Ball pythons (Python regius) are kept as pets and are often maintained in research laboratories and zoological institutions, making them one of the most common reptile species evaluated by veterinarians. Sedation or a light plane of anesthesia (light anesthesia) is occasionally necessary to enhance the quality and safety of physical examination and facilitate routine diagnostic and therapeutic procedures of snakes and other reptiles.1 Responses to commonly used sedation protocols vary across reptile taxa and can be affected by environmental conditions.2 Currently, the veterinary literature lacks systemically derived anesthetic and sedation data for snakes including ball pythons,3,4 and clinicians must extrapolate information regarding drug doses and combinations from unrelated species. This approach often results in a lack of desired clinical efficacy, prolonged induction of anesthesia or sedation, poor recovery quality, and potentially harmful adverse effects including death.2,5
Benzodiazepines, α2-adrenoceptor agonists, and more recently, neuroactive steroids such as alfaxalone are being increasingly used to induce sedation and light anesthesia in reptiles.1,2,6–8 Incorporation of those drugs into combination protocols generally reduces the dose of each drug required to achieve clinical efficacy, which aids in the prevention of deleterious dose-dependent effects.2 Effective sedation protocols involving combinations of benzodiazepines and α2-adrenoceptor agonists have been described for reptiles.5,8 Combinations of a benzodiazepine and α2-adrenoceptor agonist, such as midazolam or dexmedetomidine, are advantageous because they represent completely reversible protocols, and administration of the antagonists (flumazenil and atipamezole for benzodiazepines and α2-adrenoceptor agonists, respectively) rapidly induce reversal of sedation.8 Alfaxalone is a neuroactive synthetic steroid that enhances neuronal cell membrane chlorine ion transport through its interaction with cell surface γ-aminobutyric acid A receptors to induce sedation and anesthesia.9 Alfaxalone is advantageous for use in reptiles because it can be administered by the IM, SC, or IV route.5,8,10,11 Although administration of alfaxalone alone at high doses to reptiles has been reported to cause rapid smooth anesthesia induction, it has also been associated with apnea and prolonged recovery times, and reptiles administered lower doses of the drug appear to be reactive to stimulation.1,4,8
The goal of the study reported here was to evaluate the effectiveness of SC administration of 2 combination protocols, alfaxalone-midazolam and dexmedetomidine-midazolam, for induction of moderate sedation suitable for facilitation of various clinical procedures in ball pythons. We hypothesized that both drug combinations would result in rapid sedation and that recovery following administration of antagonist drugs would be more rapid for the dexmedetomidine-midazolam protocol than for the alfaxalone-midazolam protocol owing to the fact that the effects of both dexmedetomidine and midazolam can be reversed, whereas currently, the effects of alfaxalone cannot be reversed.
Materials and Methods
Animals
All study procedures were reviewed and approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. Twelve university-owned juvenile ball pythons (6 male and 6 female) with a mean ± SD body weight of 408 ± 148 g were used for the study. The snakes were individually housed in commercial rodent enclosures and were provided a hide box and water ad libitum. Ambient room temperature was maintained between 25.0°C and 28.0°C (77.0°F and 82.4°F). The snakes were fed frozen-thawed mice once weekly. Prior to study initiation, each snake was determined to be healthy on the basis of results of a complete physical examination, and no health abnormalities were observed in any of the snakes during the experimental trials.
Experimental design
In a randomized blinded crossover study, each snake was subcutaneously administered a combination of alfaxalonea (5 mg/kg [2.3 mg/lb]) and midazolamb (0.5 mg/kg [0.23 mg/lb]; AM protocol) and a combination of dexmedetomidinec (0.05 mg/kg [0.023 mg/lb]) and midazolam (0.5 mg/kg; DM protocol), with a washout period of at least 7 days between protocols. The 2 protocols were selected on the basis of results of unpublished pilot studies performed by our laboratory. The order in which the 2 protocols were administered to each snake was randomized by means of a random number generator. For each protocol, the 2 drugs were combined in the same syringe and injected SC over the epaxial muscles at an anatomic location associated with the observable maximum intensity of the heart. Both experimental trials were performed in a controlled environment with the ambient temperature maintained between 25.7°C and 25.8°C (78.3°F and 78.4°F).
