Methods

Results

All 20 turtles were deemed clinically healthy and PCV, lactate, and biochemistry results were within clinically acceptable limits (Table 1).^22,23 Overall, the median (range) body weight was 422 (321–494) g for males and 452 (367–562) g for females.

F-DKM resulted in significantly higher sedation scores than F-DKS and H-DKM at each 5-minute time point from 10 to 60 minutes (P < .05) (Figure 1). F-DKS resulted in higher median sedation scores than H-DKM at most time points, but no significant differences were identified. Across all time points, H-DKM resulted in significantly more variable sedation scores than F-DKS (P = .0073) or F-DKM (P = .0278). The sedation score interquartile range and range were wider for H-DKM compared to F-DKS and F-DKM at all time points from 25 to 60 minutes (Table 3). The number of turtles for which each of the 10 scored parameters was recorded as absent for at least 3 consecutive time points during each treatment period is presented (Table 4).

View Full Size

Median and 25–75th percentile cumulative sedation scores for 20 clinically healthy, captive adult eastern box turtles (Terrapene carolina carolina; 9 males, 11 females) administered IM dexmedetomidine-ketamine-0.9% sodium chloride (0.1, 10 mg/kg, and 0.2 mL/kg, respectively) in the forelimb (F-DKS) or IM dexmedetomidine-ketamine-midazolam (0.1, 10, and 1.0 mg/kg, respectively) in the forelimb (F-DKM) or hindlimb (H-DKM). A score of 0 represents no sedation and a score of 20 represents maximal sedation. *Significantly higher (P < .05) for F-DKM compared to F-DKS and H-DKM at all time points from 10 to 60 minutes.

Citation: American Journal of Veterinary Research 85, 2; 10.2460/ajvr.23.10.0226

• Download Figure
• Download figure as PowerPoint slide

Median (range) time to first effect (slowing or cessation of ambulation) was 2.4 (1.0–4.1), 1.9 (0.8–4.0), and 2.8 (0.8–10.0) minutes for F-DKS, F-DKM, and H-DKM, respectively. While these times were clinically similar, the time to first effect was significantly faster for F-DKM compared to H-DKM (P = .0170). Median (range) time to the cessation of purposeful, spontaneous movement was 5.0 (1.0–10.0), 4.8 (1.0–10.0), and 10.0 (2.5–25.0) minutes for F-DKS, F-DKM, and H-DKM, respectively; this was significantly faster for F-DKM compared to H-DKM (P = 0.0273). Additionally, this period was subjectively smooth for all turtles with no adverse responses observed.

A small, pale, well-demarcated, raised lesion was identified at the left lateral border of the glottis of a single turtle upon opening the mouth for intubation during the first study day. The etiology of the lesion was unknown, and intubation was not attempted for this turtle on any study day. As such, this turtle was not included in the intubation analysis. Of the remaining turtles, 13/19, 19/19, and 10/19 turtles in F-DKS, F-DKM, and H-DKM, respectively, had absent jaw tone for at least 3 consecutive time points, with a median (range) time to initial loss of jaw tone of 20 (10–30), 20 (5–35), and 28 (15–45) minutes, respectively. Overall, intubation was successfully achieved in 6/19, 17/19, and 2/19 turtles F-DKS, F-DKM, and H-DKM, respectively. This outcome was statistically significant for F-DKM compared to F-DKS (P = .0026) and H-DKM (P = .0003). The median (range) time to successful intubation was 28 (15–40), 30 (15–45), and 50 (50) minutes for F-DKS, F-DKM, and H-DKM, respectively. Intubation nonsuccess was attributable to glottal tone or the development of jaw tone with stimulation.

The median (range) recovery time was 10.1 (5.0–22.0), 16.0 (7.2–45.0), and 12.2 (3.5–28.3) minutes for F-DKS, F-DKM, and H-DKM, respectively. Though clinically similar, median recovery time was statistically longer for F-DKM compared to F-DKS (P = .0006); no significant differences in recovery time were detected between H-DKM and F-DKS, nor between H-DKM and F-DKM. The recovery period was subjectively smooth for all turtles with no adverse effects noted. On the first study day, 4 turtles (2 F-DKS, 1 F-DKM, 1 H-DKM) regurgitated 20 to 90 minutes after recovery and this prompted the lengthened fasting times on the remaining study days. No turtles regurgitated on subsequent study days. No other adverse reactions were observed during or after any treatment. All turtles resumed normal behaviors (eg, ambulation, foraging, and eating) after returning to outdoor enclosures (2–5 hours after recovery). All turtles remained clinically healthy for 1 week after the final study day.

