Sedation and anesthesia are necessary components of veterinary care for many reptilian species. Unlike many domesticated mammals, captive reptiles frequently require some level of chemical restraint to facilitate safe and thorough examination, diagnostic sample collection, or diagnostic imaging. Reptiles belong to one of the most phylogenetically diverse animal classes, with > 7,800 species worldwide.1 Little prospective methodical research has been published regarding the use of anesthetic agents in reptiles; thus, anesthetic management of those species is often based on anecdotal reports from clinical practice and published case reports.2–5
For snakes, anesthetic protocols generally include drugs that are administered by inhalation or injection.3 Results of a 2004 survey6 of veterinarians who work with reptiles indicate that the drugs most commonly used to induce and maintain anesthesia in reptilian species were isoflurane, ketamine, propofol, and butorphanol. Unfortunately, many of those drugs have undesirable effects, such as prolonged anesthesia induction or recovery times, cardiorespiratory depression, and inadequate anesthetic depth.2–7
Alfaxalone is a neuroactive steroid molecule believed to exert anesthetic effects through interaction with γ-aminobutyric acid A receptors, which results in CNS depression by increasing chloride conduction and hyperpolarizing cell membranes.8 Alfaxalone has been used as an anesthetic for dogs and cats since 1971.9 In dogs, IV administration of alfaxalone is characterized by a rapid onset of action, rapid redistribution, and a short terminal half-life.10 In Australian squamate species, IV administration of alfaxalone is associated with rapid induction of and smooth recovery from anesthesia.11 Intravenous administration of alfaxalone also induces LRR or a decrease in response to noxious stimuli (ie, loss of toe-pinch reflex) in various reptilian species including snakes, lizards, iguanas, turtles, tortoises, crocodiles, and chameleons.11–17
Because alfaxalone has a steroid nature and cyclodextrin formulation, it can be parenterally administered by routes other than IV (eg, IM).18–20 Intramuscular administration of alfaxalone causes variable sedation in reptiles.13,15,21–24 Alfaxalone may have minimal effects on cardiovascular and respiratory function; thus, some veterinary practitioners prefer it over other anesthetic agents such as propofol.
In snakes, IV access can be challenging owing to the small size and anatomic location of veins. Consequently, anesthetics that can be administered by the ICe route, such as alfaxalone, might be advantageous because of their ease of administration. To our knowledge, ICe administration of alfaxalone has not been described in snakes or other reptilian species.
For anesthetized mammals, anesthetic depth and efficacy are generally assessed by an objective or subjective response to the application of a stimulus.25 For anesthetized reptiles, anesthetic depth is often assessed by LRR and RRR, presence or absence of a corneal reflex, subjective measures of muscle relaxation, and response to pressure applied to a foot or tail with forceps or insertion of needles or other methods that could lead to clinically relevant tissue trauma, all of which may be associated with subjective bias.11,22–24,26 Semmes-Weinstein monofilaments are modified von Frey filaments set into handles and are the current standard for esthesiometry.27 Semmes-Weinstein monofilament sets contain 20 test filaments that can be used to apply a force ranging from 0.008 to 300 g (0.00008 to 2.9 N). In human medicine, SWMs are used to assess nerve function in patients with various neuropathies and tactile abilities in patients with cerebral palsy.27–31 In animals, SWMs have been used to assess the effectiveness of analgesics and detect mechanosensitivity thresholds in neuroanatomical studies.31–33
The objectives of the study reported here were to determine the ICe dose of alfaxalone required to achieve LRR in common garter snakes (Thamnophis sirtalis) and to evaluate the tactile stimulus response of common garter snakes that were and were not anesthetized by means of ICe administration of alfaxalone. Our hypotheses were that ICe administration of 20 mg of alfaxalone/kg would result in LRR in all snakes, use of SWMs to apply a tactile stimulus to snakes would be repeatable, and the response to tactile stimulation with SWMs would be less pronounced in anesthetized snakes than in unanesthetized snakes.