For each snake, RR, HR, and sedation variables were assessed immediately before and at 5-minute intervals after drug administration by 1 investigator (TJY). Respiratory rate was evaluated first at each assessment time because manipulation of the snake for determination of HR and level of sedation would stimulate respiration. The RR was determined by counting visible body wall excursions over 1 minute. Heart rate was measured by placement of a Doppler ultrasonographic probe on the ventral body wall against the point of visual maximum intensity of the heart. Sedation was subjectively assessed by evaluation of the righting reflex, jaw tone, superficial pain response, spontaneous body movement, tongue flicking, and head shyness. Righting reflex was determined by elongating the snake and positioning it in dorsal recumbency. Jaw tone was assessed by pulling down on the skin at the ventral aspect of the mandible in an attempt to open the snake's mouth. Superficial pain response was determined by use of a hemostat to gently pinch the skin along the dorsal midline at the level of the heart and distal tip of the tail; the tips of the hemostat were individually wrapped with white surgical tape to prevent skin trauma. Spontaneous body movement and tongue flicking were visually observed. Head shyness was defined as retraction of the head away from a stimulus and was determined by light digital palpation of the rostral aspect of the maxilla between the eyes. Tongue flicking and head shyness were scored as present or absent; all other sedation variables were scored on a 3-point scale (Appendix).
Thirty minutes after administration of the assigned protocol, each snake was evaluated to determine whether it was sufficiently sedate to allow tracheal intubation and venipuncture. The snake was gently restrained in ventral recumbency and the mouth was opened with a spatula to avoid trauma to the teeth. Following visual observation of the glottis, orotracheal intubation was attempted with an 18-gauge, 44-mm-long IV catheter with the stylet removed. Successful intubation or failure of intubation because of subject resistance was recorded, and the catheter was immediately removed. The snake was then positioned in dorsal recumbency, and a 27-gauge needle attached to a 1-mL insulin syringe was used to collect blood from the ventral tail vein. Venipuncture was considered successful if ≥ 0.1 mL of blood was obtained.
Sedation was reversed 45 minutes after administration of the assigned protocol. The AM protocol was reversed by administration of flumazenild (0.05 mg/kg, SC). The DM protocol was reversed by SC administration of a combination of flumazenil (0.05 mg/kg) and atipamezolee (0.5 mg/kg); the 2 drugs were mixed together in 1 syringe for administration. All antagonists (reversal agents) were administered over the epaxial muscles at an anatomic location associated with the observable maximum intensity of the heart.
All injections, manipulations, and assessments were performed by the same investigator (TJY) who was unaware of (blinded to) the protocols and reversal agents administered. All drugs were loaded into syringes by another person, and the syringes were partially wrapped with tape, so that the investigator performing the injections was blinded to differences in injection volume.
Respiratory plethysmography
Because of difficulties associated with visual assessment of respiratory effort after sedative administration, the experiment was repeated with some modifications for a subset of 3 ball pythons, with the most important modification being the use of plethysmography to determine RRs and respiratory characteristics for each protocol. Briefly, to detect breathing movements, each snake was placed unsedated in an airtight chamberf (inner dimensions, 20.8 × 10.9 × 5.8 cm [8.2 × 4.3 × 2.3 in]) with the inflow and outflow ports closed and a pressure transducerg attached to a separate port, which converted pressure changes to voltage signals. Airflow was initiated, and the snake was allowed 90 minutes to acclimate to the chamber. Following acclimation, breathing frequency and characterization data were collected for 30 minutes before and 90 minutes after administration of the assigned sedation protocol. The chamber was opened briefly for administration of the assigned sedation protocol and reversal agents as previously described; sedation was reversed 45 minutes after administration of the assigned sedation protocol.
