Open Veterinary Journal, (2024), Vol. 14(8): 1789–1793

Research Article

10.5455/OVJ.2024.v14.i8.6

Comparison of four different protocols using levobupivacaine for spinal anaesthesia in red-footed tortoises (Chelonoidis carbonarius)

Paulo Cesar Mendes Dos Santos Filho, Leticia Prata Juliano Dimatteu Telles, Elane Barboza da Silva, Dandara Franco Ferreira da Silva and Líria Queiroz Luz Hirano*

Hospital Veterinário, Universidade de Brasília (UnB), Brasília, Brazil

*Corresponding Author: Líria Queiroz Luz Hirano. Hospital Veterinário, Universidade de Brasília (UnB), Brasília, Brazil. Email: liriahirano [at] unb.br

Submitted: 14/04/2024 Accepted: 09/07/2024 Published: 31/08/2024

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ABSTRACT

Background: The popularity of tortoises kept in captivity is increasing and has caused concern regarding the necessity to establish safe and straightforward anaesthesia for those reptiles.

Aim: This study aimed to compare four protocols using levobupivacaine in spinal anaesthesia for the blockade of the caudal neuraxis of red-footed tortoises (Chelonoidis carbonarius).

Methods: Twenty-four tortoises were randomly assigned into four groups: G1, levobupivacaine 0.75% (1.15 mg kg⁻¹); G2, levobupivacaine 0.37% (1.15 mg kg⁻¹); G3, levobupivacaine 0.75% (2.3 mg kg⁻¹); and G4, levobupivacaine 0.75% (0.1 ml 5 cm⁻¹ of straight carapace length). Tortoises were evaluated for respiratory rate, muscle relaxation, response to hindlimb or tail pinch, and cloacal reflex.

Results: A 1.15 mg kg⁻¹ dose of levobupivacaine 0.37% appears adequate for shorter procedures, whereas a 1.15 mg kg⁻¹ dose of levobupivacaine 0.75% should be appropriate for longer procedures in red-footed tortoises.

Conclusion: Our results are the first to show the effects of levobupivacaine on spinal anaesthesia in reptiles. Weight-based doses presented more intense and more homogeneous effects than carapace length-based doses in red-footed tortoises. Spinal anaesthesia of red-footed tortoises was safe and effective with any of the weight-based protocols.

Keywords: Testudines, Loco-regional anaesthesia, Neuraxis blockade, Local anaesthetics, Reptiles.

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Introduction

The red-footed tortoises ( Chelonoidis carbonarius Spix 1824) are endemic to South America. In the Brazilian Amazon, this species mainly occurs in semi-deciduous forests and transitional areas between humid and semi-deciduous forests (Farias et al., 2007). They are potentially important seed dispersers and may be a viable ecological substitute for large frugivores in rewilding projects (Strong and Fragoso, 2006; Lautenschlager et al., 2022). Red-footed tortoises are commonly housed in zoological collections, kept as companion animals, and present to wildlife rehabilitation centres (Melo et al., 2020).

The increasing popularity of tortoises kept in captivity has caused concern regarding the necessity to establish safe and straightforward anaesthesia for those reptiles (Keller 2021; Scarabelli and Di Girolamo, 2022). The use of loco-regional nerve blocks in testudines has spread during the last years as it can improve the anaesthetic quality and reduce the doses of general anaesthetics (Chatigny et al., 2017; D’ovidio and Adami, 2019; Mones et al., 2023). Spinal (or intrathecal) anaesthesia produces satisfactory sensory and motor blockade for surgical procedures involving the cloaca, urinary bladder, genitalia, and hind limbs (Rivera et al., 2011; Dogu et al., 2015; Futema et al., 2020).

Levobupivacaine is a long-acting amide local anaesthetic, which exhibits a high affinity to plasma proteins (Heppolette et al., 2020). It is similar to its racemic form (bupivacaine) in efficacy. However, studies of mammals reported that levobupivacaine promoted shorter latency time (Steagall et al., 2017) and lower toxicity effects when compared to bupivacaine (Kanashiro, 2021). In general, the experience of spinal or epidural anaesthesia with levobupivacaine is not well documented in animals and no study has evaluated protocols with this drug for loco-regional anaesthesia in reptiles.