Materials and Methods
Animals
All study procedures were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee (protocol No. 16145). Eight mature garter snakes were obtained from a commercial sourcea for use in the study. The snakes had a mean ± SD body weight of 55.5 ± 9.0 g (range, 43.8 to 68.2 g) and length of 69.4 ± 5.1 cm (range, 62.5 to 77.5 cm). Snakes were individually housed in 76-L glass terrariums. All terrariums were kept in the same room, and all study procedures were performed in that room. The ambient daytime temperature in that room ranged from 22.8° to 25.6°C, and the humidity ranged from 15% to 38%. Ambient lighting was used to provide full-spectrum UV radiation, and there was a 12-hour light-to-dark cycle throughout the duration of the study. Snakes were fed feeder fish every 7 days. Snakes were monitored daily and determined to be healthy on the basis of results of a physical examination and observation of normal activity and feeding behavior. Snakes were acclimated for a minimum of 2 weeks before study initiation. The study consisted of 3 phases, and the same 8 snakes were used for all 3 phases. Food but not water was withheld from all snakes for 72 hours prior to initiation of the experiments for each phase.
Phase 1
Phase 1 had a randomized crossover design. Each snake received each of 3 doses (10, 20, and 30 mg/kg) of alfaxalone,b ICe. The order in which the 3 doses were administered to each snake was randomized by means of drawing paper lots from an envelope. A minimum 2-week washout period was observed between treatments, which was equal to or greater than the washout period used in other studies13,14 in which successive doses of alfaxalone were administered to reptiles.
Each dose was injected ICe by use of a 28-gauge needle affixed to a 0.5-mL tuberculin syringec with the needle directed toward the center of the body. The injection site was at the midlateral aspect of the body in the middle of the caudal third of the coelomic cavity approximately 6 cm cranial to the vent. That location was selected to minimize the potential for inadvertent injection of the drug into vital organs. For each injection, the snake was manually restrained by one investigator (DES-H) while alfaxalone was administered by another investigator (SCC-P). After injection, the snake was placed in a 14.2-L plastic containerd (dimensions, 42.6 × 30.5 × 16.8 cm) for observation. Loss of righting reflex was assessed at 30-second intervals by manually positioning the snake in dorsal recumbency. A snake was considered to have LRR when it was unable to spontaneously resume ventral recumbency within 30 seconds after being positioned in dorsal recumbency. Once LRR was observed, the snake was left in dorsal recumbency and a Doppler probee was positioned over the heart for continuous monitoring of the heart rate. The heart rate and respiratory rate were recorded at 5-minute intervals. The respiratory rate was determined by visual observation of thoracic movement. Snakes were left undisturbed until spontaneous RRR occurred. For snakes that had LRR, the durations between alfaxalone administration and LRR (time to LRR) and between onset of LRR and RRR (time to RRR) were recorded.
Phase 2
The response of unanesthetized snakes to tactile stimulation with SWMs was determined during phase 2. Snakes were restrained in customized capped PVC pipes to assess their response to tactile stimulation with SWMs. The length of each snake was measured. Then, PVC pipef (diameter, 1.3 cm) was cut into sections such that the length of each section was 10 cm shorter than the length of the snake for which it was to be used. The intent was to have the caudal portion of the snake's body exposed up to at least 2.5 cm cranial to the vent when it was restrained in the capped pipe.
Each snake was allowed to spontaneously enter the pipe. As the snake approached the opposite end of the pipe, a cap was placed over that end to prevent it from exiting. Then, a commercially available sensory evaluation kitg containing SWMs of varying diameters that could apply pressures ranging from 0.008 to 300 g was used to apply tactile stimuli to the snake. The SWMs were certified to be individually calibrated to deliver a pressure within a 5% SD of that labeled. For each snake, pressure was applied on the dorsal midline at 3 locations (1 cm cranial to the vent, at the level of the vent, and 1 cm caudal to the vent) with a single SWM, beginning with the filament with the smallest diameter. If the snake did not respond with gross purposeful movement when pressure was applied to at least 2 of the 3 locations, the test was considered negative. The process was repeated with the SWM that was the next size larger until gross purposeful movement was elicited when pressure was applied to at least 2 of the 3 locations or an applied pressure of 300 g resulted in a negative test result. The size of and corresponding pressure applied by the SWM that elicited the response was recorded, and the procedure was concluded. For this phase, gross purposeful movement was defined as movement of the exposed portion of the body in a vigorous sideways motion away from the SWM. For each snake, the procedure was performed once daily for 3 consecutive days, and the mean pressure that elicited a response was calculated. The same investigator (DES-H) performed all tactile stimulation tests.
