Evaluation of intravenous T-61 as a euthanasia method for birds

Abstract

The use of T-61 as a sole euthanasia agent for birds was investigated. Nine broiler chickens (Gallus gallus domesticus) were euthanized by intravenous T-61 and assessed for insensibility [brainstem reflexes: nictitating membrane reflex (NIC), palpebral blink reflex (PAL)], brain death [isoelectric electroencephalogram activity (EEG)], cessation of audible heartbeat, and abnormal electrocardiogram. Birds were considered dead when the heart rate was less than 180 beats/minute with an isoelectric EEG. No vocalization or wing flapping occurred. Both NIC and PAL were lost 10.5 s from start of injection and audible heartbeat ceased at 24.5 s. Latency to isoelectric activity was 16.6 s. All but 1 bird died within 60 s. Rapid induction of insensibility meant birds did not experience pain and distress within 10.5 s from start of injection and birds were not conscious during cardiac and circulatory arrest. Intravenous injection of T-61 is an effective and efficient euthanasia method for birds.

Introduction

The word euthanasia is a Greek derivative of the terms good (eu) and death (Thanatos). In animals, it is implied that death occurs with minimal fear or pain (1). The American Veterinary Medical Association’s (AVMA) Guidelines for Euthanasia of Animals, Canadian Council on Animal Care (CCAC), Euthanasia of Animals used in Science, the European Commission Euthanasia of Experimental Animals, and the Minimum Standards for Wildlife Rehabilitation describe similar criteria for euthanasia: the euthanasia method should achieve rapid loss of consciousness and death, minimize stress and pain before unconsciousness, be reliable, irreversible, safe, and esthetically acceptable for the operator. These criteria apply to all species, including avian species. Published reports evaluating euthanasia methods in avian species are most often restricted to slaughter methods for commercially raised poultry; few exist on euthanasia of individual birds (1–3). Most accounts of euthanasia in birds, other than poultry, are in non-peer-reviewed literature including book chapters, editorials, roundtable discussions and anecdotal reports (1,2,4–6). Given the diversity of avian species and the varied circumstances of use (e.g., domesticated or owned animals, wildlife rehabilitation, field studies, and basic or biomedical research), research in appropriate and effective euthanasia methods is greatly needed.

Intravenous injection of a euthanasia solution (e.g., barbiturates and barbituric acid derivatives), as stated in the AVMA guidelines, is the only acceptable method that can be used without conditions in birds (1). There are other injectable solutions for euthanasia mentioned in the AVMA guidelines including potassium chloride, tributame, ultra-potent opioids, and T-61. T-61 is a nonbarbiturate, non-narcotic combination consisting of 3 compounds: embutramide (anesthetic), mebenzonium iodide (paralytic), and tetracaine hydrochloride (potent local anesthetic) (1,7). The anesthetic depresses cerebral activity, inhibiting consciousness and the respiratory control centers in the brain, whereas the curariform (paralytic) action of the drug induces circulatory and respiratory collapse (7,8), leading to death through hypoxia. Concerns with T-61 use are whether the paralytic actions occur before unconsciousness (1). Studies in dogs and rabbits have shown that loss of consciousness and motor activity occur at the same time, but that this can be influenced by rate of injection, producing situations in which dysphoria occurs (1,7,9,10). In addition, there are reports of muscular activity and vocalization during euthanasia with T-61 that have caused distress in personnel witnessing euthanasia, withdrawal of T-61 from the market in the United States (1,9), although T-61 is currently available in Canada and other countries (1). The CCAC guidelines (11) state that T-61 is not a recommended method of euthanasia in any species, although an animal care committee can approve its use. The CCAC guidelines emphasize that it should only be used intravenously, as there are concerns about the differential rates of absorption and onset of action of the active ingredients when administered by other routes (11). However, there is no cited research to support these claims. The AVMA lists T-61 as an approved euthanasia method for companion animals and wildlife; however, sedation before use is recommended (1).

