Ventilation in reptiles is driven by blood O2 concentration rather than by CO2 concentration, wherein hypoxia serves as a stimulus for ventilation.1 Thus, high oxygen-tension environments may decrease spontaneous ventilation.1 Historically, it has been recommended that reptiles should recover from anesthesia while breathing room air rather than 100% O2 to encourage faster return to spontaneous breathing after inhalation anesthesia.2 However, there is limited scientific evidence to support this recommendation. In conscious reptiles, such as elephant trunk snakes (Acrochordus javanicus), savannah monitors (Varanus exanthematicus), and various chelonian species, exposure to 100% O2 results in a significantly reduced frequency of ventilation.3–7 However, more recent studies of Dumeril monitors (Varanus dumerili)8 and inland bearded dragons (Pogona vitticeps)9 anesthetized with inhalation anesthetics revealed that there were no significant or clinically relevant differences in physiologic variables, time to return of spontaneous ventilation, or recovery time when provided 21% O2 or 100% O2.
Sedation with injectable agents, rather than anesthesia with inhalation agents, may be more applicable in clinical settings for a variety of diagnostic and therapeutic procedures that do not require general anesthesia.10 A variety of protocols with injectable agents are used in reptiles, including protocols for dexmedetomidine-midazolam, dexmedetomidine-ketamine, and alfaxalone.10–12 However, the effect of Fio2 in sedated spontaneously breathing reptiles has not been clearly established. In Russian tortoises (Testudo horsfieldi) sedated with pentobarbital (50 mg/kg, SC), exposure to 100% O2 results in a transient reduction of ventilation frequency, a response that is abolished by transecting the vagal nerves.13 Therefore, the provision of supplemental O2 to sedated reptiles in a clinical setting may result in depression of respiration, which would be clinically undesirable because it could necessitate the need for endotracheal intubation and provision of intermittent positive-pressure ventilation in apneic animals.
The objective of the study reported here was to determine whether Fio2 has an effect on the quality of sedation, respiratory rate, and other cardiopulmonary variables of sedated, spontaneously breathing bearded dragons. We hypothesized that breathing a high concentration of O2 (100%) would decrease respiratory rates in sedated bearded dragons. In addition, reduced oxygenation secondary to inspiring room air (21% O2) would have an impact on recovery and postrecovery behavior, such as food intake.
Materials and Methods
Animals
Eight adult inland bearded dragons (5 males and 3 females) that ranged in body weight from 0.25 to 0.39 kg were obtained from a private breeder for use in the study. The animals were housed individually in glass tanks in a climate-controlled room maintained at 25° to 27°C. A cycle of 12 hours of light and 12 hours of darkness was used, and UVB light (290 to 320 nm) was provided to each tank. Animals were allowed to acclimatize to housing conditions for at least 6 weeks before the start of the experiments.
The bearded dragons were fed 4- to 6-week-old crickets (Acheta domestica), superworms (Zophobas morio), or mixed leafy greens 6 d/wk on a rotating schedule. Fresh water was available in a bowl for all bearded dragons at all times, and all bearded dragons were placed in a shallow warm water bath twice weekly. All bearded dragons were considered healthy on the basis of results of multiple physical examinations; monitoring of food intake, fecal output, and body weight; and results of hematologic and plasma biochemical analyses conducted before the study. The study was approved by the University of Wisconsin School of Veterinary Medicine Institutional Animal Care and Use Committee.
Study design
Bearded dragons were assigned by use of a random number generatora to a treatment (supplemental medical-grade compressed room air [21% O2] or 100% O2). In a complete crossover study design, the animals subsequently received the other treatment. There was a minimum washout period of 2 weeks between treatments.
Experimental procedures
On the day of an experiment, animals were transported to a procedure room, which was maintained at 25° to 27°C. Thirty minutes before sedatives were administered, baseline respiratory rate, heart rate, and sedation score were obtained for each animal. Respiratory rate was determined by observing body wall excursions. Animals then were minimally restrained, and heart rate was determined by use of Doppler ultrasonography.b Each bearded dragon was sedated by administration of alfaxalonec (20 mg/kg, SC) into the axillary region and placed in an induction chamber filled with 21% O2d or 100% O2.d Five minutes later, each bearded dragon was removed from the induction chamber, and a tight-fitting face mask was applied. Animals then were provided with the assigned O2 treatment (flow rate, 1 L/min). Respiratory rate and heart rate were monitored every 5 minutes for 100 minutes after alfaxalone administration. A pulse oximeter probee was secured to the ventral aspect of the tail of each animal and used to monitor Spo2.