Phase 3
The response of anesthetized snakes to tactile stimulation with SWMs was determined during phase 3. Each snake was anesthetized with 30 mg of alfaxalone/kg, ICe (ie, the lowest dose administered during phase 1 that resulted in LRR in all 8 snakes), as described for phase 1. Once LRR occurred, each snake was positioned in ventral recumbency for tactile stimulation testing as described for phase 2 (because the snakes were anesthetized, they did not need to be restrained in a pipe during tactile stimulation as they were in phase 2). The test procedure was repeated every 10 minutes until gross purposeful movement was elicited, which for this phase was defined as spontaneous forward motion. A tactile pressure of 300 g (highest pressure that could be applied with SWMs in the sensory evaluation kit used) was recorded if gross purposeful movement could not be elicited.
Statistical analysis
For phase 1, a 2-sided exact intrasubject McNemar test was used to assess whether the binomial proportion of snakes that developed LRR differed between alfaxalone doses. An exact McNemar test was considered appropriate because the study population was small, and the P value of an exact test is determined from the distribution of the data (in this case, a binomial distribution) rather than a large-sample approximation from a normal distribution. An intrasubject Kaplan-Meier testh was used to compare the time to LRR and time to RRR between alfaxalone doses. The effective dose of alfaxalone for 50% of the population and accompanying 95% CI were estimated from the data.i The data distributions for heart rate and respiratory rate were assessed for normality by means of the Kolmogorov-Smirnov test, and results indicated that neither variable was normally distributed. Therefore, a Friedman test was used to compare heart rate and respiratory rate among the 3 alfaxalone doses, followed by a Dunn multiple comparisons test when post hoc pairwise comparisons were necessary.
For phase 2, the data distribution for the pressure that elicited a tactile response (tactile pressure) was assessed for normality by means of the Kolmogorov-Smirnov test, and the results indicated that the data were not normally distributed. Therefore, a Kruskal-Wallis testj was used to compare the tactile pressure among the 3 days data were collected. The mean tactile pressure determined over the 3 days of tactile stimulation testing during phase 2 was calculated for the study population and designated as the baseline value for comparison with tactile pressure results obtained during phase 3.
For phase 3, the data distribution for tactile pressure was assessed for normality by means of the Kolmogorov-Smirnov test, and results indicated that the data were normally distributed. A repeated-measures ANOVAj was used to compare tactile pressure over time when snakes were anesthetized and with that at baseline (unanesthetized snakes). A Tukey-Kramer testj was used when post hoc pairwise comparisons were necessary.
Results were reported as the mean ± SD for normally distributed variables and median (range) for variables that were not normally distributed. Values of P ≤ 0.05 were considered significant for all analyses.
Results
Phase 1
Loss of righting reflex was not observed in any of the 8 study snakes following administration of the 10-mg/kg dose of alfaxalone but was observed in 5 snakes following administration of the 20-mg/kg dose and all 8 snakes following administration of the 30-mg/kg dose. The proportion of snakes that developed LRR following the 30-mg/kg dose did not differ significantly (P = 0.25) from that following the 20-mg/kg dose. Although the median time to LRR following the 30-mg/kg dose (3.8 minutes; 95% CI, 2.0 to 11.5 minutes) was significantly (P = 0.04) shorter than that following the 20-mg/kg dose (8.3 minutes; 95% CI, 1.5 to 12.0 minutes), the median time to RRR did not differ significantly (P = 0.79) between the 20-mg/kg (36.2 minutes; 95% CI, 23.1 to 37.0 minutes) and 30-mg/kg (38.8 minutes; 95% CI, 29.9 to 43.0 minutes) doses. The effective dose of alfaxalone for 50% of the population was 17.3 mg/kg (95% CI, 10.3 to 21.8 mg/kg).
