Intramuscular administration of alfaxalone in red-eared sliders (Trachemys scripta elegans) effects of dose and body temperature

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1 Veterinary Anaesthesia and Analgesia, 2013, 40, doi: /j x RESEARCH PAPER Intramuscular administration of alfaxalone in red-eared sliders (Trachemys scripta elegans) effects of dose and body temperature Michelle Kischinovsky*, Anna Duse, Tobias Wangà & Mads F Bertelsen* *Centre for Zoo and Wild Animal Health, Copenhagen Zoo, Frederiksberg C, Denmark National Veterinary Institute, Uppsala, Sweden àzoophysiology, Department of Biological Sciences, University of Aarhus, Aarhus, Denmark Correspondence: Michelle Kischinovsky, Steenwinkelsvej 3A, 1966 Frederiksberg C, Denmark. michelle_dk@hotmail.com Abstract Objective To characterise the effects of alfaxalone by intramuscular (IM) injection in red-eared slider turtles and the influence of body temperature on anaesthetic duration and depth. Study design Prospective, randomised part-blinded experimental trial. Animals Ten healthy adult female red-eared sliders. Methods Each turtle was anaesthetized four times with 10 and 20 mg kg )1 alfaxalone at 20 and 35 C respectively. Time to maximal effect and plateau and recovery periods were recorded. Skeletal muscle tone, presence of various reflexes, response to noxious stimuli, and heart rate were assessed. Results Results are given for protocols 10 mg kg )1 20 C; 20 mg kg )1 20 C; 10 mg kg )1 35 C and 20 mg kg )1 35 C, respectively: mean time (±SD) to maximal effect was 16 ± 8, 19 ± 6, 5 ± 2 and 7 ± 5 minutes; duration of the plateau phase was 13 ± 12, 28 ± 13, 8 ± 5 and 8 ± 5 minutes and recovery time was 76 ± 20, 126 ± 17, 28 ± 9 and 41 ± 20 minutes. Endotracheal intubation was successful in 80%, 100%, 0% and 30% of turtles, respectively. At 35 C, all animals retained nociceptive sensation in the front limbs, hind limbs and vent, whereas at 20 C a few turtles lost peripheral nociceptive sensation. Corneal and tap reflexes were retained in all trials. Mean heart rates were 30 ± 2 and 66 ± 4 beats minute )1 at 20 and 35 C, respectively. Conclusions and clinical relevance Alfaxalone administered IM in red-eared sliders provided smooth, rapid induction and uneventful recovery. At 35 C either dosage provided only short (5 10 minutes) and light sedation. At 20 C, 10 mg kg )1 provided sedation suitable for short non-invasive procedures. About 20 mg kg )1 provided anaesthesia of approximately 20 minutes duration, appropriate for induction of inhalational anaesthesia or for brief surgical procedures with supplemental analgesia. Keywords alfaxalone, anaesthesia, reptile, turtle. Introduction The unique anatomical, physical and physiological characteristics of chelonians impose several challenges on the anaesthetist. The use of inhalant agents is made difficult by the ability of turtles to hold their breath for extended periods of time, and retractable heads and hinged shells complicate intravenous (IV) access. Although used extensively in reptiles (Read 2004), the effects of intramuscular (IM) administration of ketamine alone, or in combination with 13

2 synergistic agents (Holz & Holz 1994), vary considerably within and amongst species and elicit adverse cardiopulmonary responses even at sedative dosages (Lock et al. 1998; Norton et al. 1998; Greer et al. 2001). Furthermore, the slow metabolism of the ectothermic animals is often associated with exceptionally long-lasting recovery (Glenn et al. 1972; Cooper 1974; Jones 1977; Read 2004). The synthetic neurosteroid anaesthetic alfaxalone is a possible alternative that can be administered IM to induce general anaesthesia in rabbits (Grint et al. 2008; Marsh et al. 2009) and iguanas (Bertelsen & Sauer 2011). Knotek et al. (2011) recently reported insufficient anaesthetic effects in red-eared sliders administered alfaxalone 10 mg kg )1 IM at a temperature between 24 and 27 C. However, this study went into very little detail and failed to investigate the effects of increased dosage. The first objective of this study was to characterise the clinical effects of alfaxalone administered IM to red-eared sliders (Trachemys scripta elegans). Because