Sedation was evaluated every 5 minutes after drug administration for 100 minutes by grading body position, palpebral reflex, jaw tone, hind limb withdrawal, response to manually being lifted from the table, and righting reflex (scale of 0 to 2, with 0 = present, 1 = diminished, and 2 = absent [unless otherwise noted]), with a maximum possible sedation score of 12 for each time point. Induction time was defined as time until loss of the righting reflex, which was assessed by placing an animal in dorsal recumbency and observing whether the animal could return to a sternal position. Withdrawal reflexes were assessed by pinching a digit of the hind limbs with a hemostat. Jaw tone was scored on a scale of 0 to 2 (0 = full jaw tone, 1 = laxity but still some resistance to opening the mouth, and 2 = mouth could be opened with no resistance). Body position was graded on a scale of 0 to 2 (0 = head and sternum lifted off the table, 1 = only the head was lifted off the table, and 2 = head and sternum were in contact with the table). Response to being manually lifted from the table was scored on a scale of 0 to 2 (0 = flailing limbs and tail movements, 1 = either tail or limb movements, and 2 = no reaction). Time to recovery was defined as the time point after induction by which the animal had regained the righting reflex.
Venous blood samples were collected 15 minutes before (baseline) and 60 minutes after administration of alfaxalone. Blood samples (0.2 mL/sample) were collected from the ventral coccygeal vein by use of a 25-gauge needle and 1-mL nonheparinized syringe,f except for collection of 3 samples when venipuncture of the coccygeal vein failed and a jugular vein was used instead. For blood gas analysis, blood samples were placed directly into a cartridgeg and analyzed with a handheld analyzer.h
Food intake was monitored and quantified to assess the effects of Fio2 on feeding behavior after recovery from sedation. Beginning 2 days before and continuing until 2 days after sedation and treatment with O2, bearded dragons were fed a diet of 5 crickets/d. The number of crickets ingested, time to ingestion of the first cricket, and time to ingestion of all 5 crickets were recorded by an observer in the room. Each bearded dragon was placed in a plastic container.i All 5 crickets were dumped into the container (in front of the bearded dragon so that they were visible to the animal), and a timer was started. Time to ingestion of the first cricket was recorded. Time required to ingest all 5 crickets was recorded, with a maximum of 5 minutes allowed for ingestion. When a bearded dragon failed to ingest all 5 crickets within the 5-minute limit, the number of crickets ingested at 5 minutes was recorded. To assess immediate postanesthetic food intake, feeding was also performed 120 minutes after each bearded dragon regained the righting reflex.
One investigator (CR) recorded all data and collected all samples. That investigator was unaware of the treatment (100% O2 or 21% O2) provided to each animal.
Statistical analysis
Data were entered into a spreadsheet for calculation of descriptive statistics. Temperature corrections with standard calibration formulas for the handheld analyzerh were performed for pH, Pco2, and Po2 to reflect the ambient temperature of 26°C as body temperature. Mean values for pulse oximetry data were calculated at 15-minute intervals for up to 60 minutes after alfaxalone injection. Commercial statistical softwarej was used for data analysis. Data were evaluated for a normal distribution by use of the Shapiro-Wilk test and for equal variance by use of the Brown-Forsythe test. A 2-way repeated-measures ANOVA was performed to evaluate respiratory rate, heart rate, sedation score, oxygen saturation, and food intake data. Data were transformed or ranked when necessary. The Holm-Sidak method was used for post hoc analysis. Blood gas data, induction time, and recovery time were compared between treatments by use of a paired t test. Values of P < 0.05 were considered significant.
Results
Respiratory rate was not affected by Fio2, and administration of 100% O2 did not result in hypoventilation. For both treatments, respiratory rate decreased over time (Figure 1). Heart rate was higher at most time points for the 21% O2 treatment, but those values did not differ significantly from the values for the 100% O2 treatment. The Spo2 was lower for the 21% O2 treatment between 15 and 60 minutes after alfaxalone injection, but those values were not significantly different from values for the 100% O2 treatment.