Although the AVMA and CCAC guidelines promote the use of barbiturates and barbituric acid derivatives in birds, there are no published studies demonstrating that these drugs meet the criteria of a good death. There are anecdotal reports of vocalizing and wing and leg muscle activity after administering barbiturate euthanasia solution to a conscious bird (6), resulting in many practitioners using sedation or general anesthesia before euthanasia with pentobarbital (5,6). Alternatives to controlled barbiturates could be extremely beneficial for humane killing of poultry, wild birds, and within animal welfare facilities. T-61 offers these advantages, as it is a schedule III drug and therefore can be administered by non-veterinarians, veterinary technicians, and others, if they are appropriately trained (1). This drug has the potential to be used as a sole agent, whereas other injectable euthanasia solutions require prior use of a sedative or general anesthesia. These factors allow for immediate euthanasia in the field and wildlife rehabilitation situations, rather than prolonged suffering during transport to a veterinarian, making T-61 ideal for use in birds. In addition, T-61 is also more esthetic than many of the physical methods (7–9).

It is vital that euthanasia methods rapidly induce a loss of consciousness (insensibility). A conscious bird is aware and able to perceive and experience stimuli from within itself and its surroundings; these include negative states such as pain and distress (12,13). When rendered insensible, due to damage or disruption of the consciousness holding regions of the brain, consciousness is abolished, inhibiting the bird’s ability to experience negative effective states that may arise with euthanasia (14,15). To minimize potential suffering prior to unconsciousness, a short interval to insensibility is necessary. Brainstem reflexes, also called cranial reflexes, are tools used in research to evaluate insensibility (16–21). These reflexes, which include the palpebral blink and nictitating membrane reflex, evaluate the functional link between the cranial nerves and the brainstem (the consciousness holding region of the brain). If the brain stem functioning is impaired or disrupted, then the brainstem reflexes will be absent, indicating that the bird has been rendered insensible (13,22,23). A short interval to insensibility and brain death, before occurrence of respiratory and cardiac arrest, is required for death by intravenous T-61 injection to be a humane death.

This study was conducted to address the lack of scientific knowledge specific to the use of T-61 as a euthanasia agent in avian species. The hope was to dispel misinformation surrounding the use of T-61 in birds, as there is no published research with evidence supporting claims that T-61 is not an appropriate euthanasia agent for birds. The objective was to demonstrate that T-61 can be used to effectively euthanize an avian species, the broiler chicken, and with sufficient efficacy and efficiency to meet the criteria for a sole injectable agent for euthanasia.

Materials and methods

Animals

The University of Saskatchewan Animal Research Ethics Board approved the research (Protocol 19940248) and all birds were cared for as specified in the Canadian Council of Animal Care’s Guide to the Care and Use of Experimental Animals. Nine healthy 5-week-old mixed-sex broiler chickens (Gallus gallus domesticus) (mean weight: 1734 g) were used because research is available for comparative purposes. Birds were housed under commercial conditions recommended by the Aviagen management guide and fed a commercial broiler diet. All birds were tested on the same day.

Experimental procedure

Each bird was weighed individually and a 0.03 mg/kg body weight (BW) dose of T-61 (labeled dose for dogs) was calculated for each animal. The birds were placed on a towel and the electrocardiogram (ECG) and electroencephalogram (EEG) recording devices were placed as described subsequently. Birds were restrained manually in lateral recumbency and T-61 was injected into the right brachial vein. Injections were given slowly (6.1 ± 1.34 s, range: 4.5 to 8.4 s) with constant observation of the vein to ensure that the T-61 was not administered subcutaneously. Baseline ECG and EEG values were collected for a minimum of 2 min before injection of T-61 and monitoring was continued after the end of injection until total cessation of cardiac activity on the ECG.