metabolism generally increases two to threefold with a 10 C rise in body temperature (Schmidt-Nielsen 1983), the rate of breakdown and excretion of drugs is expected to increase similarly when temperature increases. Thus, a second objective was to evaluate how body temperature affects depth of alfaxalone anaesthesia and subsequent recovery. Materials and methods Turtles Ten adult female red-eared sliders (Trachemys scripta elegans) were used in this study with the approval of the Danish Animal Experiments Inspectorate. Their mean body mass (±SD) was 1.4 ± 0.4 kg (range: kg). The turtles were deemed healthy on the basis of a clinical examination and blood samples collected prior to the study to verify normal plasma protein values. The turtles were obtained from a zoological collection where they had been kept as a group for several years. Husbandry The turtles were housed in two groups in cm plastic water tubs. Each tub was equipped with a dry basking area under which was a dark aquatic retreat. The basking area was partly exposed to a radiant heat source and artificial lighting was provided 14 hours a day (7:00 21:00). Water temperature was maintained at C, while temperature in the basking area ranged from 30 to 40 C. Room temperature was between 23 and 26 C. Diet consisted of earthworms, mussels, super worms (Zophobas morio), mice, chicks and smelt. The turtles were fasted for 48 hours prior to each trial. Study design and procedure The study was conducted as a prospective, randomised trial. Based on a pilot study, alfaxalone (Alfaxan-CD RTU; Jurox, Australia) was administered IM at dosages of 10 or 20 mg kg )1. Each turtle was anaesthetized four times: 10 mg kg )1 at 20 C; 20 mg kg )1 at 20 C; 10 mg kg )1 at 35 C and 20 mg kg )1 at 35 C with no <7 days between each trial. The two temperatures were selected based on a preferred body temperature of active redeared sliders of C (Hammond et al. 1988). The investigator performing the clinical assessment (MK) was unaware of the administered dose at the time of the trial. One to 4 hours before anaesthesia, the animal was placed in a tub containing water at target temperature and was maintained there until its cloacal temperature was within 2 C of the desired temperature. Heart rate was measured immediately before administration of alfaxalone by ultrasonography (1101 Merlin, B-K Medical, Denmark) via the cervico-brachial acoustic window. Respiratory rate was not assessed. Activity level and resistance to manipulation of the neck and limbs were assessed at this time. Alfaxalone was administered with a 25 gauge, 16mm needle into the brachial muscles, with half the dose deposited into each forelimb. Drawback was carried out in order to ensure that intravenous injection had not occurred. Anaesthetic monitoring parameters and scoring Parameters monitored were the presence or absence of spontaneous movement, reflexes, and response to pain. Muscle tone was scored as described below. Spontaneous movement was defined as the animal performing purposeful and coordinated movement. Monitored reflexes were palpebral, corneal and tap, defined as the rapid limb retraction in response to tapping of the dorsocaudal portion of the metacarpus and metatarsus with a light object (Ziolo & Bertelsen 2009). Nociceptive response was elicited by a hard pinch with two fingernails on the vent or 14 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 40, 13 20

3 interdigital skin of the front and hind limbs. Skeletal muscle tone was scored in the neck, forelimbs, hind limbs and jaw by assessing resistance to manual manipulation. A subjective scale was used, where 3 represented tone in the conscious animal; 2, partially reduced strength; 1, markedly reduced strength; and 0, no strength/withdrawal. Observations of all the variables were made every 60 seconds for the first 10 minutes following drug administration and then every 5 minutes until recovery. Heart rate was recorded prior to injection, at 10 minutes post-injection and then every 5 minutes until recovery. Endotracheal intubation with a 14-gauge catheter was attempted when no spontaneous movement was present and jaw tone score was equal to or <1. Intubation was considered successful, if