Plethysmography signals were digitalized with a data acquisition system and analyzed offline with computer software.h Upward deflections indicated an increase in pressure within the chamber (ie, expiration), whereas downward deflections indicated a decrease in pressure (ie, inspiration). Respiratory data were recorded continuously during sequential 20-minute closed-chamber sessions that were separated by 2-minute open-chamber periods, during which time oxygen (concentration, 100%) airflow was used to flush the chamber. In a previous study12 conducted by our laboratory group, the respiratory frequency of ball pythons remained unchanged when the chamber was closed for 20 minutes; therefore, keeping a snake in a closed chamber for 20 minutes was unlikely to result in marked carbon dioxide accumulation or hypoxia. Breathing frequency was determined by counting the number of upward (expiratory) deflections during the 20-minute closed-chamber sessions. Obvious, irregular movement artifacts were not included in that count. The mean RR was determined over a 15-minute period. Changes in tidal volume were estimated by calculation of the mean amplitude (ie, distance between the highest and lowest point of a breathing trace) for 10 respiratory peaks during the 30 minutes before administration of the assigned sedation protocol (presedation period) and comparing that value with up to 10 respiratory peaks (when available) within 15-minute intervals during the 90 minutes after administration of the assigned sedation protocol (postsedation period). Snakes were monitored for adverse effects throughout and after the study.
Statistical analysis
Time to first sedative effect was defined as the duration between administration of the assigned protocol and the first assessment time when no spontaneous movement was observed (spontaneous movement score, 2). The time to loss of jaw tone was defined as the duration between administration of the assigned protocol and the first assessment time when the investigator was able to open the snake's mouth (jaw tone score, 1). Time to recovery was defined as the duration between administration of the reversal agent or agents and the first assessment time when all sedation variables had returned to a score of 0.
Data were analyzed with commercial statistical analysis software.i Data distributions for continuous variables were assessed for normality by use of the Shapiro-Wilk test. The Wilcoxon signed rank test was used to compare times to loss and recovery of selected sedation variables between protocols. The RR and HR were recorded every 5 minutes, and the data were consolidated into 15-minute intervals. For both variables, the mean for the 3 recorded data points within each 15-minute interval was calculated and used for analysis purposes. The Brown-Forsythe test was used to evaluate those data for equal variance. Data that did not meet the assumptions for ANOVA were transformed or ranked prior to further analysis. The effects of sedation protocol and time on RR and HR were assessed with a 2-way ANOVA for repeated measures. The Holm-Sidak method was used for post hoc pairwise comparisons when necessary. A Fisher exact test was used to compare the superficial pain response between sedation protocols. Results were reported as the median and interquartile (25th to 75th percentile) range for non-normally distributed variables and the mean ± SEM for normally distributed variables unless otherwise noted. Values of P < 0.05 were considered significant.
Results
Snakes
All snakes maintained adequate body condition and appetite throughout the study, and no adverse effects were observed following administration of either sedation protocol or the reversal agents. No tissue damage was observed at any of the injection sites.
Sedative effects
Both protocols resulted in a similar depth of sedation, which was characterized by loss of righting reflex, jaw tone, tongue flicking, spontaneous movement, and head shyness. For the AM protocol, the times to first sedative effect and loss of jaw tone were significantly shorter and the duration of righting reflex absence and time to recovery were significantly longer, compared with the corresponding values for the DM protocol (Table 1). The proportion of snakes that remained reactive to superficial pain stimulation following administration of the AM protocol (8/12) was significantly (P = 0.001) greater than that following administration of the DM protocol (0/12). For both protocols, tracheal intubation and blood collection were successful 30 minutes after protocol administration in all snakes. Following administration of the AM protocol, the majority (9/12) of snakes exhibited spontaneous open-mouth behavior and subsequent chewing that was not associated with respiratory effort or any observed external stimulation.