Data collection

Reflexes

Latency to insensibility was visually assessed using brainstem reflexes measured from start and end of T-61 injections to the time of their absence. The 2 brainstem reflexes assessed were the palpebral blink reflex and nictitating membrane reflex; these were assessed every 5 s. Palpebral blink reflex tested for a blink (closing of the eyelids) in response to the approach or touching of the cornea (22). The nictitating membrane reflex tested the closing of the nictitating membrane (3rd eyelid) across the eye in response to the approach or touching of the cornea and the medial canthus (22). Brainstem reflexes were reassessed every 30 s to confirm absence of reflexes and to ensure reflexes did not return. When all brainstem indicators were absent, the brainstem was rendered non-functional and thus unable to maintain consciousness. The duration and latency to cessation of heartbeat was assessed by auscultation with a stethoscope (Littmann Classic, 3M; London, Ontario), with the lack of audible heartbeat recorded as the time of cessation of heartbeat. The birds were monitored for any activity such as vocalization, wing flapping, and muscle tremors or contractions. An ethogram with definitions of the reflexes is available in Table 1.

Table 1.

Ethogram of reflexes and clinical indicators measured antemortem after T-61 injection.

Measure	Type of indicator	Meaning of indicator	Description
Palpebral blink	Cranial nerve V/VII	Brainstem dysfunction	Closing of eyelids (blinking) in response to approach or touching of cornea
Nictitating membrane	Cranial nerve V/IV	Brainstem dysfunction	Closure of the nictitating membrane in response to approach or touching of cornea and medial canthus
Cessation of heartbeat	Clinical indicator	Cardiac arrest	No audible heartbeat with auscultation

Adapted from references 13,21–23.

Electroencephalogram

The EEG recording electrodes, 27-gauge subcutaneous electrodes, were positioned immediately lateral to the comb and the reference electrodes were placed caudal to the external auditory meatus on the same side, in accordance with published techniques (24). The ground electrode was placed along the lateral aspect of the pelvis. Topical lidocaine (20 mg/mL; Zoetis Canada, Kirkland, Quebec), 1 or 2 drops, were applied to skin 10 min before electrode placement, to control pain.

Electroencephalograms were recorded using a portable neurodiagnostic system (Sierra Sumitt, Cadwell, Washington, USA). The high frequency filter was set to 100 Hz and the low frequency filter was set to 1 Hz. Baseline EEGs were recorded in all birds before the T-61 injection. Time to continuous isoelectric activity relative to the start and end of the injection was recorded in all birds. The EEG recordings were continued for a minimum of 10 s after isoelectric activity occurred, to ensure this was continuous and thus representative of brain death, and there was no sudden burst of high frequency activity after a transient period of isoelectric activity (24–26). Isoelectric activity was defined as the absence of observable positive or negative deflections from baseline, as determined by visual, qualitative assessment of recorded traces (24–27).

Electrocardiogram

Heart rate and electrical activity of the heart were monitored and recorded using Lead II with reuseable ECG electrodes (MLA700) on a Powerlab system and analyzed using LabChart 7.0 (AD Instruments, Colorado Springs, Colorado, USA). The ECG leads were attached to the patagium bilaterally and to the skin of the stifle joint on both legs, in accordance with published techniques (10). The skin and clips were covered with alcohol to enhance electrical conduction. Continuous ECG monitoring began before the injection of T-61, with the start and end times of injection recorded.

The ECG waveform data were analyzed using LabChart 7.0 (AD Instruments). Visual inspection of the ECG traces was used to determine normal and abnormal ECG, and heart rate in each bird. Heart rate was determined for each bird at 20, 40, 60, and 80 s. The ECG of 1 bird was removed from analysis of heart rate, as the trace was unreadable after 40 s. To determine heart rate, the waveforms were counted 5 s before the time stamp and multiplied by 12. Similar to the results of Coenen et al (28), the bird was considered to be dead when a heart rate less than 180 beats/min (bpm) measured by ECG was reached in conjunction with isoelectric EEG.