Box-and-whisker plots of respiratory rate (A), line graph of mean ± SEM heart rate (B), and bar graph of mean ± SEM Spo2 (C) for 8 inland bearded dragons (Pogona vitticeps) sedated with alfaxalone (20 mg/kg, SC) and provided 21% O2 (gray symbols) or 100% O2 (white symbols) in a complete crossover study (minimum washout period between treatments was 2 weeks). Baseline (BL) values were determined 30 minutes before alfaxalone injection; respiratory and heart rates were determined at 5-minute intervals after alfaxalone injection, whereas mean values for Spo2 were calculated for 15-minute intervals after alfaxalone injection. In panel A, each box represents the interquartile (25th to 75th percentile) range, the horizontal line in each box represents the median, the whiskers represent the range, and the dots represent outliers.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.129
The HCO3− concentration and tCO2 content were significantly higher for the 21% O2 treatment, compared with results for the 100% O2 treatment (Table 1). Values for pH, Pco2, Po2, Spo2, lactate concentration, and base excess were not significantly different between treatments. When comparing baseline blood gas values with the 60-minute values within a treatment, HCO3− concentration, tCO2 content, and base excess were significantly increased for the 21% O2 treatment at 60 minutes. No significant differences between baseline and 60-minute blood gas values were found for the 100% O2 treatment.
Mean ± SD venous blood gas values for 8 inland bearded dragons (Pogona vitticeps) sedated with alfaxalone (20 mg/kg, SC) and provided 21% O2 or 100% O2 in a complete crossover study, with a minimum washout period between treatments of 2 weeks.
| 100% O2 | 21% O2 (room air group) | |||||
|---|---|---|---|---|---|---|
| Variable | Baseline | 60 minutes | Difference | Baseline | 60 minutes | Difference |
| pH | 7.63 ± 0.14 | 7.62 ± 0.12 | −0.01 | 7.56 ± 0.21 | 7.62 ± 0.15 | 0.06 |
| Po2 (mm Hg) | 24.8 ± 10.4 | 32.4 ± 14.2 | 7.6 | 28.9 ± 18.2 | 33.0 ± 13.5 | 4.0 |
| Pco2 (mm Hg) | 22.4 ± 3.9 | 23.0 ± 4.5 | 0.6 | 25.7 ± 8.4 | 24.6 ± 4.3 | −1.1 |
| BE (mmol/L) | 2.6 ± 9.5 | 3.0 ± 9.8 | 0.4 | −3.3 ± 4.8 | 1.1 ± 5.6† | 4.4 |
| HCO3− (mmol/L) | 26.6 ± 7.8 | 26.9 ± 8.6 | 0.3 | 26.6 ± 12.3 | 29.5 ± 11.5*† | 2.9 |
| tCO2 (mmol/L) | 27.3 ± 8.4 | 27.7 ± 9.4 | 0.4 | 23.7 ± 2.8 | 26.7 ± 4.0*† | 3.0 |
| Spo2 (%) | 90.8 ± 6.1 | 89.8 ± 12.3 | −1.0 | 89.5 ± 10.1 | 85.7 ± 2l.9 | −3.8 |
| Lactate (mmol/L) | 2.8 ± 2.6 | 2.5 ± 2.3 | −0.3 | 4.8 ± 3.5 | 2.4 ± 2.5† | −2.4 |
Blood samples were collected 15 minutes before (baseline) and 60 minutes after injection of alfaxalone. Difference was calculated as the value at 60 minutes minus the baseline value.
Value differs significantly (P < 0.05) from the value at 60 minutes for the 100% O2 treatment.
Within a treatment, value differs significantly (P < 0.05) from the baseline value.
Cumulative sedation score at each time point was not significantly different between treatments. Similarly, mean ± SD time to maximum sedation (100% O2, 23.1 ± 10.7 minutes; 21% O2, 25 ± 10.7 minutes [P = 0.77]), loss of righting reflex (100% O2, 11.7 ± 7.5 minutes; 21% O2, 10.8 ± 5.8 minutes [P = 0.82]), and return of righting reflex (100% O2, 80.0 ± 24.5 minutes; 21% O2, 83.0 ± 36.4 minutes [P = 0.56]) were not significantly different between treatments.
Analysis of food intake data revealed no significant or clinically relevant differences between treatments for the time to ingestion of the first cricket, time to finish eating all 5 crickets, or total number of crickets eaten at any time during the study (Table 2). All bearded dragons ate 120 minutes after recovery from sedation for both treatments.