For snakes that developed LRR, heart rate was recorded immediately after LRR and every 5 minutes thereafter until just before RRR. The median heart rate did not differ significantly at any time for snakes that developed LRR following the 20-mg/kg dose of alfaxalone and those that developed LRR following the 30-mg/kg dose. All 8 snakes developed LRR after administration of the 30-mg/kg dose of alfaxalone, and the median heart rate at LRR (80 bpm; range, 80 to 96 bpm) did not differ significantly from that at 20 (76 bpm; range, 60 to 80 bpm) and 15 (74 bpm; range, 56 to 80 bpm) minutes before RRR but was significantly greater than the median heart rate at 10 (58 bpm; range, 44 to 80 bpm; P < 0.05) and 5 (52 bpm; range, 40 to 80 bpm; P < 0.01) minutes before RRR and at RRR (54 bpm; range, 28 to 60 bpm; P < 0.001). The mean ± SD respiratory rate (7 ± 3 breaths/min) did not differ at any time following administration of any of the 3 doses or among doses.
Phase 2
The mean ± SD tactile pressure required to induce purposeful movement in unanesthetized snakes did not differ significantly (P = 0.16) among the 3 consecutive days during which tactile stimulation testing was performed (Figure 1). The mean ± SD baseline tactile pressure was 16.9 ± 14.3 g (0.16 ± 0.14 N).
Mean ± SD tactile pressure applied to the dorsum of 8 unanesthetized healthy mature garter snakes (Thamnophis sirtalis) that resulted in purposeful movement on each of 3 consecutive days (phase 2). During tactile stimulation testing, each snake was restrained in a customized capped PVC pipe (diameter, 1.3 cm), the length of which was 10 cm less than the length of the snake. Each snake was allowed to spontaneously enter the pipe. As the snake approached the opposite end of the pipe, a cap was placed over that end to prevent it from exiting. Then, SWMs of varying diameters (pressure range, 0.008 to 300 g [0.00008 to 2.9 N]) were used to apply tactile stimuli to the snake. For each snake, pressure was applied on the dorsal midline at 3 locations (1 cm cranial to the vent, at the level of the vent, and 1 cm caudal to the vent) with a single SWM, beginning with the filament with the smallest diameter. If the snake did not respond with gross purposeful movement when pressure was applied to at least 2 of the 3 locations, the test was considered negative. The process was repeated with the SWM that was the next size larger until gross purposeful movement was elicited when pressure was applied to at least 2 of the 3 locations or an applied pressure of 300 g resulted in a negative test result. For this phase, purposeful movement was defined as movement of the exposed portion of the body in a vigorous sideways motion away from the SWM. For each snake, the procedure was performed once daily for 3 consecutive days. The same investigator performed all tactile stimulation tests. The mean ± SD tactile pressure (16.9 ± 14.3 g [0.17 ± 0.14 N]) for the study population was calculated and used as the baseline tactile pressure for comparison with data for anesthetized snakes in phase 3.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.144
Mean ± SD tactile pressure applied to the dorsum of 8 unanesthetized healthy mature garter snakes (Thamnophis sirtalis) that resulted in purposeful movement on each of 3 consecutive days (phase 2). During tactile stimulation testing, each snake was restrained in a customized capped PVC pipe (diameter, 1.3 cm), the length of which was 10 cm less than the length of the snake. Each snake was allowed to spontaneously enter the pipe. As the snake approached the opposite end of the pipe, a cap was placed over that end to prevent it from exiting. Then, SWMs of varying diameters (pressure range, 0.008 to 300 g [0.00008 to 2.9 N]) were used to apply tactile stimuli to the snake. For each snake, pressure was applied on the dorsal midline at 3 locations (1 cm cranial to the vent, at the level of the vent, and 1 cm caudal to the