Statistical analysis

All reflex and clinical indictor data, and EEG data were checked for normality using the Shapiro-Wilk Normality test (PROC Univariate, SAS 9.4; SAS, Cary, North Carolina, USA) before analyses; none of the data were abnormal. Statistical comparison for differences between time to isoelectric activity, and to cessation of brainstem reflexes and audible heartbeat were performed using paired Student’s t-test via PROC t-test. Differences were considered significant when P ≤ 0.05 and trends were noted when P ≤ 0.10. Means and standard deviation were reported.

Results

Reflexes

No vocalization, wing flapping, muscle tremors, or contractions occurred during the experiment. The mean duration (as measured from start of injection) until occurrence of variables was nictitating membrane reflex ceased at 10.5 s (SD = 1.9, χ̄ = 10, range: 8 to 13 s) and palpebral blink reflex at 10.5 s (SD = 1.9, χ̄ = 10, range: 7 to 13 s), followed by the occurrence of continuous isoelectric activity at 16.5 s (SD = 1.9, χ̄ = 17, range: 14 to 20 s), and the loss of audible heartbeat occurred at 24.5 s (SD = 13.2, χ̄ = 25, range: 10 to 50 s).

Latency to the absence of nictitating membrane reflex was shorter than latency to isoelectric activity; t_o = −6.00 s, P < 0.01 (Table 2). Similarly, the latency to absence of palpebral blink reflex was shorter than the latency to isoelectric activity; t_o = −6.00 s, P < 0.01. The latencies of both nictitating membrane reflex and palpebral blink reflex were also shorter than the time to cessation of audible heartbeat; t_o = −13.94 s, P < 0.01 and t_o = −13.94 s, P < 0.01, respectively. A trend for a difference was seen for time from end of injection to isoelectric activity being shorter than the time from end of injection to cessation of audible heartbeat (n = 8); t_o = 8.70 s, P = 0.08.

Table 2.

Difference in latency to cessation of brainstem reflexes, audible heart beat and time to isoelectric activity (s) for 5-week-old broiler chickens (Gallus gallus domesticus) (N = 9) euthanized by intravenous T-61 injection.

Variables	Mean of difference	Standard deviation of difference	Standard error of difference	t-test	P-value
From start of injection
Nictitating membrane
Isoelectric activity	−6.00	2.96	0.986	−6.09	< 0.01
Heartbeat	−13.94	12.34	4.115	−3.39	< 0.01
Palpebral blink
Isoelectric activity	−6.00	2.87	0.957	−6.27	< 0.01
Heartbeat	−13.94	12.05	4.016	−3.47	< 0.01
Heartbeat
Isoelectric activity	7.94	13.17	4.389	1.81	0.11
From end of injection
Heartbeat
Isoelectric activity	8.70	13.05	4.350	2.00	0.08

Electroencephalogram

The mean duration of the baseline recording was 56 s (SD = 29.3, χ̄ = 45, range: 29 to 126 s). The mean injection time was 6.9 s (SD = 1.8, χ̄ = 7, range: 4 to 9 s). The mean time to continuous isoelectric activity from the start of the injection was 16.6 s (SD = 1.9, χ̄ = 17, range: 14 to 20 s) and from the end of the injection was 9.7 s (SD = 2.1, χ̄ = 10, range: 6 to 13 s). In 1 bird, the needle dislodged from the vein during the injection and the time to continuous isoelectric activity from the end of the injection could not be determined. No burst of high frequency activity occurred after isoelectric activity was first recorded. A representative EEG recording is depicted in Figure 1.

Figure 1.

Electroencephalogram recording of a 5-week-old broiler chicken during euthanasia with T-61. The recording from the left side of the skull (L) is above the recording from the right (R). The gray arrow indicates when the injection was started and the black arrow indicates when the injection was completed.