Median (range) food intake data for 8 inland bearded dragons (Pogona vitticeps) sedated with alfaxalone (20 mg/kg, SC) and provided 21% O2 or 100% O2 in a complete crossover study.
| Time to ingestion of first cricket (s) | Time to ingest all 5 crickets (s) | Total No. of crickets eaten in 5 minutes | ||||
|---|---|---|---|---|---|---|
| Day | 100% O2 | 21% O2 | 100 O2 | 21% O2 | 100% O2 | 21% O2 |
| −2 | 2.5 (1–6) | 2 (1–7) | 22 (8–85) | 29 (14–63) | 5 (3–5) | 5 (5) |
| −l | 2 (1–5) | 1 (1–3) | 13.5 (9–37) | 13 (6–77) | 5 (0–5) | 5 (5) |
| 0 | 3 (1–8) | 4 (1–10) | 48.5 (42–53) | 53.5 (28–70) | 4.5 (0–5) | 4.5 (0–5) |
| 1 | 1 (1–4) | 1 (1–4) | 27 (10–67) | 23 (11–67) | 5 (1–5) | 5 (5) |
| 2 | 1 (1) | 1.5 (1–4) | 22 (10–60) | 30 (16–47) | 5 (1–5) | 5 (5) |
Five crickets were placed in front of each bearded dragon, and a timer was started. Time to ingestion of the first cricket and time to ingest all 5 crickets were recorded; a maximum of 5 minutes was allowed for ingestion. Day 0 was the day of sedation and O2 treatment; minimum washout period between treatments was 2 weeks.
Discussion
Physiologic variables, blood gas values, sedation quality, and recovery times were assessed in the study reported here to determine clinically important differences in bearded dragons receiving room air (21% O2) or 100% O2 during sedation. Only calculated blood gas values were significantly different between treatments, whereas heart rate, respiratory rate, Spo2, and postsedation food intake did not differ significantly or clinically.
Few studies have been conducted to investigate the role of Fio2 when controlling respiration in reptiles, and most are older physiology reports. Those studies3–5,7 predominantly revealed hypoventilation of conscious reptiles in hyperoxic environments. However, similar to results of other recent studies8,9 of lizards, we did not detect a significant difference in respiratory rate regardless of Fio2 in sedated bearded dragons in the present study. Neither treatment resulted in hypoventilation or apnea; therefore, supplemental administration of O2 should be considered a safe procedure in spontaneously breathing sedated bearded dragons.
In the study reported here, there was no significant difference between treatments in Po2 measured via pulse oximetry or venous blood gas analysis. This is in contrast to results of a previous study9 in which anesthetized, intubated bearded dragons receiving 21% O2 had a significantly lower mean ± SD Spo2 of 74.7 ± 10.6%, compared with the value for bearded dragons receiving 100% O2 (90.04 ± 9.17%). The mean Spo2 for the sedated bearded dragons when spontaneously breathing 21% O2 in the present study was > 85% at all time points. The same pulse oximetry instrumentation was used in that previous study9 and in the study reported here; however, pulse oximetry has not been validated as an accurate means of measuring Spo2 in bearded dragons and may be unreliable. The animals in the present study were not intubated; instead, a face mask was placed on each bearded dragon so that they breathed spontaneously instead of being assisted with intermediate positive-pressure ventilation.
The method of oxygen administration may alter Fio2.14 The discrepancy between 21% O2 and 100% O2 administered via an endotracheal tube may be more pronounced than the discrepancy between 21% O2 and 100% O2 delivered via a face mask because there is more dead space, leakage, and mixing of O2 with surrounding air when provided via a face mask. This factor may have contributed to the differences found in other studies8,9 in which lizards inhaled 21% O2 through an endotracheal tube.
Venous blood samples can reflect Pco2 and Po2, but values for venous samples may differ significantly from values for arterial samples because of the fact that venous blood is impacted by metabolic activity and tissue perfusion and may not provide an adequate reflection of ventilation.15 In anesthetized mice that received 100% O2 versus 21% O2, ventilation-perfusion mismatching and respiratory acidosis were identified by use of blood gas analysis for the 100% O2 treatment, without significant changes to respiratory or heart rates.16 Mixing of oxygenated blood from the lungs and less-oxygenated blood returning from the systemic circulation within the reptilian heart may have accounted for the failure to detect a significant difference in Po2 and Pco2 in the present study. Bearded dragons had a higher HCO3−concentration and tCO2 content when breathing 21% O2 than when breathing 100% O2; however, the clinical importance of these results is unclear because there was no detectable difference in pH between treatments.