vent) with a single SWM, beginning with the filament with the smallest diameter. If the snake did not respond with gross purposeful movement when pressure was applied to at least 2 of the 3 locations, the test was considered negative. The process was repeated with the SWM that was the next size larger until gross purposeful movement was elicited when pressure was applied to at least 2 of the 3 locations or an applied pressure of 300 g resulted in a negative test result. For this phase, purposeful movement was defined as movement of the exposed portion of the body in a vigorous sideways motion away from the SWM. For each snake, the procedure was performed once daily for 3 consecutive days. The same investigator performed all tactile stimulation tests. The mean ± SD tactile pressure (16.9 ± 14.3 g [0.17 ± 0.14 N]) for the study population was calculated and used as the baseline tactile pressure for comparison with data for anesthetized snakes in phase 3.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.144
Mean ± SD tactile pressure applied to the dorsum of 8 unanesthetized healthy mature garter snakes (Thamnophis sirtalis) that resulted in purposeful movement on each of 3 consecutive days (phase 2). During tactile stimulation testing, each snake was restrained in a customized capped PVC pipe (diameter, 1.3 cm), the length of which was 10 cm less than the length of the snake. Each snake was allowed to spontaneously enter the pipe. As the snake approached the opposite end of the pipe, a cap was placed over that end to prevent it from exiting. Then, SWMs of varying diameters (pressure range, 0.008 to 300 g [0.00008 to 2.9 N]) were used to apply tactile stimuli to the snake. For each snake, pressure was applied on the dorsal midline at 3 locations (1 cm cranial to the vent, at the level of the vent, and 1 cm caudal to the vent) with a single SWM, beginning with the filament with the smallest diameter. If the snake did not respond with gross purposeful movement when pressure was applied to at least 2 of the 3 locations, the test was considered negative. The process was repeated with the SWM that was the next size larger until gross purposeful movement was elicited when pressure was applied to at least 2 of the 3 locations or an applied pressure of 300 g resulted in a negative test result. For this phase, purposeful movement was defined as movement of the exposed portion of the body in a vigorous sideways motion away from the SWM. For each snake, the procedure was performed once daily for 3 consecutive days. The same investigator performed all tactile stimulation tests. The mean ± SD tactile pressure (16.9 ± 14.3 g [0.17 ± 0.14 N]) for the study population was calculated and used as the baseline tactile pressure for comparison with data for anesthetized snakes in phase 3.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.144
Phase 3
All snakes received 30 mg of alfaxalone/kg, ICe, during phase 3, and the mean ± SD tactile pressure required to elicit purposeful movement in unanesthetized (baseline [as calculated in phase 2]) and anesthetized snakes at LRR and every 10 minutes thereafter for 1 hour were plotted (Figure 2). Purposeful movement was not elicited by tactile stimulation testing in any of the 8 snakes at 10 and 20 minutes after LRR. Tactile stimulation did elicit a response in 4 snakes at 30 minutes after LRR (tactile pressure range in snakes that had a response, 0 to 180 g [0 to 1.77 N]), 5 snakes at 40 minutes after LRR (tactile pressure range in snakes that had a response, 0 to 26 g [0 to 0.25 N]), and all 8 snakes at 50 (tactile pressure range, 0 to 15 g [0 to 0.15 N]) and 60 (tactile pressure range, 0 to 4 g [0 to 0.04 N]) minutes after LRR. The mean tactile pressure required to elicit purposeful movement was significantly greater than that at baseline at 10, 20, and 30 minutes after LRR but did not differ significantly from baseline at LRR and at 40, 50, and 60 minutes after LRR.
