Anaesthesia with sevoflurane in pigeons: minimal anaesthetic concentration determination and investigation of cardiorespiratory variables at 1 MAC Julie Botman1 (BSc), Fabien Gabriel1 (DVM), Alexandra Dugdale2 (DVM, PhD, Dip.ECVAA), Jean-Michel Vandeweerd1 (DVM, PhD, DECVS) 1Department of Veterinary Medicine, University of Namur, Namur Research Institute for Life Sciences, rue de Bruxelles, 61, 5000, Namur, Belgium. 2Faculty of Health and Life Sciences, University of Liverpool, School of Veterinary Science, Chester High Road, Neston CH64 7TE, United Kingdom. The authors have no conflicts of interest. Corresponding author: Dr. Alex Dugdale Email: [email protected] Abstract Objective. To determine the minimal anaesthetic concentration (MAC) of sevoflurane in pigeons, and to investigate the effects of 1 MAC sevoflurane anaesthesia on cardiovascular and respiratory variables compared with the awake state. Design. Prospective, experimental study. Animals. Seven healthy adult pigeons. Procedure. After acclimatisation to handling, heart rate (HR), heart rhythm, respiratory rate (fR), end-expired carbon dioxide tension (PE’CO2), inspired CO2 tension (iCO2), indirect systolic arterial blood pressure (SAP) and cloacal temperature (T) were measured to determine baseline, ‘awake’ values. Pigeons were then anaesthetised with sevoflurane and MAC was determined by the “bracketing” method. The same variables were monitored during a 40 minute period at 1.0 MAC sevoflurane for each bird. Results. Mean MAC was 3.0 ± 0.6% for SEVO. During maintenance of anaesthesia at 1.0 MAC, SAP decreased significantly (P < 0.001) without any significant change in HR. Although PE’CO2 increased significantly (P = 0.001) despite an increase in fR, awake PE’CO2 values were unexpectedly low. Sinus arrhythmias were detected in 2 birds under sevoflurane anaesthesia. The times to tracheal intubation and to recovery were 2.5  0.7 min and 6.4  1.7 min, respectively. Recovery was rapid and uneventful in all birds. Conclusions and clinical relevance. Sevoflurane is suitable for anaesthesia in pigeons. List of abbreviations fR= respiratory rate HR= heart rate iCO2= inspired CO2 tension ISO = isoflurane PE’CO2 = end-expired carbon dioxide tension SAP= indirect systolic arterial blood pressure SEVO = sevoflurane T= clocal temperature Introduction Although avian anaesthesia can be performed with either injectable or inhalation agents, inhalation anaesthesia is often preferred because of rapid anaesthetic induction and recovery, rapid adjustments in anaesthetic depth, minimal biotransformation and minimal myocardial depression (Naganobu and Hagio, 2000; Rival, 2002; Escobar and others, 2009). Sevoflurane (SEVO) has been used for avian anaesthesia, where it is characterized by less airway irritation (Granone and others, 2012) and a lower blood solubility (Edling, 2006) than isoflurane (ISO), and is associated with faster anaesthetic induction and recovery times that could justify its wider use (Korbel, 1998; Joyner and others, 2008; Granone and others, 2012; Phair and others, 2012). In the pigeon, anaesthetic protocols with SEVO have been described where the delivered concentrations (according to vaporiser dial setting) were 8% for induction and 4% for maintenance of anaesthesia (Korbel, 1998). As bird lungs contain air capillaries instead of alveoli, the term minimal alveolar concentration, as reported in mammals, is inappropriate. Instead, the term minimal anaesthetic concentration (MAC) is used. The MAC of SEVO is unknown in pigeons, as well as cardiorespiratory parameters under SEVO. In contrast, there is more information about the effects of ISO (Fitzgerald and Blais, 1991; Korbel, 1998; Touzot-Jourde and others, 2005; Botman and others, 2016). Protocols administering 3.0– 5.0% ISO for induction and 1.5–3.0% for maintenance of anaesthesia have been described (Korbel, 1998; Touzot-Jourde and others, 2005; Botman and others, 2016), and two MAC values of ISO have been reported: 1.51 ± 0.15% (Fitzgerald and Blais, 1991) and 1.8 ± 0.4% (Botman and others, 2016). In addition, ISO anaesthesia in pigeons can result in hypercapnia, hypotension, mild hypothermia and second- and third-degree atrioventricular blocks (Botman and others, 2016). The objectives of the current study were to determine the MAC of sevoflurane and the effects of sevoflurane anaesthesia on the cardiorespiratory variables at 1 MAC sevoflurane. Materials and methods Study design This was a prospective, experimental study; the experimental protocol (Figure 1; 14/209/VA) was approved by the ethical