This study aimed to compare four protocols using levobupivacaine in spinal anaesthesia for the blockade of the caudal neuraxis of red-footed tortoises.

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Materials and Methods

Animals

Twenty-four adult male red-footed tortoises were included in this study. Animals had a mean weight (± standard deviation, SD) of 5.52 ± 1.81 kg (range 2.8–8.45 kg) and a mean straight carapace length of (±SD) of 43.4 ± 6.39 cm (range 36–49 cm). Before inclusion in the study, all tortoises were determined to be healthy based on history and physical examination findings. Throughout the course of the experiment, room temperature was maintained at a mean of 27.65°C ± 1.54°C (81.77 ± 34.77°F).

Loco-regional anaesthesia

The choice of local anaesthetic and its dose were based on previous reports of spinal anaesthesia with bupivacaine in red-footed tortoises (Carvalho, 2004; Santos et al., 2011). Animals were physically restrained and positioned in dorsal recumbency. Before administration of the local anaesthetic, the tail was aseptically prepared using 70% alcohol. Then, it was pulled cranio-ventrally, and the dorsal vertebral processes were palpated for identification of intervertebral spaces. The needle was inserted on the midline between two vertebral processes. If blood was sampled, the needle was retracted gently. The injection was given when no resistance was perceived. All injections of levobupivacaine (Novabupi, Cristália) were performed with 3-ml syringes and 13 × 4.5 mm (26 G) needles.

The tortoises were divided into 4 different treatments. Treatments were dosed as follows: 1.15 mg kg⁻¹ of levobupivacaine 0.75% (G1) (Santos et al., 2011); 1.15 mg kg⁻¹ of levobupivacaine 0.37% (G2, levobupivacaine 0.75% diluted with sterile water for injection, 1:1 dilution rate); 2.3 mg kg⁻¹ of levobupivacaine 0.75% (G3); and 0.1 ml 5 cm⁻¹ of straight carapace length of levobupivacaine 0.75% (G4) (Carvalho, 2004). After injection, the animals were kept in dorsal recumbency for two minutes facilitating the drug diffusion between the spinal space. Then, they were returned to standing position.

The following variables were assessed: respiratory rate (RR); muscle relaxation; response to hindlimb or tail pinch; cloacal reflex; and level of consciousness. RR was assessed by observation of skin movement in the region between the neck and shoulder or the femoral fossa. To prevent stress-related alterations, all the RR evaluations were done at a distance of at least 0.5 m between the evaluator and the tortoises. Muscle tone in the hindlimbs was evaluated by assessing resistance to manual manipulation (gentle limb retraction). The hindlimb withdrawal response was tested by pinching a digit and the skin bilaterally on each limb; the tail-pinch response was tested in the same manner. The cloacal reflex was tested by pinching the skin in the perineal area and observing cloacal contractions. A hemostat was used to apply increasing amounts of subjectively determined pressure until a positive response was obtained or for a maximum duration of 1 minute. No skin damage was noted using the pinch test. Assessment of muscle relaxation, response to hindlimb or tail pinch, and cloacal reflex were scored using a 3-point scale: score 0 (full response); score 1 (diminished response); and score 2 (no response). The animals’ response to external stimuli (manipulation and pinch) was used to evaluate consciousness.

The monitoring variables were assessed prior to (baseline parameters) levobupivacaine administration. After drug administration, the variables were repeated at 5-minutes intervals for 20 minutes, and then at 10-minutes intervals until the tortoises had recovered from loco-regional anaesthesia.

Latency time was defined as the time between the levobupivacaine injection and the onset time of motor and/or sensory blockade (score 1). The plateau phase was defined as the stable phase in which all measured variables reached score 2. Partial recovery was considered when any measured variables returned to score 1. Finally, complete recovery was considered when the tortoises achieved full motor and sensory recovery (score 0). Additionally, the times of exposure and retraction of the phallus were recorded.