Mean ± SD tactile pressure that resulted in purposeful movement for the snakes of Figure 1 before (baseline; obtained during phase 2) and at various times after they were anesthetized with alfaxalone (30 mg/kg, ICe; phase 3). Following anesthesia induction and LRR, snakes were positioned in ventral recumbency and tactile stimulation testing was performed as described in Figure 1 (except snakes were not restrained in PVC pipes) every 10 minutes for 1 hour. For anesthetized snakes, purposeful movement was defined as spontaneous forward motion. A tactile pressure of 300 g (highest pressure that could be applied with an SWM in the sensory evaluation kit used) was recorded if purposeful movement could not be elicited. *Mean differs significantly (P < 0.01) from that at baseline. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.144
Mean ± SD tactile pressure that resulted in purposeful movement for the snakes of Figure 1 before (baseline; obtained during phase 2) and at various times after they were anesthetized with alfaxalone (30 mg/kg, ICe; phase 3). Following anesthesia induction and LRR, snakes were positioned in ventral recumbency and tactile stimulation testing was performed as described in Figure 1 (except snakes were not restrained in PVC pipes) every 10 minutes for 1 hour. For anesthetized snakes, purposeful movement was defined as spontaneous forward motion. A tactile pressure of 300 g (highest pressure that could be applied with an SWM in the sensory evaluation kit used) was recorded if purposeful movement could not be elicited. *Mean differs significantly (P < 0.01) from that at baseline. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.144
Mean ± SD tactile pressure that resulted in purposeful movement for the snakes of Figure 1 before (baseline; obtained during phase 2) and at various times after they were anesthetized with alfaxalone (30 mg/kg, ICe; phase 3). Following anesthesia induction and LRR, snakes were positioned in ventral recumbency and tactile stimulation testing was performed as described in Figure 1 (except snakes were not restrained in PVC pipes) every 10 minutes for 1 hour. For anesthetized snakes, purposeful movement was defined as spontaneous forward motion. A tactile pressure of 300 g (highest pressure that could be applied with an SWM in the sensory evaluation kit used) was recorded if purposeful movement could not be elicited. *Mean differs significantly (P < 0.01) from that at baseline. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.144
Discussion
In the present study, the efficacy of ICe administration of alfaxalone to common garter snakes at doses of 10, 20, and 30 mg/kg for induction of LRR was assessed as was the tactile response to stimulation by use of SWMs in snakes that were and were not anesthetized by means of ICe administration of alfaxalone. Results indicated that only the 30-mg/kg dose of alfaxalone induced LRR in all 8 study snakes; thus, our hypothesis that ICe administration of alfaxalone at a dose of 20 mg/kg would reliably induce LRR in common garter snakes was rejected. However, we failed to reject our hypotheses that tactile stimulation with SWMs would induce repeatable results in snakes and that the response to tactile stimulation with SWMs would be less pronounced in anesthetized snakes than in unanesthetized snakes.
To our knowledge, the present study was the first to describe ICe administration of alfaxalone to snakes. Among the 8 snakes of the present study, LRR was induced in 0, 5, and 8 snakes after ICe administration of alfaxalone at doses of 10, 20, and 30 mg/kg, respectively. That finding suggested that alfaxalone has a positive dose-effect relationship following ICe in snakes. In dogs, a similar positive dose-effect relationship is observed following IV administration of alfaxalone at doses of 2, 6, and 20 mg/kg.18 In turtles, a significant dose-dependent effect on time to anesthetic recovery is observed following IM administration of alfaxalone at doses of 10 and 20 mg/kg.21
Alfaxalone has a unique steroid molecular structure that, in conjunction with solubilizing factors, allows for good tissue absorption following administration by various routes.8 That property makes alfaxalone particularly attractive for use in nontraditional veterinary species such as reptiles in which venous access can be difficult to attain. However, optimal doses and routes of administration of alfaxalone for immobilization and anesthesia have yet to be elucidated for most reptilian species and have been investigated in only a few. In native Australian lizards and snake species, alfaxalone (9 mg/kg, IV) results in rapid anesthetic induction with maintenance of spontaneous ventilation.11 In green iguanas (Iguana iguana), alfaxalone (5 mg/kg, IV) results in rapid anesthetic induction and good muscle relaxation and allows for endotracheal intubation.14 Conversely, IM administration of alfaxalone at doses of 10 and 20 mg/kg results in inconsistent sedation and anesthesia in red-eared sliders (Trachemys scripta elegans).21,22 In green iguanas, IM administration of alfaxalone at a dose of 10 mg/kg induces light sedation with continued response to noxious stimuli, whereas IM administration of doses of 20 and 30 mg/kg provides more consistent sedation and generally allows for endotracheal intubation.13 In ball pythons (Python regius), IM administration of alfaxalone at doses of 20 to 30 mg/kg (depending on injection site), but not 10 mg/kg, is sufficient to allow endotracheal intubation.24