Descriptive statistics for sedation variables assessed for 12 healthy juvenile ball pythons (Python regius) that were subcutaneously administered a combination of alfaxalone (5 mg/kg [2.3 mg/lb]) and midazolam (0.5 mg/kg [0.23 mg/lb]; AM protocol) and a combination of dexmedetomidine (0.05 mg/kg [0.023 mg/lb]) and midazolam (0.5 mg/kg; DM protocol) in a randomized crossover design with a washout period of at least 7 days between protocols.
| AM protocol | DM protocol | ||||
|---|---|---|---|---|---|
| Variable | Median (IQR) | Range | Median (IQR) | Range | P value |
| Time to first sedative effect (min) | 5.0 (5.0-5.0) | 5.0-5.0 | 7.5 (5.0-10.0) | 5.0-10.0 | 0.031 |
| Time to loss of jaw tone (min) | 5.0 (5.0-10.0) | 5.0-10.0 | 15.0 (11.3-20.0) | 10.0-25.0 | < 0.001 |
| Time to loss of righting reflex (min) | 5.0 (5.0-10.0) | 5.0-10.0 | 10.0 (5.0-13.8) | 5.0-20.0 | 0.164 |
| Duration of absent righting reflex (min) | 80.0 (56.3-85.0) | 50.0-90.0 | 50.0 (42.5-55.0) | 30.0-55.0 | 0.007 |
| Time to recovery (min) | 42.5 (23.8-50.0) | 15.0-50.0 | 15.0 (11.3-15.0) | 5.0-15.0 | < 0.001 |
Forty-five minutes after protocol administration, sedation was reversed by SC administration of flumazenil (0.05 mg/kg) alone or in combination with atipamezole (0.5 mg/kg; DM protocol only). Time to first sedative effect was defined as the duration between administration of the assigned protocol and the first assessment time when no spontaneous movement was observed. Time to loss of jaw tone was defined as the duration between administration of the assigned protocol and the first assessment time when the investigator was able to open the snake's mouth. Time to recovery was defined as the duration between administration of the reversal agent or agents and the first assessment time when all sedation variables had returned to presedation levels.
Values of P < 0.05 were considered significant.
IQR = Interquartile (25th to 75th percentile) range.
Heart rate decreased significantly following administration of both sedation protocols (Figure 1). However, the decrease in HR was significantly greater following administration of the DM protocol, and the mean HR following administration of the DM protocol was significantly less than that following administration of the AM protocol at all postadministration assessment times. The RR also decreased significantly following administration of both sedation protocols, with periods of apnea observed at 30 and 45 minutes after protocol administration.
Mean ± SEM HR (A) and box-and-whisker plots of RR (B) for 12 healthy juvenile ball pythons (Python regius) before (−5 minutes) and after SC administration of a combination of alfaxalone (5 mg/kg [2.3 mg/lb]) and midazolam (0.5 mg/kg [0.23 mg/lb]; AM protocol; white bars) and a combination of dexmedetomidine (0.05 mg/kg [0.023 mg/lb]) and midazolam (0.5 mg/kg; DM protocol; gray bars) in a randomized crossover design with a washout period of at least 7 days between protocols. Protocol administration was designated as time 0. Forty-five minutes after protocol administration, sedation was reversed by SC administration of flumazenil (0.05 mg/kg) alone or in combination with atipamezole (0.5 mg/kg; DM protocol only). For each box-and-whisker plot, the solid line within each box represents the median, the lower and upper limits of the box represent the interquartile range (25th and 75th percentiles, respectively), the whiskers delimit the range, and circles represent outliers.
Citation: Journal of the American Veterinary Medical Association 256, 5; 10.2460/javma.256.5.573
*Within an assessment time, mean or median value differs significantly (P < 0.05) between protocols.Plethysmographic findings
Representative plethysmographic tracings for snakes before and after administration of the AM and DM protocols are provided (Figure 2). The mean plethysmographically determined RR following administration of the AM and DM protocols to the 3 evaluated snakes was plotted. The RR tended to decrease after protocol administration. However, the decrease in RR was more profound following administration of the DM protocol, and all 3 snakes underwent periods of apnea beginning as soon as 15 minutes after administration of that protocol. The mean change in estimated tidal volume amplitude from that during the presedation period was significantly greater for the DM protocol than for the AM protocol between 15 and 45 minutes after drug administration, with the greatest increase in tidal volume amplitude observed for the DM protocol 45 minutes after administration.