Statistical analysis was performed using Bioestat 5.3 (Ayres et al., 2007). The normal distribution of data was determined using the Shapiro-Wilk test. The parametric Tukey test was used to compare RR measurements over time. It was also employed to compare time measurements (latency, plateau phase, partial, and complete recovery) between the treatments. The level of significance was set at 5% (p < 0.05). A binomial test was used to compare the proportion of animals that reached the plateau phase and the phallus exposure in each treatment.

Ethical approval

The study was approved by the ethics committee for the use of animals of Universidade de Brasília (CEUA/UnB, protocol # 56-2019) and by the Sistema de Autorização e Informação em Biodiversidade (SISBIO, protocol # 68741-1).

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Results

Table 1 compares the mean (±SD) of the final volume simulating the data of the 24 testudines, for each protocol. Tortoises were conscious at all times and exhibited no signs of local anaesthetic toxicity (i.e., neurotoxicity signs such as seizures or loss of consciousness).

Proportions of tortoises that reached the plateau phase were lower using the straight carapace length-based dose (G4) than doses of 1.15 mg kg⁻¹ (G1) and 2.3 mg kg⁻¹ (G3) ( p= 0.0209) of levobupivacaine 0.75%. Additionally, G3 had a higher number of animals with phallus exposure compared to G1 and G4 (p=0.0455 and p=0.0143, respectively).

RRs remained constant throughout the study period and did not diverge significantly (p > 0.05) from baseline values. Mean (± SD) baseline RR was 10.33 (± 4.37) breaths/min for G1; 11.57 (± 6.19) breaths/min for G2; 7.5 (± 3.15) breaths/min for G3; and 7 (± 3.61) breaths/min for G4. Additionally, mean (± SD) cross time across individuals’ RR at the other intervals was 7.31 (± 3.82) breaths/min for G1; 8.5 (± 4.01) breaths/min for G2; 6.46 (± 3.35) breaths/min for G3; and 6.87 (± 3.12) breaths/min for G4.

Table 1. Mean (± standard deviation) of final volume (ml) calculated for C. carbonarius (n=24) administered spinal levobupivacaine 0.75%, 1.15 mg kg⁻¹ (G1); 0.37%, 1.15 mg kg⁻¹ (G2); 0.75%, 2.3 mg kg⁻¹ (G3); and 0.75%, 0.1 ml 5 cm⁻¹ of straight carapace length (G4).

Table 2. Mean (± standard deviation) times (minutes) to the start of latency, plateau phase, partial and total recovery, exposure and retraction of the phallus in C. carbonarius administered spinal levobupivacaine 0.75%, 1.15 mg kg⁻¹ (G1); 0.37%, 1.15 mg kg⁻¹ (G2); 0.75%, 2.3 mg kg⁻¹ (G3); and 0.75%, 0.1 ml 5 cm⁻¹ of straight carapace length (G4).

We considered the time of latency as 5 minutes, because it was the first evaluation, and all the tortoises showed a reduction in motor and/or sensory response at this moment (Table 2). Using the dose of 1.15 mg kg⁻¹ of levobupivacaine, the mean time of partial recovery was significantly longer with a concentration of 0.75% (G1), compared with 0.37% (G2) ( p= 0.0317).

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Discussion

Results of the present study indicated that the use of levobupivacaine for spinal anaesthesia in red-footed tortoises promoted sensory and motor blockade, without RR reduction. Additionally, the RR (approximately 7 breaths/minutes) registered after levobupivacaine injection was similar to the mean values of 10 breaths/ minutes reported for adult red-footed tortoises at 25ºC (Oliveira et al., 2023).

Most studies report the use of spinal anaesthesia alone for surgical procedures in testudines (Rivera et al., 2011; Dogu et al., 2015; Futema et al., 2020); however, it is important to assess animal welfare and to evaluate the need of associating regional anaesthesia with sedative or general anaesthetics drugs, like alfaxalone (Zec et al., 2024).