Intracoelomic administration of alfaxalone may be beneficial for small reptiles in which venous access is difficult and the muscular mass may be inadequate for IM injection. The doses of alfaxalone (10, 20, and 30 mg/kg) chosen for ICe administration during phase 1 of the present study were similar to the alfaxalone doses (10 and 20 mg/kg) used for IM administration in reptilian species of other studies.13–15,21–24 We chose to evaluate the 30-mg/kg dose because we suspected that uptake of alfaxalone from the coelomic cavity following ICe injection may differ from the uptake of the drug from muscle following IM injection. During phase 1, LRR was achieved in all snakes after ICe administration of alfaxalone only at the 30-mg/kg dose. That finding suggested that higher doses of the drug may be required for ICe administration, compared with IM administration, to achieve therapeutic drug concentrations in target tissues, likely owing to quicker elimination of the drug by the renal-portal system or an increase in the extent of first-pass metabolism by shunting directly to the liver after ICe administration in the caudal portion of the body. This supposition is supported by results of a study24 involving ball pythons, in which a higher dose of alfaxalone was required for successful anesthetic induction when the drug was injected IM at a caudal site (epaxial muscle in the posterior third of the body) relative to that when the drug was injected IM at a cranial site (1 cm cranial to the heart). Intracoelomic injection of alfaxalone to snakes at a site more cranial than that (6 cm cranial to the vent) used in the present study may have consistently induced LRR at a lower dose (< 30 mg/kg). However, the cranial aspect of the coelomic cavity contains vital organs, and ICe injection of a drug into that part of the coelomic cavity must be done with extreme caution to avoid inadvertent puncture of one of those organs. The coelomic cavity has less vasculature than skeletal muscle. Therefore, drug uptake from the coelomic cavity may be decreased relative to that of skeletal muscle, which may contribute to the need for administration of alfaxalone at a higher dose ICe than IM to achieve the same therapeutic drug concentration at target tissues, although we could not find any published reports to support that theory.
In the present study, the heart rate of snakes that developed LRR decreased by approximately 30 bpm from onset of LRR to immediately before RRR. Hence, a decrease in heart rate may be a useful clinical indicator of decreasing anesthetic depth in snakes immobilized with alfaxalone. The heart rate of reptilian species is affected by various factors including temperature, body size, metabolic rate, presence or absence of noxious stimuli, and parasympathetic tone.34,35 Amphibians and reptiles appear to have a baroreflex similar to that of mammals such that heart rate decreases as systolic pressure increases via vagal mediation.36 For the snakes in the present study that developed LRR, the decrease in heart rate immediately prior to RRR might have reflected an increase in sympathetic activity as anesthetic recovery approached. The heart rate of ectothermic reptilian species is also affected by body temperature. For example, in black and white tegu lizards (Tupinambis merianae), heart rate decreases in a linear manner as the body temperature decreases.37 Body temperature was not assessed for the snakes of the present study owing to concerns about potentially stimulating the snakes and biasing study results; therefore, it is unknown to what extent the heart rate changes observed were caused by changes in body temperature or anesthetic depth. In the present study, all snakes were housed in and all phases of the study were performed in the same climate-controlled room, and the ambient temperature remained unchanged throughout the study. Thus, heart rate should have been unaffected by ambient temperature. Handling of snakes may have affected heart rate. All snakes were manually restrained for alfaxalone injection, and the stress associated with that handling may have accounted for the fairly high heart rate observed immediately after LRR, compared with that immediately before RRR. The snakes of this study were quite active, and a true resting (baseline) heart rate could not be obtained because to do so would require manual restraint for application of the Doppler monitor and the stress associated with the restraint would have likely caused an increase in heart rate. It is important to note that a decrease in heart rate as a clinical indicator of anesthetic depth of snakes is unlikely to be valid in circumstances that differ from those evaluated in this study because the heart rate of snakes will respond differently to other anesthetics or analgesics or surgical stimulation.
