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Anaesthesia in tortoises may be challenging because intravenous (IV) access is difficult and induction with inhaled anaesthetic agents may be impeded by breath-holding. Anaesthetic induction by the intramuscular (IM) route circumvents these problems, but the number of drugs available for the purpose is limited. Alfaxalone is a synthetic neuroactive steroid registered for IM and IV anaesthesia in cats and IV use in dogs (Jurox 2011). Its potential for use as an intramuscular anaesthetic agent in reptiles has been described in green iguanas (Iguana iguana) (Bertelsen & Sauer 2011) and red-eared sliders (Trachemys scripta elegans) (Kischinovsky et al. 2012). The systemic analgesic effect of alfaxalone has been shown to be minimal in rats and cats (Gilron & Coderre 1996; Pathirathna et al. 2005a,b; Murison & Taboada 2010), and highly questionable in turtles (Kischinovsky et al. 2012). Moreover, its intramuscular administration in reptiles is hampered by excessive injection volumes (Kischinovsky et al. 2012). Medetomidine has sedative and analgesic properties (Posner & Burns 2009), and has been used for IM sedation in several chelonian species including the desert tortoise (Gopherus agassizii) (Sleeman & Gaynor 2000) and for anaesthesia in combination with ketamine in red-eared sliders (Greer et al. 2001). A combination of medetomidine and alfaxalone might, therefore, confer some advantages compared to alfaxalone alone.
The objective of this study was to characterise some of the clinical and physiological effects of intramuscularly administered alfaxalone alone and in combination with medetomidine in a terrestrial chelonian, the Horsfield's tortoise (Agrionemys horsfieldii), one of the most commonly kept species of tortoises in captivity.
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Median total anaesthesia/sedation time, time to plateau phase, duration of plateau phase and recovery are presented in Table 1. The total anaesthesia/sedation time produced by alfaxalone was significantly increased (p < 0.05) by the addition of medetomidine. There were no significant differences regarding time to plateau phase and duration of plateau phase.
Table 1. Mean ±SD and the 25th/75th percentile in brackets for time to plateau phase, duration of plateau phase, recovery time and total time. Number of tortoises intubated, lack of response to the 30 and 60 minutes IM injection, and number of tortoises with loss of peripheral nociceptive sensation in thoracic limbs, pelvic limbs and tail respectively
| ||Anaesthetic protocol|
|10 mg kg−1 A||10 mg kg−1 A + 0.1 mg kg−1 M||20 mg kg−1 A||20 mg kg−1 A + 0.05 mg kg−1 M|
|Time to plateau phase (minutes)||15 ± 5 (10/18.50)||15 ± 6 (10/18.50)||14 ± 6 (8/18.50)||13 ± 4 (10/14)|
|Plateau phase (minutes)||46 ± 23 (25/65)||99 ± 74 (45/159)||83 ± 36 (58.5/93)||103 ± 49 (68/138)|
|Recovery time (minutes)||55 ± 40 (22/97)d||110 ± 40 (27.5/127)||90 ± 45 (50/127)||160 ± 99a (82.5/232)|
|Total time (minutes)||116 ± 39b,d (87/157.5)||223 ± 85a (152.5/292)||187 ± 30d (162.5/217.5)||275 ± 108a,c (205/325)|
|Ability to intubate||0/9b,c,d||6/9a||5/9a||8/9a|
|Time to intubation (minutes)||–||17.4 ± 2.9||23.7 ± 16.0||23.2 ± 16.5|
|Time to extubation (minutes)||–||136.2 ± 59.2||69.8 ± 18.4||125.8 ± 44.5|
|Lack of response to injection (30 minutes)||0/9b,d||5/9a||2/9||6/9a|
|Lack of response to injection (60 minutes)||0/9||4/9||0/9||4/9|
|Loss of peripheral nocicep-tive sensation: thoracic limbs||0/9b,d||7/9a||2/9d||8/9a,c|
|Loss of peripheral nocicep- tive sensation: pelvic limbs||0/9b||5/9a,c||0/9b||4/9|
|Loss of peripheral nocicep-tive sensation: tail||0/9||2/9||0/9||4/9|
|Loss of peripheral nocicep-tive sensation: limbs and tail||0/9||2/9||0/9||3/9|
The pre-anaesthetic HR was 53 ± 6 beats minute−1. With all protocols, there was a significant decrease in HR following injection (p < 0.001). From 6 to 30 minutes a significantly lower HR (p < 0.05) was seen in the protocols including medetomidine (Fig. 1). After atipamezole administration at 60 minutes, the HR increased for the two protocols that included medetomidine. Heart rate increased more slowly when using 10 mg kg−1 alfaxalone + 0.1 mg kg−1 medetomidine compared to 20 mg kg−1 alfaxalone + 0.05 mg kg−1 medetomidine. This increase in HR was only transient, with further slow decreases observed approximately 120 minutes after injection of 10 mg kg−1 alfaxalone + 0.1 mg kg−1 medetomidine, and 150–180 minutes after 20 mg kg−1 alfaxalone + 0.05 mg kg−1 medetomidine (Fig. 1).