This study provides an unprecedented comparison of the two methods used to determine the dose of spinal anaesthetics in testudines (weight-based vs. straight carapace length-based) (Hirano et al., 2012; Futema et al., 2020). Here, red-footed tortoises of weight-based treatments (G1, G2, and G3) presented more intense and more homogeneous effects than tortoises of carapace length-based treatment (G4). The more domed carapace shape, and consequent variation in vertebral column length, may result in lower doses of levobupivacaine for red-footed tortoises. In contrast, a study with dogs reported that the vertebral column length was directly related to the bupivacaine volume (Freitag et al., 2023). This direct relationship between dose and carapace shape was also observed in other species of testudines, like the green turtle (Futema et al. 2020), and highlights the need for more controlled studies addressing the appropriate method of dose determination in various species of testudines.

Previous studies of humans have suggested that it is possible to obtain the same sensory blockade with a low concentration of local anaesthetics while keeping the same volume (Lyons et al., 2007; Suzuki et al., 2022). Despite the absence of statistical differences, the use of levobupivacaine 0.37% (G2) in red-footed tortoises reduced the recovery time of spinal anaesthesia when compared with the same volume of levobupivacaine 0.75% (G3). These findings are in agreement with results reporting decreased time to recovery and efficient analgesia by use of lower concentration of local anaesthetics in dogs and humans (Kim et al., 2009; Nejamkin et al., 2020; Suzuki et al., 2022).

In G3, there was used the double volume but with the same concentration of G1. Previous studies with horses and goats showed that the volume of injectate has a significant role in drug distribution, directly influencing the cranial spread of a solution injected spinally (Johnson et al., 1996; Floriano et al., 2023). Although 2.3 mg kg⁻¹ levobupivacaine 0.75% (G3) promoted earlier plateau phase time than 1.15 mg kg⁻¹ levobupivacaine (G1), as was observed in G2, the amount of the anaesthetic was not enough to extend the effects to the forelimb or prolongate the recovery time, and no benefits were seen with the higher dose.

Despite, the loss of muscle tone in the hindlimbs and the tail were noted immediately after injection, the first evaluation of the tortoises was carried out at 5 minutes after levobupivacaine administration. Because of this, any differences between the latency were obscured by the measurement interval. In Geoffroy’s side-necked turtle ( Phrynops geoffroanus Schweigger 1812) and Amazonian freshwater turtle ( Podocnemis expansa Schweigger 1812), the use of lidocaine 2% and bupivacaine 0.5% for spinal anaesthesia also promoted a short latency, with mean time between 0.29 and 0.9 minutes (Carvalho, 2004; Nascimento, 2013).

Regarding the complete recovery, similar results were observed among the four protocols, with a mean time interval between 180 (G2) and 275 (G3) minutes. The complete recovery time of the present study was longer than the results obtained in P. geoffroanus (61.5 minutes) and D’Orbigny’s slider turtles (Trachemys dorbigni) (68 minutes) with 1.15 mg kg⁻¹ of bupivacaine (Andrade, 2010; Ribeiro, 2011).

This is the first study to evaluate the use of spinal levobupivacaine in reptiles, but similar to the present study, the levorotatory enantiomer promoted longer sensory blockade when compared with bupivacaine for epidural anaesthesia in humans (Kopacz et al., 2000).

Our results are the first to show the effects of levobupivacaine on spinal anaesthesia in reptiles. Weight-based doses presented more extensive and more homogeneous effects than carapace length-based doses in red-footed tortoises. Spinal anaesthesia of red-footed tortoises was safe and effective with any of the weight-based protocols. A 1.15 mg kg⁻¹ dose of levobupivacaine 0.37% appears adequate for shorter procedures, whereas a 1.15 mg kg⁻¹ dose of levobupivacaine 0.75% should be appropriate for longer procedures in red-footed tortoises.

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Acknowledgment

Not applicable.

Conflict of interest

The authors declare that there is no conflict of interest.

Authors’ contributions

LQLH set the conception as well as the design of the study. PCMSF, LPJDT, and EBS collected the data. All the authors analyzed the data and wrote the original manuscript. DFFS did the statistical analyses and wrote the final version of the manuscript. All authors have approved the final manuscript.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Data availability

All data are provided in the manuscript.

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