the tube was placed uneventfully or when only a minor cough response was elicited. Intubation was defined as unsuccessful when intubation stimulated crawling or evoked a bite or strong cough. Cloacal temperature was determined using a thermometer probe (Testo 925, Testo GmbH, Austria). It was measured at regular intervals and they were maintained at 20 ± 2.5 C or 35 ± 2.5 C by means of a heat lamp or a refrigerated ice pack (5 10 C) placed above or beneath the turtle, respectively. Time to maximal effect was defined as the time elapsed from administration of alfaxalone until all of the measured parameters were at their minimum in that particular anaesthetic event, but not necessarily zero. The plateau phase was defined as the time from maximal effect until any one of the parameters increased. Recovery time was the period between this time point and until data collection was terminated when all reflexes were present, neck and jaw muscle reached a score of 2 or above and limb muscle tone reached a score of 1 or above. At this point, the turtle was able to fully extend its neck, hold its head above water and swim unassisted. All turtles, however, were placed in a dry holding container at room temperature until complete pre-anaesthetic skeletal muscle strength and activity level had returned. All assessments were made by the same individual. Data analysis Results from the four different combinations of anaesthetic dosage and temperature were compared. Heart rate and variables involving time measurements were compared using a linear mixed model. The latter were transformed to the natural logarithmic scale to meet the assumptions of normally distributed residuals and equal variances. The mixed procedure in SAS statistical analysis software (SAS, version 9.1, SAS Institute Inc, NC) was used to conduct these analyses. For calculation of sequences in loss and regain of skeletal muscle tone, loss was defined as the time used for reaching score 1 or lower, and regain as the time of the first increased value. The McNemar test was used to investigate a relationship between loss of the palpebral reflexes and loss of nociceptive response in the front limbs, hind limbs and vent, respectively. Both tests were conducted using the frequency procedure in SAS. The effect of dosage and temperature on the magnitude of the maximal effect, defined as the overall minimum muscle score, was investigated using the GLIMMIX procedure in SAS with multinomial distribution and a cumulative logit link. Turtle ID was included as a random effect. Differences corresponding to p < 0.05 were considered significant. The temperature coefficient for a 10 C change in temperature (Q10) was calculated for the time to maximal effect and recovery time as Q10 = (R 1 /R 2 ) 10/(T2)T1) where R 1 and R 2 are the rates at temperature T1 and T2, respectively. Results Mean time to maximal effect as well as the duration of the plateau phase, recovery phase and total anaesthesia are presented in Table 1. Time to maximal effect (p < ), plateau phase (p < ), recovery phase (p < ) and total anaesthetic duration (p < ) were all significantly longer at the lower temperature. Duration of the plateau phase (p = 0.024), recovery phase (p < ) and total anaesthetic duration (p < ) were significantly longer for turtles receiving 20 compared to 10 mg kg )1, while duration of time to maximal effect did not differ (p = ). There was significant interaction between dosage and temperature in the total anaesthetic duration (p = ). The magnitude of the maximal effect, defined as the overall minimum muscle score, was significantly affected by both dosage and temperature. At 20 C, a significantly higher maximal effect was obtained when the high dosage was administered (mean minimum score = 0.03 versus 0.45; p = 0.003). Similarly, at 35 C, a significantly Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 40,