Figure 1. Mean (±SD) heart rate (beats minute−1) of nine Horsfield's tortoises during anaesthesia with four different protocols. During the first 30 minutes, heart rate in protocols 1 and 2 was significantly different to protocols 3 and 4. After atipamezole administration at 60 minutes (dashed vertical line), the heart rate in the two protocols including medetomidine increased. Squares: 10 mg kg−1 alfaxalone; triangles: 10 mg kg−1 alfaxalone + 0.1 mg kg−1 medetomidine; lines: 20 mg kg−1 alfaxalone; circles: 20 mg kg−1 alfaxalone + 0.05 mg kg−1 medetomidine.
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Two of the tortoises developed periods of extreme bradycardia (no cardiac activity for 10 minutes), diagnosed with cardiac ultrasound, when administered the protocols which included medetomidine. In three of these four episodes, the tortoises were intubated and mechanical ventilation was instigated, but no exhaled carbon dioxide could be detected with capnography. In the fourth episode, the tortoise resisted intubation. During each of these episodes, tap, palpebral and corneal reflexes were present and in three of the four episodes, pelvic limb withdrawal reflexes were also preserved. Cardiac function returned spontaneously on each occasion; heart rate gradually increased and subsequently followed the pattern of the other tortoises in that experimental group. In three of the four episodes, cardiac arrest occurred at 65–80 minutes after the first injection (5 and 20 minutes after atipamezole administration), while in the fourth case it occurred during the first 30 minutes.
The RR showed a significant decrease 2 minutes after injection with all four protocols (p < 0.0001). Between 8 and 29 minutes, mean RR was ≤1 breath minute−1 for all the protocols, and with protocols 2, 3 and 4, mean RR fluctuated around 1 breath minute−1 until the end of the recovery period.
The proportion of successfully intubated tortoises in each group is presented in Table 1. Significantly fewer tortoises could be intubated with protocol 1 than with 2, 3 and 4 (p < 0.03). For the animals that were intubated, there was no significant difference between groups regarding times to intubation or extubation (Table 1).
The time to reach a score of ≤ 1 in muscle tone in the neck, jaw and limbs ranged from 9 to 11 minutes with all four protocols. The pattern of loss and return of muscle tone is illustrated in Fig. 2. Muscle tone was lost significantly (p < 0.05) faster in the pelvic limbs than in the thoracic limbs, neck and jaw, but there was no clear pattern in the loss of muscle tone among thoracic limbs, neck and jaw. The return of muscle tone occurred in a cranio-caudal direction. In almost half of the experiments, muscle tone transiently fluctuated at the end of the plateau phase.
Figure 2. Combined mean skeletal muscle tone (jaw, neck, thoracic and pelvic limbs) for 9 Horsfield's tortoises using four different drug protocols. Scores were defined as follows: 3 – full muscle tone of the un-anaesthetised tortoise; 2 – intermediate tone; 1 – weak tone; and 0 – no muscle tone. Each recording ends when the tortoise was returned to its enclosure (at a score of 2 in muscle tone). Squares: 10 mg kg−1 alfaxalone; triangles: 10 mg kg−1 alfaxalone + 0.1 mg kg−1 medetomidine; lines: 20 mg kg−1 alfaxalone; circles: 20 mg kg−1 alfaxalone + 0.05 mg kg−1 medetomidine.