Representative whole-body closed-chamber Plethysmographie tracings for a healthy juvenile ball python before (baseline) and after administration of the AM and DM protocols described in Figure 1 (A), and column scatterplots of the RR (B) and fold increase in estimated tidal volume amplitude (C) for a subset of 3 of the 12 snakes of Figure 1 after repeated administration of the AM (white circles) and DM (black circles) protocols. A—Downward deflections represent inspiration and upward deflections represent expiration.
Citation: Journal of the American Veterinary Medical Association 256, 5; 10.2460/javma.256.5.573
See Figure 1 for remainder of key.Discussion
Few prospective studies3,4,13 of injectable sedation protocols for snakes have been published. In the present study, SC administration of a combination of alfaxalone (5 mg/kg) and midazolam (0.5 mg/kg; AM protocol) or a combination of dexmedetomidine (0.05 mg/kg) and midazolam (0.5 mg/kg; DM protocol) resulted in sedation sufficient for tracheal intubation and blood sample collection in ball pythons.
Both the AM and DM protocols caused rapid immobilization, although the median time to the first sedative effect (duration between protocol administration and the first assessment time when no spontaneous movement was observed) for the AM protocol (5 minutes) was slightly shorter than that for the DM protocol (7.5 minutes), most likely owing to the more rapid onset of action for alfaxalone relative to dexmedetomidine. For the ball pythons of the present study, the sedation induction times for the AM and DM protocols were slightly shorter than those for leopard geckos (Eublepharis macularius) of another study8 that were administered higher doses of each drug in the respective combinations. The study8 involving leopard geckos was conducted in our laboratory under conditions comparable to those used for the present study; thus, the observed differences in sedation induction between the 2 studies emphasizes the presence of dose-dependent or species-specific differences in drug efficacy.
The median duration of righting reflex absence for ball pythons following administration of the AM protocol (80 minutes) was significantly longer than that following administration of the DM protocol (50 minutes). That finding was similar to results for leopard geckos8 and was expected because there is currently no antagonist available to reverse the effects of alfaxalone, whereas antagonists are available for both dexmedetomidine and midazolam and were administered to the snakes of the present study. The fact that both dexmedetomidine and midazolam can be reversed may make the DM protocol more advantageous than the AM protocol in clinical situations that require patient immobilization for only a short period of time. However, for the snakes of the present study, cardiovascular changes induced by the DM protocol were more evident than those induced by the AM protocol, and the potential adverse effects associated with those changes should be considered when choosing between the 2 protocols.
Nine of the 12 snakes of the present study developed spontaneous mouth opening and chewing behavior after administration of the AM protocol. Excitement, disorientation, violent movements, paddling, and muscle twitching have been described in mammalian species following administration of alfaxalone alone or in combination with a benzodiazepine.9 The mechanism of action leading to those types of movements has yet to be elucidated, but coadministration of an α2-adrenoceptor agonist or opioid with alfaxalone appears to decrease the incidence of spontaneous abnormal movements.9
Both sedation protocols assessed in the present study were administered by the SC route. Historically, in reptiles, SC administration of sedatives and anesthetics has been associated with prolonged and unreliable induction times relative to IV or IM administration.14 In the present study, SC administration of both protocols resulted in rapid induction of sedation that was comparable to induction following IM or IV administration of sedation protocols to reptiles.1,2 Compared with intracardiac or IM injections in reptiles, SC injections are safer and cause less discomfort. Another advantage associated with SC drug administration observed in the present study was the ability to deliver larger volumes more quickly, compared with IM administration. Further research to compare the pharmacokinetics and pharmacodynamics of sedatives and anesthetics following SC, IV, and IM administration to reptiles is necessary.
In the present study, the HR of snakes decreased after administration of both the AM and DM protocols, but the decrease in HR was more profound for the DM protocol. It was likely that the magnitude of the decrease in HR observed for the snakes of this study was exaggerated because of a restraint-induced increase in HR immediately prior to injection, but that effect should have been similar for both protocols. Decreases in HR have been associated with administration of α2-adrenoceptor agonists and alfaxalone in other reptile species.10,13,15 In the previously described study,8 leopard geckos also had a more profound decrease in HR after administration of dexmedetomidine-midazolam than after administration of alfaxalone-midazolam.