4 Table 1 Mean duration ± SD (minutes) of the anaesthetic phases (induction, plateau, recovery and total anaesthetic duration) in 10 red-eared sliders administered a single dose of alfaxalone (10 or 20 mg kg )1 ) intramuscularly at low or high temperature (20 or 35 C) Anaesthetic protocol 10 mg kg )1 20 C 20 mg kg )1 20 C 10 mg kg )1 35 C 20 mg kg )1 35 C Time to maximal effect 16 ± 8 cd 19 ± 6 cd 5±2 ab 7±5 ab Plateau 13 ± 12 b 28 ± 13 acd 8±5 b 8±5 b Recovery 76 ± 20 bcd 126 ± 17 acd 28 ± 9 abd 41 ± 20 abc Total time 105 ± 22 bcd 172 ± 15 acd 41 ± 8 abd 56 ± 18 abc a Significantly different from 10 mg kg )1 20 C (p < 0.01); b significantly different from 20 mg kg )1 20 C (p < 0.01); c significantly different from 10 mg kg )1 35 C (p < 0.01); d significantly different from 20 mg kg )1 35 C (p < 0.01). higher maximal effect was obtained when the higher dosage was administered (mean minimum score = 0.57 versus 0.88; p = 0.006). Endotracheal intubation was successful in 80%, 100%, 0% and 30% of turtles in protocols 10 mg kg )1 20 C; 20 mg kg )1 20 C; 10 mg kg )1 35 C and 20 mg kg )1 35 C, respectively. The proportion of animals that lost the palpebral reflex was 60%, 90%, 10% and 40% in protocols 10 mg kg )1 20 C; 20 mg kg )1 20 C; 10 mg kg )1 35 C and 20 mg kg )1 35 C, respectively. The corneal and tap reflex was retained in all turtles in all four protocols. The mean duration of loss of spontaneous movement was 46, 101, 15 and 18 minutes for protocols 10 mg kg )1 at 20 C; 20 mg kg )1 at 20 C; 10 mg kg )1 at 35 C and 20 mg kg )1 at 35 C, respectively. The duration of loss of spontaneous movement was significantly longer at 20 C than at 35 C (p < ). Regardless of temperature, the turtles receiving 20 mg kg )1 had a significantly longer loss of spontaneous movement than those receiving the 10 mg kg )1 (p = 0.015). However, at 35 C there was no significant difference between high and low dosage. At 20 C, 10 mg kg )1 resulted in loss of nociceptive response in the front limbs, hind limbs and vent of 20%, 20% and 10%, respectively, whereas 20 mg kg )1 caused 40%, 20% and 20% loss of nociceptive response. At 35 C, no animals lost this response. The McNemar s test indicated that there was a significant relationship between the loss of palpebral reflex and the loss of sensation to noxious stimuli of the front limbs (p < ), hind limbs (p < ) and vent (p < ). Mean times to first and last score 1 for the skeletal muscle tone of the neck, jaw, front and hind limbs are presented in Table 2. Mean scores for the combined (neck, front and hind limbs) skeletal muscle tone over time are presented in Fig. 1. Each data series ends when the first turtle reached the criteria for recovery, as described above. The loss of muscle tone in the groups assessed occurred rapidly and nearly simultaneously with no clear pattern in sequence of loss. Although the regain of muscle tone during recovery took longer, there was no consistent sequence in the gain of functions. Transient phases with fluctuations of muscle scores (one score up or down) and reflexes were observed in the plateau phases of 25% of animals anaesthetized. These fluctuations, lasting from 4 to 35 minutes, were observed at both dosages and temperatures and occurred in all muscle groups. Heart rates remained constant throughout anaesthesia with mean rates of 30 ± 2 and 66 ± 4 beats minute )1 at 20 and 35 C, respectively. Rates did not diverge substantially from values obtained prior to anaesthesia, which were 28 ± 2 and 60 ± 2 beats minute )1 at 20 and 35 C, respectively. Increasing the dosage did not significantly alter heart rate; however, a markedly higher heart rate was observed in turtles at the higher core body temperature. Q10 for time to maximal effect with 10 and 20 mg kg )1 was 2.1 and 2.0, respectively. For recovery time Q10 was 1.9 and 2.1, respectively. Discussion Alfaxalone administered IM to red-eared sliders provided a rapid, smooth dose- and temperaturedependent induction of anaesthesia. Only a 16 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 40, 13 20