Electrocardiogram

All birds were observed to have an abnormal ECG (including but not limited to widening of QRS interval, ST segment elevation, T-wave inversion) within 3 s after the end of T-61 injection. No recovery of normal ECG complexes occurred after the start of ECG abnormalities. Within 20 s after the end of injection, 2 out of 8 birds had a heart rate of ≤ 180 bpm. At 40 s, 6 out of 8 birds had a heart rate of 180 bpm or less. By 60 s, all but 1 bird had a heart rate of ≤ 180 bpm, and the remaining bird had a heart rate < 180 bpm at 80 s post-injection.

Discussion

This study showed that intravenous injection of T-61 as a sole agent is an effective, fast, and humane euthanasia agent for an avian species. The brainstem was rendered non-functional and the birds, therefore, were insensible around 10 s after the start of injection and 4 s after the end of injection, as measured by palpebral blink and nictitating membrane reflex. This indicates that birds were no longer conscious and thus insensible to distress and the possible paralytic effects (1) of T-61 by 10 s after the start of injection. Permanent absence of cerebral activity (brain death) was detected by continuous isoelectric activity on the EEG recording and occurred 16.6 s after beginning the injection and within 10 s after completing the injection. This is similar to times reported in horses euthanized with pentobarbital sodium (25). Loss of nictitating and palpebral blink reflex occurred earlier than isoelectric activity, indicating that birds were insensible for a period before brain death. Audible heartbeat ceased by 25 s, after both insensibility and brain death had occurred, indicating that the birds were not conscious of the occurrence of cardiac arrest and circulatory collapse. All but 1 bird were considered dead within 60 s, with the final bird dead within 80 s (as measured by the most conservative measure of a heartbeat < 180 bpm). The depression action of T-61 initially renders the brainstem incapable of maintaining consciousness and then causes complete, irreversible cessation of cerebral activity, rendering vital centers non-functional. These actions occurred before the combined effect of depression and paralytic action causing the heart to stop. Furthermore, when assessing brain electrophysiologic activity during euthanasia with pentobarbital sodium in horses, Aleman et al (25) hypothesized that the continuous isoelectric pattern of the brain occurred before the absence of electrocardiogram activity, therefore suggesting that the euthanasia method would be an effective and humane procedure. Our results showed that the sustained isoelectric EEG of brain death occurred before the absence of ECG activity, implying that T-61 as a euthanasia method in birds is an effective and humane procedure. Some concern has been shown about the occurrence of paralysis with T-61. Muscle activity was not measured in our experiment; however, using EEG and electromyography (EMG), Hellebrekers et al (9) reported that loss of muscle activity and brain death occurred at the same time in dogs and rabbits that were euthanized by T-61. The authors concluded that the presence of the muscle relaxant in this drug was not an ethical problem (9).

When evaluating euthanasia methods, the AVMA Panel on Euthanasia takes many factors into consideration, including the emotional effects on observers or operators. Use of T-61 has been reported to cause distress to operators euthanizing dogs, and because of these concerns, the drug was withdrawn from the market in the United States (1). T-61 has also been reported to induce convulsions in larger birds or poultry following intravenous, intracardiac, intrapulmonary, or intramuscular administration (7,29). However, during this study, each bird was observed for activity that may cause undue distress to observers and operators such as vocalizations, wing flapping, convulsions, or tremors, but no bird showed any of these clinical signs during or after the administration of T-61. An overdose of T-61 has been suggested to lead to overexcitement and/or convulsions, in mammals (8). In the authors’ clinical experience, convulsions have not been observed in any species of bird euthanized by intravenous T-61 injection and better clinical efficacy has been noted with higher doses (closer to 0.4 to 0.5 mg/kg BW). It should also be mentioned that safe and appropriate disposal of carcasses previously euthanized with T-61 is necessary, as secondary toxicity can occur in animals that consume the remains of animals euthanized with T-61 (1). Accidental intoxication with T-61 can occur in humans, with oral ingestion having the potential to cause digestive disorders and coma associated with cardiorespiratory failure (9).