Median (range) baseline heart rate on the second and third study days was 41 (26–50) and 34 (24–44) bpm, respectively. Heart rate decreased over time compared to baseline for turtles in all groups (Figure 2). Statistical comparisons were not performed for heart rates at the 5-minute time point, as many turtles were minimally sedate and resistant to Doppler assessment at this time point (n = 13, n = 8, and n = 16 for F-DKS, F-DKM, and H-DKM, respectively). Median heart rate was consistently lower with H-DKM compared to F-DKM over the 60-minute monitoring period; this difference was only statistically significant at the 30-minute time point (Figure 2). In both intubated and nonintubated turtles, spontaneous ventilation was rarely observed (either visually or via capnograph) after cessation of spontaneous movement. As a result, all intubated turtles were manually ventilated for the duration of intubation. Median (range) end-tidal carbon dioxide concentration during intubation was 18.7 (8.4–35.4), 18.6 (7.0–35.9), and 19.7 (12.4–22.9) mmHg for turtles in F-DKS, F-DKM, and H-DKM, respectively.

View Full Size

Median and 25–75th percentile heart rates (bpm) for 20 clinically healthy, captive adult eastern box turtles (Terrapene carolina carolina) administered IM dexmedetomidine-ketamine-0.9% sodium chloride (0.1, 10 mg/kg, and 0.2 mL/kg, respectively) in the forelimb (F-DKS) or IM dexmedetomidine-ketamine-midazolam (0.1, 10, and 1.0 mg/kg, respectively) in the forelimb (F-DKM) or hindlimb (H-DKM). ^aSignificantly different (P < .05) for F-DKS compared to H-DKM at 15 minutes. ^bSignificantly different (P < .05) for F-DKM compared to H-DKM at 30 minutes.

Citation: American Journal of Veterinary Research 85, 2; 10.2460/ajvr.23.10.0226

• Download Figure
• Download figure as PowerPoint slide

Cloacal temperatures remained within 2 °C of room temperature for turtles in all groups. A single male turtle experienced marked bradycardia at the 45-minute time point on each study day. The heart rate steadily decreased from 24–26 bpm at baseline to 8–10 bpm at 45 minutes; the concurrent sedation scores at the latter time point were 11, 17, and 12, for F-DKS, F-DKM, and H-DKM, respectively. On each day, antagonists were promptly administered, and the remainder of the trial was aborted. The turtle recovered uneventfully each day. Data for this turtle were excluded from analysis for the 50-minute time point onward.

Sedation scores were not significantly different between blinded investigators at any time point for F-DKM and H-DKM. For F-DKS, sedation scores were only significantly different at a single time point (30 minutes) (P = .0465).

Discussion

Administration of dexmedetomidine-ketamine-midazolam (0.1, 10, and 1.0 mg/kg, respectively) in the hindlimb of clinically healthy eastern box turtles resulted in reduced and significantly more variable anesthetic effects compared to administration in the forelimb. This was supported by significantly lower median cumulative sedation scores and wider interquartile and minimum-maximum ranges at the majority of time points after hindlimb compared to forelimb administration. Additionally, the median time to cessation of spontaneous movement for turtles in H-DKM was approximately twice as long as turtles receiving either forelimb treatment. Given the current knowledge of chelonian vascular anatomy¹⁷ and that all 3 drugs are primarily metabolized in the liver,²⁰ the decreased level of sedation achieved with hindlimb administration supports a hepatic first-pass effect in the current study. Alternative explanations for the decrease in efficacy include differences in drug absorption, muscle perfusion, fat stores, and/or blood flow between forelimb and hindlimb injection sites and individual differences in response to drugs unrelated to the hepatic portal system.