The only variables measured with the cartridge were pH, Pco2, Po2, and lactate concentration; the remainder of the values were calculated from results for these variables. This meant that the significant differences detected in HCO3− concentration and tCO2 content may have indicated differences in the CO2 and HCO3− equilibrium between the 2 treatments. However, pH or Pco2 was not clinically or significantly different; therefore, differences in the HCO3− concentration and tCO2 content should not be overinterpreted. In addition, use of an analyzer intended for analysis of human blood at 37°C17,18 further limited our ability to interpret results of the present study.
All blood gas values, except for lactate concentration, that were significantly different at 60 minutes, compared with the baseline values, for bearded dragons when breathing 21% O2 were calculated values and had the same aforementioned limitations. Lactate concentration was measured directly; thus, it was relatively unaffected by temperature and could be directly interpreted.18 However, baseline lactate concentration was not equivalent between treatments; therefore, the differences between the treatments should not be overinterpreted. On the basis of results of the study reported here, not providing 100% O2 to sedated bearded dragons did not result in clinically important lactatemia. Rather, it was found that the blood lactate concentration decreased over the sedation period. Because lactate is produced via anaerobic metabolism, increased lactate concentrations could be a theoretical concern for animals receiving a lower concentration of O2, but this concern would appear to be unfounded in bearded dragons on the basis of results of the present study.
It has anecdotally been advocated to allow reptiles to recover from anesthesia while breathing room air (approx 21% O2) as opposed to 100% O2 because it has been believed that 100% O2 would result in slower recovery and slower return to spontaneous breathing in apneic reptiles recovering from inhalation anesthesia.2,10,19 However, in 2 studies (one of Dumeril monitors8 and the other of bearded dragons9), animals received inhalation anesthetics at various Fio2 values, and induction and recovery times were measured. No significant differences were reported regarding return of spontaneous ventilation and recovery from anesthesia between 21% O2 and 100% O2 in those studies.8,9 In the study reported here, there was no detectable difference in recovery time after sedation; bearded dragons had a return of the righting reflex at similar times regardless of the Fio2 provided. The animals in the present study breathed spontaneously throughout sedation, as opposed to requiring intermittent positive-pressure ventilation during general anesthesia, and supplementation with 100% O2 did not result in a reduced respiratory rate at any time point. Similarly, the quality and duration of sedation were unaffected by the Fio2.
Recovery from sedation was directly assessed by return of the righting reflex; however, prolonged effects of sedation are more difficult to evaluate. Therefore, feeding behavior after sedation was monitored to determine whether provision of 21% O2 versus 100% O2 resulted in metabolic derangements affecting the bearded dragons. Although sedation caused a transient decrease in food ingestion 120 minutes after the administration of alfaxalone in all bearded dragons regardless of Fio2, no effects on feeding behavior were detected 24 to 48 hours after sedation regardless of Fio2
In the study reported here, supplementation of spontaneously breathing sedated bearded dragons with 100% O2 did not have a negative effect on respiratory rate. Therefore, supplementation with 100% O2 can be recommended without the risk of inducing hypoventilation or apnea. Supplementation with 100% O2 may have merit for apneic bearded dragons because results of the present study suggested that apnea would not be induced by exposing sedated bearded dragons to 100% O2. Inspiration of room air (21% O2) did not result in clinically important negative effects in healthy sedated bearded dragons; therefore, the lack of O2 supplementation did not appear to have clinically important consequences.
Acknowledgments
Supported by Abaxis Global Diagnostics Inc.
ABBREVIATIONS
| Fio2 | Fraction of inspired oxygen |
| Spo2 | Oxygen saturation as measured by pulse oximetry |
| tCO2 | Total carbon dioxide |
Footnotes
Research Randomizer, version 4.0, Geoffrey C. Urbaniak and Scott Plous, Middletown, Conn. Available at: www.randomizer.org. Accessed Oct 2, 2017.
digiDop, Digicare Animal Health, Boynton Beach, Fla.
Alfaxan, Jurox Pty Ltd, Rutherford, NSW, Australia.
Airgas USA LLC, Radnor, Pa.
Masimo Radical, Masimo Corp, Irvine, Calif.
Terumo Medical Corp, Elkton, Md.
CG4+ cartridge, Abaxis North America, Union City, Calif.
i-STAT, Abaxis North America, Union City, Calif.
N40 large mouse cage, Ancare Corp, Bellmore, NY.
SigmaPlot, version 13.0, Systat Software Inc, San Jose, Calif.
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