5 Table 2 Mean times ± SD (minutes) to first and last score 1 of skeletal muscle tone (neck, jaw, front and hind limbs) in 10 red-eared sliders administered a single dose of alfaxalone (10 or 20 mg kg )1 ) intramuscularly at low or high temperature (20 C or 35 C) Anaesthetic protocol 10 mg kg )1 20 C 20 mg kg )1 20 C 10 mg kg )1 35 C 20 mg kg )1 35 C Time to first score 1 Time to last score 1 Time to first score 1 Time to last score 1 Time to first score 1 Time to last score 1 Time to first score 1 Time to last score 1 Neck 5 ± 2 d3 56 ± 16 bcd 2 3±1 d 113 ± 22 acd 234 3± ± 12 ab 3 2±2 ab 29 ± 8 ab 2 Jaw 5 ± 3 cd 32 ± 11 bcd 134 4±2 c 61 ± 16 acd 134 3±1 ab ± 12 ab 2±1 a 16 ± 8 ab 1 Front 6 ± 3 bcd 1 65 ± 34 cd 2 4±2 acd 77 ± 10 cd 12 4±2 ab ± 18 ab 24 3±2 ab 27 ± 18 ab Hind 5 ± 3 bd 65 ± 37 cd 2 4±2 acd 81 ± 22 cd 12 4±2 b12 31 ± 14 ab 3 3±2 ab 25 ± 14 ab a Significantly different from 10 mg kg )1 20 C (p < 0.05); 1 significantly different from neck (p < 0.05). b Significantly different from 20 mg kg )1 20 C (p < 0.05); 2 significantly different from jaw (p < 0.05). c Significantly different from 10 mg kg )1 35 C (p < 0.05); 3 significantly different from front limb (p < 0.05). d Significantly different from 20 mg kg )1 35 C (p < 0.05); 4 significantly different from hind limb (p < 0.05). Figure 1 Combined (neck, front limbs and hind limbs) mean skeletal muscle tone (0 3) over time in 10 red-eared sliders administered a single dose of alfaxalone (10 or 20 mg kg )1 ) intramuscularly at low or high temperature (20 or 35 C). Each data series ends when the first turtle recovers. About 10 mg kg )1 20 C (circle); 20 mg kg )1 20 C (dot); 10 mg kg )1 35 C (triangle) and 20 mg kg )1 35 C (diamond). marginal difference in time to maximal effect was noted between high and low dosages within each temperature trial. However, the temperature had profound effects on both duration and level of anaesthesia. It is generally recommended that reptiles be maintained at or near their preferred body temperature (PBT) during anaesthesia and recovery (Mosley 2005; Bertelsen 2007) to maintain optimal immune response and metabolic rate. When reptiles have been anaesthetized at temperatures below their PBT, they have appeared more deeply sedated (Cooper 1974), the duration of both anaesthesia and recovery have been prolonged and smaller dosages of anaesthetic have been required to produce sedation and anaesthesia (Arena et al. 1988). To our knowledge, only Preston et al. (2010) have described the effects of elevating core body temperature to the high end of the PBT. In red-sided garter Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 40,

6 snakes (Thamnophis sirtalis parietalis) anaesthetized with methohexital sodium, the recovery time at 36 C (upper range of PBT) was half that at 21 C (low end of PBT). However, in accordance with our study, the snakes rarely reached a level of anaesthesia suitable for surgery at the high temperature. The Q10 for recovery time very close to two in this study is in remarkable agreement with the study of recovery following methohexital anaesthesia in garter snakes, where Q10 for C, C and C was 2.1, 1.9 and 1.8, respectively (Preston et al. 2010). This twofold difference in duration of anaesthesia is similar to the twofold rise in oxygen uptake within this temperature range (Jackson 1971; Glass et al. 1983). This may reflect that increased metabolism capacity, most probably of the liver, to metabolise the anaesthetics and account for the larger dosage required at high temperature. Perhaps more interestingly, exactly the same Q10 was found for the time to maximal effect, this time reflecting not metabolism, but rather increased circulation time at the higher temperature. At 35 C, either dose of alfaxalone provided only a short (5 10 minutes) and light sedation, whereas at 20 C, administration of 10 mg kg )1 alfaxalone provided sedation suitable for short non-invasive procedures such as clinical examination, blood and biopsy sampling, etc. Few turtles lost muscle strength and only in the hind part. Animals given 20 mg kg )1 at 20 C reached deep anaesthesia with good muscle relaxation lasting approximately 20 minutes, sufficient for brief surgical procedures, with supplementation of appropriate analgesia. Although additional doses of IM or IV alfaxalone or even supplementation through continuous infusion are possible, such techniques have not yet been investigated properly, so endotracheal intubation and supplementation with inhalational anaesthesia is recommended for maintenance. At either temperature the time to maximal effect was longer with 20 than 10 mg kg )1. Although immediately counterintuitive, this