetized with ketamine.
        Pesq Vet Bras. 2009; 29 (Article in Portuguese. English abstract obtained online on November 21, 2012): 969-973
        • Chapman CR
        • Casey KL
        • Dubner R
        • et al.
        Pain measurement: an overview.
        Pain. 1985; 22: 1-31
        • DeJong RH
        • Hershey WN
        • Wagman IH
        Nerve conduction velocity during hypothermia in man.
        Anesthesiology. 1966; 27: 805-810
        • Ferre PJ
        • Pasloske K
        • Whittem T
        • et al.
        Plasma pharmacokinetics of alfaxalone in dogs after intravenous bolus of Alfaxan-CD RTU.
        Vet Anaesth Analg. 2006; 33: 229-236
        • Hunsaker D
        • Lansing RW
        Electroencephalographic studies of reptiles.
        J Exp Zool. 1962; 49: 21-32
        • International Species Information System (ISIS)
        Physiological Reference Ranges for Trachemys scripta (Common Slider). Apple Valley, MN, USA2002
        • Johnson R
        The Use of Alfaxalone in Reptiles. Proceedings of the Australian Veterinary Association, Broadbeach, QLD, Australia2005: 1-4 (Conference)
        • Lawrence K
        • Hawkey C
        Seasonal variations in haematological data from Mediterranian tortoises (Testudo graeca and Testudo hermanni) in captivity.
        Res Vet Sci. 1986; 40: 225-230
        • Leece EA
        • Girard NM
        • Maddern K
        Alfaxalone in cyclodextrin for induction and maintenance of anaesthesia in ponies undergoing field castration.
        Vet Anaesth Analg. 2009; 36: 480-484
        • Maddern K
        • Adams VJ
        • Hill NAT
        • et al.
        Alfaxalone induction dose following administration of medetomidine and butorphanol in the dog.
        Vet Anaesth Analg. 2005; 37: 7-13
        • Mader DR
        Metabolic bone disease.
        in: Mader DR Reptile Medicine and Surgery. 2nd edn. Saunders Elsevier, St. Louis, USA2005: 841-851
        • Martin BJ
        Evaluation of hypothermia for anesthesia in reptiles and amphibians.
        ILAR J. 1995; 37: 186-190
        • McArthur SD
        • Wilkinson RJ
        • Meyer J
        Medicine and Surgery of Tortoises and Turtles. Blackwell Publishing, Oxford, UK2004
        • McKune CM
        • Brosnan RJ
        • Dark MJ
        • et al.
        Safety and efficacy of intramuscular propofol administration in rats.
        Vet Anaesth Analg. 2008; 35: 495-500
        • Mosley CAE
        Anesthesia and analgesia in reptiles.
        Semin Avian Exot Pet Med. 2005; 14: 243-262
        • Muir W
        • Lerche P
        • Wiese A
        • et al.
        Cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in dogs.
        Vet Anaesth Analg. 2008; 35: 451-462
        • Muir W
        • Lerche P
        • Wiese A
        • et al.
        The cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in cats.
        Vet Anaesth Analg. 2009; 36: 42-54
        • Pasloske K
        • Sauer B
        • Perkins N
        • et al.
        Plasma pharmacokinetics of alfaxalone in both premedicated and unpremedicated Greyhound dogs after single, intravenous administration of Alfaxan at a clinical dose.
        J Vet Pharmacol Ther. 2009; 32: 510-513
        • Read MR
        Evaluation of the use of anesthesia and analgesia in reptiles.
        J Am Vet Med Assoc. 2004; 224: 547-552
        • Reyes C
        • Milsom WK
        Circadian and circannual rhythms in the metabolism and ventilation of red-eared Sliders (Trachemys scripta elegans).
        Physiol Biochem Zool. 2010; 83: 283-298
        • Rosenberg ME
        Temperature and the nervous conduction in the tortoise.
        J Physiol (London). 1977; 270: 50P-51P
        • Rosenberg ME
        Thermal relations of nervous conduction in the tortoise.
        Comp Biochem Physiol. 1978; 60A: 57-63
        • Rupprecht R
        • Holsboer F
        Neuropsychopharmaco-logical properties of neuroactive steroids.
        Steroids. 1999; 64: 83-91
        • Santos ALQ
        • Bosso ACS
        • Alves Jr, JRF
        • et al.
        Pharmacological restraint of captivity giant Amazonian turtle Podocnemis expansa (Testudines, Podocnemididae) with xylazine and propofol.
        Acta Cir Bras. 2008; 23: 270-273
        • Sladky KK
        • Miletic V
        • Paul-Murphy J
        • et al.
        Analgesic efficacy and respiratory effects of butorphanol and morphine in turtles.
        J Am Vet Med Assoc. 2007; 230: 1356-1362
        • Sladky KK
        • Kinney ME
        • Johnson SM
        Analgesic efficacy of butorphanol and morphine in bearded dragons and corn snakes.
        J Am Vet Med Assoc. 2008; 15: 267-273
        • Strachan FA
        • Mansel JC
        • Clutton RE
        A comparison of microbial growth in alfaxalone, propofol and thiopental.
        J Small Anim Pract. 2008; 49: 186-190
        • Tortorici MA
        • Kochanek PM
        • Polovac SM
        Effects of hypothermia on drug disposition, metabolism, and response: a focus of hypothermia-mediated alterations on the cytochrome P450 enzyme system.
        Crit Care Med. 2007; 35: 2196-2204
        • White FN
        • Somero G
        Acid-base regulation and phospholipid adaptations to temperature. Time courses and physiological significance of modifying the milieu for protein function.
        Physiol Rev. 1982; 62: 40-90
        • Whittem T
        • Pasloske KS
        • Heit MC
        • et al.
        The pharmacokinetics and pharmocodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan at clinical and supraclinical doses.
        J Vet Pharmacol Ther. 2008; 31: 571-579
        • Wilkinson R
        Clinical pathology.
        in: McArthur S Wilkinson R Meyer J Medicine and Surgery of Tortoises and Turtles. Blackwell Publishing, Ames, IA, USA2004: 141-186
        • Zaki S
        • Ticehurst KE
        • Miyaki Y
        Clinical evaluation of Alfaxan-CD® as an intravenous anaesthetic in young cats.
        Aust Vet J. 2009; 87: 82-87