committee for animal welfare of the University of Namur. Birds and baseline monitoring parameters Seven adult racing pigeons (Columbia livia) were used. They had a mean ± SD (standard deviation) weight of 443 ± 60g (1.0 ± 0.1 lb). They were housed in an aviary (3 x 2 x 2 m) between interventions. Food and water were available ad libitum. All pigeons were acclimatized to handling during a 1 month period before this study. They were selected for good health based on a physical examination and for absence of cardiac arrhythmias in the awake state. One week prior to the study, baseline heart rate and rhythm were assessed with an electrocardiograph (ECG) (Mindray; PM-9000vet, China) , and systolic blood pressure was determined by Doppler plethysmography (Doppler Vet BP; Mano Medical, France) (Figure 2). Furthermore, capnography (Scio Four Oxi Plus; Dräger, Germany) was used to measure respiratory rate, inspiratory carbon dioxide tension and end-expired carbon dioxide tension (PE’CO2), and cloacal temperature was measured (Mindray; PM-9000vet, China). These measurements were performed in a quiet environment without tranquilization. Pigeons were covered with a light towel. Flat clip electrodes were directly connected to the skin at the patagium of each wing and at the skin fold proximal to the left stifle joint. Pigeons were gently restrained for 15 minutes before recording measurements. To reduce stress, they were kept in an upright position (Lopez Murcia and others, 2005). For capnography, pigeons breathed from a sectioned blind end of a latex glove finger fixed around the beak proximal to the nares (Botman and others, 2016) (Figure 3). The other end of the glove was used to administer a fresh gas flow of 100% oxygen delivered at 500mL/min via a non-rebreathing Bain system. Inspired and expired gases (for measurement of inspired CO2 and PE’CO2 tension) were sampled at a sidestream sampling rate of 200 mL/min via a 20-gauge non-blunted needle, inserted into the glove close to the beak and connected, via the sampling tubing, to the capnograph (measuring to 1 decimal place). Measurements were recorded separately over a 10 minute period. Induction and maintenance of anaesthesia Before general anaesthesia, birds were fasted for 6 hours. After 5 minutes of preoxygenation with a facemask (100% oxygen, 1L/min), anaesthesia was induced with SEVO (Sevorane; Abbott, North Chicago, Ill) at an initial vaporiser setting of 7 %, delivered in 100% oxygen (1L/min) via a Bain non-rebreathing system. This SEVO percentage was chosen on the basis of the work of Joyner and others (2008), who compared 4% ISO and 7% SEVO in bald eagles; and we have previously reported the use of 4% ISO in pigeons (Botman and others, 2016). The vaporiser (Tec 7 Vaporizer, Datex-Ohmeda, Finland) was serviced regularly and its calibration was checked before and after the study. Once the bird was sufficiently relaxed, the facemask was removed, the bird’s trachea was intubated with a 3 mm noncuffed endotracheal tube and it was positioned in dorsal recumbency (Figure 4). Sevoflurane was then delivered at 4% for a 15 min stabilization period before MAC determination began. Oxygen delivery was adjusted to a flow rate of 500mL/min for the remainder of the procedure. Birds were allowed to breathe spontaneously. An insulated mattress and an infrared light were used to maintain body temperature between 40.6 and 41.0°C (105.1- 105.8°F). Monitoring The ECG was performed as in awake animals, except that birds were in dorsal recumbency. Conductive gel was applied sparingly, to increase electrode conductivity, but to minimize any cooling effect. To collect end-expired gas samples, the tip of a 20-gauge non-blunted needle was inserted into the lumen of the endotracheal tube near its connection with the breathing system, and its hub was connected to the gas sampling line of a gas analyzer (Scio Four Oxi Plus; Dräger, Germany) for continuous monitoring of the SEVO concentration, inspired CO2, PE’CO2 and fR. The fresh gas flow of oxygen was set at 4 x minute ventilation (minute ventilation was estimated as 200mL/kg/min, such that 100 % oxygen was delivered at 500mL/min via a calibrated oxygen flowmeter on the anaesthetic machine and then through a non-rebreathing Bain system). Gas samples were withdrawn at a sidestream sampling rate of 200 mL/min; withdrawn samples were not returned to the breathing system but were ducted to the waste anaesthetic agent scavenger system. Before the study began, the calibration of the gas analyzer was checked against a standard gas mixture (Quick