For the snakes of the present study, the respiratory rate did not change over time following ICe administration of alfaxalone, onset of LRR, and RRR. However, tidal volume and arterial blood gas tensions were not evaluated, so it was unknown whether there were alterations in minute volume. The absence of a change in respiratory rate following ICe administration of alfaxalone to the snakes of this study was consistent with findings of a study11 involving Australian lizards and snakes, in which alfaxalone administration rapidly induced anesthesia while spontaneous ventilation was maintained. In dogs, alfaxalone administration is associated with a decrease in respiratory rate, which leads to an increase in Paco2.38 Differences between mammals and reptiles regarding the effects of alfaxalone on respiratory function are likely a reflection of interspecies differences in the function and location of chemoreceptors and mechanoreceptors.35 In mammals, Paco2 is the primary driver of ventilation owing to its effect on the pH of the CNS, whereas in most aquatic reptiles, Pao2 is the primary driver of ventilation with Paco2 and environmental temperature having only minor roles in mediating ventilation.39,40 In fact, for many reptiles, the stimulus to breathe results from low blood oxygen concentration, as evidenced by the fact that supplementation of reptiles with 100% oxygen significantly decreases minute ventilation.40,41 Because the snakes of the present study were not supplemented with oxygen, it is likely that their Pao2 remained fairly constant, which allowed the respiratory rate to remain consistent throughout the observation period.
Results of the present study suggested that SWMs might be a useful noninvasive and quantitative method to assess the tactile sensation in unanesthetized and anesthetized snakes and possibly other reptiles. Baseline tactile pressure measurements were obtained in unanesthetized snakes over 3 consecutive days, and results did not differ significantly among those 3 days, which suggested that tactile pressure measurements obtained by SWMs were repeatable. The same investigator performed all tactile stimulation tests in the present study to eliminate the potential for inter-investigator technique differences. Prior to performing tactile stimulation testing on the study snakes, the investigator practiced applying pressure with the SWMs on multiple synthetic surfaces to develop familiarity and repeatability with the filaments. To use an SWM, one end of the filament is placed on the surface to be tested and pressure is applied to the opposite end of the filament until it develops a slight curve, which indicates that the maximum pressure applicable by that filament has been achieved. Continued application of force to the SWM will result in additional bending of the filament but does not change the force applied to the surface being tested,42 although overbending might add a lateral component that can alter the stimulus,27 so care was taken to ensure that the initial bend was not exceeded. During phase 3 of the present study, anesthetized snakes did not respond when the maximum pressure (300 g) was applied with the SWM at 10 and 20 minutes after LRR, which suggested that anesthetic depth was greatest between 10 and 30 minutes after LRR. Thus, for alfaxalone-anesthetized snakes, stimulating procedures should be performed between 10 and 30 minutes after LRR. It is important to note that the window for performing stimulating procedures likely differs among anesthetic protocols and stimulation with SWMs likely differs from surgical stimulation. Consequently, additional studies are necessary to assess the anesthetic efficacy of alfaxalone in combination with other anesthetics or analgesics during surgical procedures on snakes.
In the present study, ICe administration of 30 mg of alfaxalone/kg to common garter snakes induced LRR and a decrease in the response to the application of tactile pressure by SWMs in all snakes. Intracoelomic administration of alfaxalone might be an effective method for anesthetizing snakes.
Acknowledgments
The authors thank John Cwaygel for technical assistance.
ABBREVIATIONS
| bpm | Beats per minute |
| CI | Confidence interval |
| ICe | Intracoelomic |
| LRR | Loss of righting reflex |
| PVC | Polyvinyl chloride |
| RRR | Return of righting reflex |
| SWM | Semmes-Weinstein monofilament |
Footnotes
Sailfin Pet Shop, Champaign, Ill.
Alfaxan, Jurox Inc, Kansas City, Mo.
Monoject, Covidien, Mansfield, Mass.
Hefty, HMS Manufacturing Co, Troy, Mich.
Ultrasonic Doppler Flow Detector Model 811, Parks Electronics Laboratory, Beverton, Ore.
JM Eagle, Los Angeles, Calif.
Touch-Test Sensory Evaluator, North Coast Medical Inc, Gilroy, Calif.
SAS, version 9.4, SAS Institute Inc, Cary, NC.
SPSS, IBM Corp, Armonk, NY.
InStat, GraphPad Software, La Jolla, Calif.
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