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The proportions of animals in each group demonstrating lack of withdrawal response to injection at 30 and 60 minutes, and loss of peripheral nociceptive sensation in the limbs and tail are presented in Table 1. For the protocols including medetomidine, significantly (p < 0.03) fewer tortoises responded to injection at 30 minutes. The response to injection at 60 minutes was not significantly different between protocols. For the protocols including medetomidine, there was significantly (p < 0.03) less peripheral nociceptive sensation in both thoracic and pelvic limbs than with alfaxalone alone.
The palpebral and corneal reflexes were present in all animals at all time points except for one animal in protocol 2, which lost palpebral and corneal reflexes for 79 and 59 minutes, respectively.
Following recovery, most tortoises showed decreased activity for the rest of the day, but were observed eating and exhibiting normal behaviour the following day. Those tortoises that developed transient cardiac arrest were notably slower to resume normal activity in comparison to the others. However, neither tortoise displayed long-term behavioural changes after these episodes.
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The results of this study suggest that the effects of alfaxalone used alone and in combination with medetomidine for IM sedation or anaesthesia in terrestrial tortoises are rapid in onset and are dose-dependent. There are, however, variable individual effects. A dose of 10 mg kg−1 alfaxalone resulted in light to moderate sedation with no analgesia and endotracheal intubation was not possible; 20 mg kg−1 resulted in moderate to deep sedation with minimal analgesic effect. The addition of medetomidine resulted in deep sedation or anaesthesia with significantly increased total anaesthetic time; its addition also significantly reduced peripheral nociceptive sensation compared to the protocols using alfaxalone alone. This is similar to cats and rats, in which alfaxalone has been shown to have either no or minimal analgesic effect (Gilron & Coderre 1996; Pathirathna et al. 2005a,b; Murison & Taboada 2010), whereas medetomidine does provide analgesia (Posner & Burns 2009).
Red-eared sliders kept at 20 °C and anaesthetised with alfaxalone at the same dosages as in the present study were more deeply sedated, easier to intubate and had less peripheral nociceptive sensation (Kischinovsky et al. 2012). Although this may be partly explained by the slightly lower temperature, it may be that red-eared sliders are more sensitive to alfaxalone than Horsfield's tortoises.
Only male tortoises were used in this study, so the results potentially may be gender-biased. In one study, female rats were four times more sensitive than males to intraperitoneally administered alfaxalone/alfadolone (Fink et al. 1982), whereas no difference could be detected in mice, rats and rabbits following intravenous injection (Child et al. 1971; Ferre et al. 2006). No data are available on potential gender differences in alfaxalone sensitivity in chelonians.
Muscle tone was lost in the pelvic limbs first but there was no clear pattern of loss between the thoracic limbs, neck and jaw. However, as muscle tone was assessed only at 2, 8 and 12 minutes in this study (to allow time for other measurements) more than one parameter was often decreased at the same time, blurring the sequence. If the muscle tone had been assessed at shorter intervals, a directional loss might have been observed. The return of muscle tone occurred in a cranio-caudal direction. For lizards and red-eared sliders anaesthetised with alfaxalone, no clear pattern in loss of muscle tone was observed, although it was regained in a cranio-caudal direction (Bertelsen & Sauer 2011; Kischinovsky et al. 2012). For medetomidine-ketamine and propofol anaesthesia in red-eared sliders, muscle tone was lost in a cranio-caudal direction and regained in the opposite direction (Greer et al. 2001; Ziolo & Bertelsen 2009), indicating that the direction of return of muscle tone is more dependent on the protocol used than the species, and that alfaxalone appears to uniformly result in a cranio-caudal return of tone.
In this study, almost half of the tortoises had transient fluctuations in muscle tone at the end of the plateau phase. This phenomenon has also been observed in loggerhead turtles (Caretta caretta) anaesthetised with medetomidine-ketamine and maintained on sevoflurane. Here several periods of strong, coordinated movements for 1–2 minutes were followed by inactivity during recovery (Chittick et al. 2002). In red-eared sliders anaesthetised with alfaxalone, fluctuations in muscle tone and reflexes lasting from 4 to 35 minutes were seen during the plateau phase (Kischinovsky et al. 2012). These fluctuations have been observed in several species of chelonians anaesthetised with several drug classes, and deserve further investigation.
As the tap reflex was present in all tortoises, even during extreme bradycardia, it is not a reliable reflex for anaesthetic monitoring in Horsfield's tortoises.