Cal calibration gas: GE Healthcare) by the service engineers. In addition, before and after each anaesthetic, the analyzer’s calibration was checked against room air (zero CO2). A Doppler blood flow probe and occlusive cuff with sphygmomanometer (Doppler Vet BP; Mano Medical, France), were used to monitor indirect SAP. A blood pressure cuff (size 2.5cm) (Pedisphyg, CAS Medical Systems Inc., USA) was positioned between the tibiotarsal- tarsometatarsal joint and the stifle joint. The width of the blood pressure cuff was approximately equivalent to 40-50 % of the limb circumference. The Doppler probe was placed, using ultrasound coupling gel to ensure adequate contact, distal to the blood pressure cuff, over the metatarsal artery. To measure the body temperature an electronic temperature probe (Mindray; PM-9000vet, China) was inserted into the cloaca. Minimal Anaesthetic Concentration (MAC) determination After 15 minutes of stabilization and placement of the monitoring instruments, physiological variables were recorded at baseline (start of the period for MAC determination) and observed every five minutes, during MAC determination (Figure 1). The MAC was determined by using the “bracketing” method described by Ludders and others (1989). Briefly, after stabilization at a predetermined end-expired anaesthetic concentration, the jaws of a Rochester-Carmalt forceps were clamped to the first ratchet lock on a digit until either a gross purposeful movement occurred (kicking of the limbs or moving of the wings), or for up to 60 seconds. The end-expired SEVO concentration was decreased by 10% if no motor response occured. After 15 minutes of equilibration at each new constant end-expired SEVO concentration, this procedure was repeated until the bird reacted to the stimulus. The end-expired SEVO concentration was then increased by 10%, followed by 15 minutes of equilibration, until the reaction disappeared. End- expired SEVO concentration was determined as the average of four samples. The time between tracheal intubation and the first stimulation for MAC determination (stabilization period) was 30 minutes. The MAC was defined and calculated as “the median value between the maximal end-expired concentration that allowed movement and the minimal end-expired concentration that prevented movement” (Naganobu and Hagio, 2000). Temperature and inspired CO2 were observed during the whole procedure to ensure that no hypothermia and CO2 inspiration occurred during MAC determination. Recording of cardiorespiratory and other variables Immediately after the MAC was determined, the end- expired SEVO concentration was maintained at 1.0 MAC for a period of 15 minutes for stabilization, followed by a period of 40 minutes during which monitored variables were recorded at 1.0 MAC (Figure 1). HR (beats/min), fR (breaths/min), iCO2 (mmHg), PE’CO2 (mmHg), SAP (mmHg) and T (°C) were recorded 3 times over each 5 minute epoch and a mean value was determined. ECG was recorded for the entire anaesthetic period, including the earlier periods of stabilization and MAC determination. Several intervals of interest were measured. Time to induction of anaesthesia was defined as the time from initial delivery of volatile anaesthetic agent to successful tracheal intubation (Mercado and others, 2008). The time between tracheal intubation and the first stimulation for MAC determination (stabilization period) was recorded, as was the time from the start of inhalant anaesthetic administration to successful MAC determination. After the last monitored variable was measured, the vaporiser was turned off and 100% oxygen was administrated until the endotracheal tube was removed. Time to tracheal extubation was defined as the time from cessation of volatile agent administration (vaporiser turned off) to the time of coughing, swallowing or shaking of the head, resulting in removal the endotracheal tube (Granone and others, 2012). Recovery time was defined as the time from cessation of volatile anaesthetic administration (vaporiser turned off) until the bird was able to stand and walk unassisted (Botman and others, 2016). The time from the start of anaesthetic administration to the time of cessation of anaesthetic administration was defined as the total anaesthetic time (Phair and others, 2012). Data analysis Data were collected in Microsoft Excel and were analyzed using IBM® SPSS® Statistics, version 21.0. Shapiro-Wilk tests were used to examine the normality of data. Parametric data were analysed using the paired Student t-test. The paired Wilcoxon test was used for non