simply reflects that longer time was required to reach a deeper level of sedation with the higher dosage. Anaesthetic depth can be difficult to assess in chelonians. Loss of palpebral reflexes was correlated with anaesthetic depth as judged by muscle tone, reaction to noxious stimuli and the ability to intubate. The fact that all turtles exhibited response to pinching at 35 C probably reflects faster drug redistribution and metabolism at the elevated temperature, as discussed above. The palpebral reflex was always lost when pain sensation was absent, but a remarkably large proportion of turtles lost the palpebral reflex, while pain sensation was still present. The palpebral reflex cannot, therefore, be recommended as a sole indicator for anaesthetic depth, although it correlates well with other parameters such as loss of muscle strength and ability to intubate. Furthermore, muscle relaxation does not correlate with loss of pain sensation in front limbs, hind limbs and vent in any of the four protocols, and invasive surgical procedures would not be advised without proper analgesic coverage. The retained corneal and tap reflexes in all trials indicate that an excessively deep plane of anaesthesia has been reached if these reflexes are lost, which was also concluded in a study using propofol (Ziolo & Bertelsen 2009). In the present study it was not possible to demonstrate a consistent sequence in loss and regain of skeletal muscle strength. In contrast, skeletal muscle tonewaslostinacranio-caudaldirectionandregained in the opposite direction during recovery in red-eared sliders anaesthetized with medetomidine-ketamine (Greer et al. 2001) and propofol (Ziolo & Bertelsen 2009). A similar phenomenon has been described in varanid lizards during induction with various inhalation agents (Bertelsen et al. 2005). It is possible that a similar sequence of loss of functions was masked in the present study by the ability of alfaxalone to produce a rapid induction. Though a specific craniocaudal or caudo-cranial direction could not be found in all four muscle groups, it is still noteworthy that in all protocols a majority of turtles regained muscle strength in the cranial half of the body first. This is in accordance with a study in green iguanas (Bertelsen & Sauer 2011) in which a clear pattern in the loss of muscle tone during induction could also not be demonstrated; however, during recovery, tone returned in a cranio-caudal direction. As these patterns have now been observed in different species, there are reasons to believe that this may be a characteristic of the drug rather than the species investigated; however, this requires further investigation. Transient fluctuations of muscle scores and presence of reflexes were observed in all protocols, indicating that this phenomenon is independent of dose and temperature, but rather related to an innate feature of turtles. The authors have no 18 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 40, 13 20

7 explanation for these observations, but a similar phenomenon was seen in anaesthetized red-eared sliders recovering from ketamine, ketamine/xylazine and ketamine/midazolam combinations, respectively (Holz & Holz 1994) and is not uncommonly observed in chelonians sedated with propofol (Bertelsen, personal observation). There are grounds for further research into this subject. In the red-eared slider, alfaxalone shares many of the desired qualities with propofol, likely due to the same mode of action. They both provide a smooth and rapid onset of anaesthesia, a relatively fast recovery and an uneventful course (Ziolo & Bertelsen 2009). A clear difference is that alfaxalone can be administered intramuscularly; however, dosages required to reach surgical anaesthesia may result in very large injection volumes for some animals. Combining alfaxalone with benzodiazepines or alpha-2-adrenergic agonists, as is often done with ketamine (Sleeman & Gaynor 2000; Greer et al. 2001; Dennis & Heard 2002), would seem a logical avenue to explore. In conclusion, intramuscular administration of alfaxalone provides reliable anaesthesia in red-eared sliders, highly dependent on body temperature. Supplemental analgesia and/or inhalational