The effect of injection sites on the pharmacology of anesthetic drugs has been studied in other reptile species with mixed results. In leopard geckos (Eublepharis macularius), a similar effect of injection site (forelimb vs hindlimb) was documented with IM administration of dexmedetomidine-ketamine.¹² In red-eared sliders (Trachemys scripta elegans), SC hindlimb administration of another hepatically metabolized drug, buprenorphine, resulted in significantly lower plasma concentrations compared to forelimb administration.¹⁰ Finally, decreased anesthetic efficacy was reported in yellow-bellied sliders (Trachemys scripta scripta) and ball pythons (Python regius) after IM or IV administration of alfaxalone (also hepatically metabolized) in the caudal half of the body.^13–15 In contrast, a recent study detected no significant pharmacokinetic or pharmacodynamic differences between forelimb and hindlimb administration of IM alfaxalone in bearded dragons (Pogona vitticeps).²⁴ Similarly, no significant differences in anesthetic effects were noted with hindlimb rather than forelimb administration of a xylazine-ketamine combination in broad-snouted caimans (Caiman latirostris).²⁵ These results in combination with the present study highlight the potential for species-, drug-, and dose-based differences with regards to the injection site, but support a likely hepatic first-pass effect in chelonians and eastern box turtles, specifically.

Turtles amongst all test groups demonstrated inter-individual variability in anesthetic effects (Table 3) and this was likely influenced by alternative explanations above (eg, variable bioavailability, metabolic differences). That said, variability in sedation scores was significantly greatest amongst turtles in H-DKM, underscoring the potential clinical variability associated with a hepatic first-pass effect. Compromised hepatic or renal function could alternatively explain the individual variability; however, given that all turtles were clinically healthy with normal biochemistry results, this seems unlikely. Similarly, while possible, it is unlikely that inter-investigator variability contributed to these findings, as sedation scores generated by each blinded investigator were statistically and clinically similar save for a single time point in 1 treatment.

IM administration of dexmedetomidine (0.1 mg/kg) and ketamine (10 mg/kg) in the forelimb of clinically healthy eastern box turtles produced significantly greater sedation when midazolam (1.0 mg/kg) was added. This was supported by significantly higher median cumulative sedation scores and a higher intubation success rate (89% vs 32%) in F-DKM compared to F-DKS. This finding is not unexpected as both published literature and clinical anecdotes support the efficacy of midazolam as a sedative in numerous reptile species^26–29 and it is routinely used in clinical practice.^3–5 In the current study, F-DKM permitted intubation and abolished muscle tone and forelimb withdrawal response in the majority of turtles; hindlimb and tail withdrawal response was largely maintained. As such, this protocol could be considered for use in clinical situations in which heavy sedation or a light plane of anesthesia are desired. While not definitively proven in eastern box turtles, ketamine and dexmedetomidine likely also provide some degree of analgesia, an added benefit if performing a painful procedure or in a turtle with a source of pain.^{1,3–5,30,31}

The clinical effects of F-DKM administered to eastern box turtles in this study were largely similar to those reported with the same protocol in a related species, the ornate box turtle.² In both species, F-DKM resulted in observable first effects within 5 minutes, provided clinically relevant sedation lasting at least 40 minutes, and permitted smooth and clinically rapid recoveries (<30 minutes) after antagonist administration. While cumulative sedation scores were not calculated for ornate box turtles, many of the same parameters were evaluated and reported as present, reduced, or absent, permitting a qualitative comparison of anesthetic depth between species. Generally speaking, F-DKM resulted in a shorter time to peak effects and marginally deeper sedation in ornate box turtles relative to eastern box turtles in the present study. Additionally, F-DKM permitted intubation in only 89% of eastern box turtles (17/19) with a median (range) time to successful intubation of 30 (15–45) minutes, while all ornate box turtles (16/16) were intubated within 15 minutes of drug administration.² These variations may reflect true species differences; however, the possible contribution of environmental (eg, room temperature, time of year) or other population-based differences (eg, amenability to handling, baseline health status, age) cannot be ruled out. It is also important to note that, in contrast to the present study, assisted positive pressure ventilation was not performed for ornate box turtles in the aforementioned study despite observed hypoventilation and/or apnea.² Thus, consequences of hypoventilation (eg, hypoxemia, hypercapnia, acid-base imbalance, and/or tissue hypoxia) could have impacted the level of apparent anesthetic depth or response to stimulation.