Dose-dependent respiratory depression following administration of alfaxalone or α2-adrenoceptor agonists has been reported in dogs,16,17 cats,18 green iguanas (Iguana iguana),6 Horsfield tortoises (Testudo horsfieldii),7 and desert tortoises (Gopherus agassizii).15 Moreover, Horsfield tortoises administered alfaxalone alone and in combination with the α2-adrenoceptor agonist medetomidine, developed apnea that lasted from 10 to 220 minutes.7
For the snakes of the present study, the RR decreased significantly following administration of both the AM and DM protocols, and visual assessment of the RR became difficult as the level of sedation increased. As a result, the experimental procedure was repeated for 3 of the 12 snakes, with plethysmography used to assess the RR and respiratory characteristics. Plethysmographic results indicated that the mean RR for the 3 evaluated snakes was significantly lower following administration of the DM protocol than following administration of the AM protocol. All 3 snakes also developed transient apnea that lasted for 5 to 10 minutes after administration of the DM protocol but not after administration of the AM protocol. Interestingly, the estimated tidal volume amplitude increased markedly after administration of both the AM and DM protocols, which suggested that the snakes compensated for a decrease in RR by increasing tidal volume while sedated regardless of the protocol administered. Further studies in which arterial blood gases are measured are necessary to assess ventilation and oxygenation of ball pythons following sedation with the AM and DM protocols evaluated in the present study.
The present study had some limitations. Although no adverse reactions were observed in any of the snakes aside from transient apnea, the study population was small. Also, all snakes of the present study were healthy, and we caution use of either of the evaluated sedation protocols in debilitated ball pythons.
Results of the present study indicated that SC administration of a combination of alfaxalone (5 mg/kg) and midazolam (0.5 mg/kg; AM protocol) or a combination of dexmedetomidine (0.05 mg/kg) and midazolam (0.5 mg/kg; DM protocol) to healthy juvenile ball pythons resulted in sufficient sedation for tracheal intubation and blood sample collection and should provide adequate sedation and immobilization for performance of similarly or less invasive procedures. No adverse effects aside from transient self-limiting apnea were observed in association with sedation protocol administration. Both protocols induced significant decreases in RR and HR, which were more profound after administration of the DM protocol. Therefore, these protocols should be used with caution, especially in snakes with compromised cardiopulmonary function. An advantage of the DM protocol relative to the AM protocol is that antagonists are available for both dexmedetomidine and midazolam; an antagonist is not currently available for alfaxalone. Thus, the sedative effects for the DM protocol are completely reversible, whereas those for the AM protocol are only partially reversible.
ABBREVIATIONS
| HR | Heart rate |
| R | Respiratory rate |
Footnotes
Alfaxan, Jurox Inc, Kansas City, Mo.
Midazolam, West-Ward Pharmaceuticals Corp, Eatontown, NJ.
Dexdomitor, Pfizer Animal Health, New York, NY.
Flumazenil, West-Ward Pharmaceuticals Corp, Eatontown, NJ.
Atipamezole, Zoetis, Kalamazoo, Mich.
Pelican 1060 case, Pelican Products Inc, Torrance, Calif.
DigiData 1200, Axon Instruments, Sunnyvale, Calif.
pClamp software, Axon Instruments, Sunnyvale, Calif.
SigmaPlot, version 13, Access Softek, Berkeley, Calif.
References
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Appendix
Description of the subjective scoring system used to assess level of sedation in 12 healthy juvenile ball pythons.
| Score | |||
|---|---|---|---|
| Variable | 0 | 1 | 2 |
| Righting reflex | Snake rights itself immediately and effortlessly | Snake rights itself with effort | Snake is unable to right itself |
| Jaw tone | Unable to open mouth | Able to open mouth but jaw tone still present | Readily able to open mouth owing to absence of jaw tone |
| Superficial pain response | Rapid avoidance of stimulus | Reduced response to stimulus | No response to stimulus |
| Spontaneous body movement | Actively moving | Reduced and small uncoordinated movements | No spontaneous movements |
| Tongue flicking | Present | Absent | — |
| Head shyness | Present | Absent | — |
— = Not applicable.