anaesthesia is advised for longer or more invasive procedures. In turtles anaesthetized at 20 C, recovery time can be shortened by elevating core body temperature toward the upper range of the PBT at the end of the procedure. Acknowledgements This study was funded by the Novo Nordisk Foundation. The Alfaxalone used was supplied by Jurox, Pty Ltd., Australia. The authors wish to thank the reptile staff at Copenhagen Zoo, particularly Lars Jensen, as well as Helle Bernstorf Hydeskov, Carsten Grøndahl and Helle Flaga. References Arena PC, Richardson KC, Cullen LK (1988) Anaesthesia in two species of large Australian skink. Vet Rec 123, Bertelsen MF (2007) Squamates (snakes and lizards). In: Zoo Animal and Wildlife Immobilization and Anesthesia. West G, Heard D, Caulket N (eds). Blackwell Publishing, Ames, IA, USA pp Bertelsen MF, Sauer CD (2011) Clinical efficacy of Alfaxalone in green iguanas (Iguana iguana). Vet Anaesth Analg 38, Bertelsen MF, Mosley CAE, Crawshaw GJ et al. (2005) Inhalation anesthesia in Dumeril s monitor (Varanus dumerili) with isoflurane, sevoflurane and nitrous oxide: effects of inspired gasses in induction and recovery. J Zoo Wildl Med 36, Cooper JE (1974) Ketamine hydrochloride as an anesthetic for East African reptiles. Vet Rec 95, Dennis PM, Heard DJ (2002) Cardiopulmonary effects of a medetomidine-ketamine combination administered intravenously in gopher tortoises. J Am Vet Med Assoc 220, Glass ML, Boutlier RG, Heisler N (1983) Ventilatory control of Arterial PO 2 in the turtle Chrysemys picta bellii: effects of temperature and hypoxia. J Comp Physiol 151, Glenn J, Straight R, Snyder CC (1972) Clinical use of ketamine hydrochloride as an anesthetic agent for snakes. Am J Res 33, Greer LL, Jenne KJ, Diggs HE (2001) Medetomidine-ketamine anesthesia in red-eared slider turtles (Trachemys scripta elegans). Contemp Top Lab Anim Sci 40, Grint NJ, Smith HE, Senior JM (2008) Clinical evaluation of alfaxalone in cyclodextrin for the induction of anaesthesia in rabbits. Vet Rec 163, 395. Hammond KA, Spotila JR, Standora EA (1988) Basking behavior of the turtle pseudemys scripta: effects of digestive state, acclimation temperature, sex, and season. Physiol Zool 61, Holz P, Holz RM (1994) Evaluation of ketamine, ketamine/ xylazine, and ketamine/midazolam anesthesia in redeared sliders (Trachemys scripta elegans). J Zoo Wildl Med 25, Jackson DC (1971) The effect of temperature on ventilation in the turtle, Pseudemys scripta elegans. Respir Physiol 12, Jones DM (1977) The sedation and anesthesia of birds and reptiles. Vet Rec 101, Knotek Z, Hrda A, Knotkova Z (2011) Anaesthesia of the red-eared slider (Trachemys scripta elegans) by intramuscular injection of alfaxalone. Veterinarni klinika 8, Lock BA, Heard DJ, Dennis P (1998) Preliminary evaluation of medetomidine-ketamine combinations for immobilization and reversal with atipamezole in three tortoises. Bull Assoc Reptilian Amphibian Vet 8, 6 9. Marsh MK, McLeod SR, Hansen A et al. (2009) Induction of anaesthesia in wild rabbits using a new alfaxalone formulation. Vet Rec 164, Mosley CAE (2005) Anesthesia and analgesia in reptiles. Sem Av Ex Pet Med 14, Norton TM, Spratt J, Behler J et al. (1998) Medetomidine and ketamine anaesthesia with atipamezole reversal in private free-ranging gopher tortoises, Gopherus polyphemus. Bull Assoc Reptilian Amphibian Vet 8, Preston DL, Mosley CAE, Mason RT (2010) Sources of variability in recovery time from methohexital sodium anesthesia in snakes. Copeia 3, Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 40,

8 Read MR (2004) Evaluation of the use of anesthesia and analgesia in reptiles. J Am Vet Med Assoc 224, Schmidt-Nielsen K (1983) Animal Physiology: Adaptations and Environment (3rd edn). Cambridge University Press, Cambridge, UK pp Sleeman JM, Gaynor J (2000) Sedative and cardiopulmonary effects of medetomidine and reversal with atipamezole in desert tortoises (Gopherus agassizii). J Zoo Wildl Med 31, Ziolo MS, Bertelsen MF (2009) Effects of propofol administered via the supravertebral sinus in red-eared sliders. J Am Vet Med Assoc 234, Received 7 October 2011; accepted 1 December Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 40, 13 20