All 3 protocols in the current study produced hypoventilation and in many instances apnea, as spontaneous ventilation was rarely observed. While this was subjectively assessed in nonintubated turtles and potentially an imperfect metric, if spontaneous ventilation was present in these turtles, it was likely shallow at best. Additionally, while it is possible that the capnograph utilized in the present study was not sensitive enough in the face of a shallow, spontaneous breath, end-tidal carbon dioxide concentrations were successfully measured with every assisted breath. Recall that in F-DKS and H-DKM, turtles permitted intubation in only 6 and 2 of 38 total trials, respectively; thus, many turtles in these groups were likely hypoventilated and were not able to be provided with assisted ventilation. Although chelonians are hypoxia-tolerant in comparison to mammals and no turtles in the current study demonstrated any clinically apparent side effects of hypoventilation, any acute or chronic effects are unknown. While anecdotally not always practiced, intubation and positive pressure ventilation are the arguable standard of care for sedated or anesthetized reptiles exhibiting hypoventilation, including chelonians.^32–35 As such, F-DKS or H-DKM should be used with caution, especially in turtles with compromised cardiorespiratory function or other comorbidities that would render them less tolerant to sequelae of hypoventilation.

Turtles in all groups demonstrated a decrease in heart rate over the study period. While this was statistically significant between groups at 2 time points (Figure 2), these singular inter-group differences were not clinically relevant. The observed bradycardia is likely largely a result of the cardiovascular effects of dexmedetomidine, which causes peripheral vasoconstriction and a subsequent reflex bradycardia.³⁶ While this can result in decreased cardiac output, the net cardiovascular consequences of the tested drug combinations in the current study are unknown. Comparable decreases in heart rate were observed in the aforementioned study in ornate box turtles and a study using the same protocol in a larger chelonian species, the red-footed tortoise (Chelonoidis carbonaria).^2,21

All 3 protocols permitted smooth and clinically rapid (< 30 minutes) recoveries, with 1 exception (45 minutes for a single turtle in F-DKM). No turtles experienced resedation and, aside from the isolated incidents of regurgitation on the first study day, the recovery period was uneventful, and turtles swiftly resumed normal behaviors. As prolonged recovery time continues to be a challenge of chelonian anesthesia, the consistent and clinically favorable recovery periods observed in the present study lends support to the suitability of the tested protocols in eastern box turtles.

Challenges associated with cardiopulmonary assessment in chelonians presented several limitations in the present study. Pulse oximetry, electrocardiography, and blood pressure monitoring are standard of care for anesthetized veterinary species; however, pulse oximetry has not been validated for reptiles⁴ and electrocardiography was precluded in the present study due to detection limits of available equipment. Oscillometric blood pressure monitoring has been investigated in boids and iguanas but was not an accurate substitute for direct measurements.^37,38 Direct blood pressure monitoring has not previously been reported in eastern box turtles, and it would be challenging given the small patient size. Similarly, while blood gas analysis would have provided additional, clinically useful information, the inherent challenges of repeated venipuncture in small reptiles discouraged its use in the current study. Mainstream capnography was utilized in this study; however, the accuracy in using end-tidal carbon dioxide (ETCO₂) values to estimate arterial or venous carbon dioxide concentration has not been established in reptiles, though it may be useful for monitoring trends.^4,39 Lastly, although the sedation scoring system utilized in the present study quantitatively reflected the subjective clinical differences observed between treatments and was modeled after previous chelonian sedation studies,^2,21 it has not been formally validated and, thus, may be a confounding factor. That said, to the authors’ knowledge, no validated sedation scoring systems exist for chelonians.

In conclusion, forelimb dexmedetomidine-ketamine resulted in clinically relevant anesthetic effects in eastern box turtles that were heightened with the addition of midazolam. Additionally, hindlimb rather than forelimb IM administration of dexmedetomidine-ketamine-midazolam resulted in significantly reduced and more variable anesthetic effects in eastern box turtles. This finding is suggestive of a clinically relevant hepatic first pass that should be considered when administering the above combination or any hepatically metabolized anesthetic or analgesic drugs in box turtles. For all 3 evaluated protocols, recovery times were favorable after antagonist administration.

Disclosures

The authors declare no conflicts of interest. No AI-assisted technologies were used in the generation of this manuscript.

Funding

The authors have nothing to disclose.