All tortoises experienced periods of apnoea lasting from 10 to 220 minutes; if intubated, the lungs were ventilated only once every 5 minutes so hypocapnia-induced ventilatory depression could be avoided. Dose-dependent respiratory depression after administration of alfaxalone has been observed in dogs (Muir et al. 2008), cats (Muir et al. 2009) and green iguanas (Bertelsen & Sauer 2011). Similarly, medetomidine administration is associated with respiratory depression in sheep (Bryant et al. 1996; Celly et al. 1997), dogs (Lerche & Muir 2004) and desert tortoises (Gopherus agassizii) (Sleeman & Gaynor 2000). It is therefore not surprising that significant respiratory depression was observed with all four protocols. However, ventilatory depression was no worse with medetomidine-alfaxalone than with alfaxalone alone, and administration of atipamezole did not notably increase the respiratory rate. A similar lack of effect on the respiratory rate after administration of atipamezole was observed in desert tortoises sedated with medetomidine (Sleeman & Gaynor 2000).
Although the baseline heart rate was undoubtedly elevated as a result of stress during pre-anaesthetic handling for ultrasonography of the heart, all four protocols significantly decreased the heart rate, and medetomidine clearly potentiated the bradycardic effect of alfaxalone. This potentiating effect was clearly demonstrated following administration of atipamezole, after which the heart rate increased to values observed with alfaxalone alone. This correlates well with medetomidine's depressive effect on heart rate (Sleeman & Gaynor 2000; Greer et al. 2001), and the ability of atipamezole to reverse this effect (Savola 1989; Vainio & Vähä-Vahe 1990). Transient cardiac arrest occurred in two tortoises when administered the protocols containing medetomidine. Although an electrocardiogram may have revealed cardiac arrhythmias or other cardiac problems, no damage to the heart could be clinically observed after these incidents. These transient episodes of cardiac arrest were followed by an unusually long period of anaesthesia. Remarkably, both tortoises showed normal behaviour and appetite 48 hours after anaesthesia, and no long-term effects were observed over the subsequent 10 months. Such periods of transient cardiac arrest will be unnoticed in the absence of monitoring and such episodes may be more frequent than is generally thought.
As three of the four episodes of severe bradycardia occurred 5–20 minutes after atipamezole administration, this drug may have influenced the heart rate. We have no plausible explanation for this, and it warrants further research. Until more information is available, atipamezole should be used with some caution in chelonians. Consequently, the combination of alfaxalone and medetomidine should be used with caution as it may increase the risk of anaesthesia: a sick or otherwise compromised tortoise might not be able to tolerate the severe bradycardic events that could occur.
Administration of atipamezole 0.4 mg kg−1 one hour after injection of alfaxalone and medetomidine did not seem to effectively antagonise the sedation/anaesthesia. Heart rate increased just after its administration, but slowly decreased again 1–2 hours afterwards. This suggests that the effects of medetomidine outlast those of atipamezole. Re-sedation has been described in dogs (Vainio & Vähä-Vahe 1990), ponies (Di Concetto et al. 2007) and impalas (Aepyceros melampus) (Bush et al. 2004) when alpha-2 agonist sedation was has been antagonised with atipamezole; consequently, either higher doses, or repeated administration of atipamezole, may be required. Interestingly, this observation was made when tortoises were given atipamezole at either four- (protocol 2) or eight- (protocol 4) times the dose of medetomidine. There was, however, a difference in the rapidity of the increase in heart rate increase and in the extent of the subsequent decrease (Fig. 1). This apparent lack of antagonism of the sedative effects of medetomidine is in contrast to findings in desert tortoises administered 0.15 mg kg−1 medetomidine, in which effective antagonism was achieved with 0.75 mg kg−1 atipamezole (five-times the dose of medetomidine) (Sleeman & Gaynor 2000).
In conclusion, IM administration of either alfaxalone, or alfaxalone in combination with medetomidine, can be used in terrestrial tortoises for sedation or anaesthesia for minor procedures, or possibly for induction of anaesthesia prior to maintenance with inhalational agents. Alfaxalone has either no or minimal analgesic effect, but addition of medetomidine partly alleviates this deficit. Observed side effects were significant respiratory depression and bradycardia, including periods of transient cardiac arrest, which may limit the use of this combination for routine anaesthesia in tortoises.