Previous studies have recorded brainstem auditory evoked responses (BAERs) during euthanasia rather than just relying on the EEG to indicate brain death (25). This is because many agents used in euthanasia, such as barbituates and propofol, can cause transient periods of isoelectric activity, followed by periods of high frequency activity (burst suppression) (30), which could be mistaken for brain death. In our study, it was not possible to record concurrent BAERs due to the physical limitations of evaluating chickens. The recordings were continued for a minimum of 10 s after the isoelectric activity was first recorded; however, no subsequent bursts of activity were noted. Furthermore, T61 contains embutramide, a potent opioid agonist, rather than a gamma-aminobutyric acid (GABA) agonist such as propofol and barbiturates (9). It is thus reasonable to conclude that an absence of cerebral activity and thus brain death was truly achieved.

Electrocardiographic activity continued in all birds after conversion to isoelectric EEG and past the detection of audible heartbeat, although always with an abnormal rate, rhythm, or waveform. Paul-Murphy et al (3) reported similar findings when sparrows were euthanized by intraosseous pentobarbital and intrathoracic compression. Aleman et al (25) suggested that ECG activity occurring after brain death and undetectable heart sounds represent ineffective contraction with no cardiac output. No birds were observed to have recovery of normal ECG complexes after the onset of ECG abnormalities. Laying hens euthanized by pentobarbital showed brain death, defined by continuous isoelectric activity, within 20 s of euthanasia attempt (31), whereas in our study T-61 isoelectric activity occurred within 16.5 s. When comparing time to brain death in poultry species, time to brain death was shorter when euthanized by T-61 than with pentobarbital (31). This has also been reported in other species, with the time to brain death, as measured by isoelectric activity, being shorter in dogs euthanized by T-61 than by pentobarbital (9). When compared within the same species, T-61 results in brain death earlier than pentobarbital. Notably, neither the terminal/agonal gasp nor the vocalizations or wing and leg muscle activity reported to occur with pentobarbital euthanasia were observed for birds euthanized with T-61 (6,7,32).

Thoracic compression, also called rapid cardiac compression, a field technique used to euthanize small birds, was recently recommended as an effective euthanasia method for small birds (33). However, brain death with thoracic compression takes 19.0 to 88.5 s depending on the species (3), whereas with T-61 brain death occurred 16.5 s from the start of injection. Therefore, T-61 results in brain death earlier than thoracic compression, indicating T-61 is a more efficacious euthanasia method for birds.

Compared to commonly used methods for euthanizing chickens on-farm, T-61 results in brain death faster than gaseous euthanasia or manual cervical dislocation (MCD). Gaseous euthanasia with nitrogen or a combination of nitrogen and carbon dioxide resulted in isoelectric activity after 48.9 s and 34.2 s (32), whereas MCD took 171 s to isoelectric activity (31). These are both longer than the 16.5 s to isoelectric activity noted with T-61 in our study. The times to absence of the nictitating membrane and palpebral blink reflex, indictors of insensibility, for 5-week-old broiler chickens euthanized by MCD were 25.3 and 14.5 s, respectively (34), which indicates that T-61 is more effective in inducing both insensibility and brain death than MCD. Another on-farm euthanasia method, a non-penetrating captive bolt (the Zephyr), is faster at inducing insensibility, with both brainstem reflexes lost within 1.8 s (34). However, this method has a longer time to death (126 s) than T-61, measured as the amount of time until total cessation of movement (34). It is important to recognize that loss of consciousness likely occurred before brain death was detected in both these previous reports and in the current study. Further comparative studies using methods such as spectral analysis are warranted to investigate the relationship between loss of consciousness relative to brain death in euthanasia protocols. Additionally, convulsion and/or wing flapping occur with Zephyr, MCD and gaseous euthanasia (34), but do not occur with T-61 injection. Convulsions can be unpleasant for the operator and thus should be a factor when choosing an appropriate euthanasia method. Convulsion can also affect the condition of the cadaver and tissues of the bird post-euthanasia, which needs to be considered if using the body or tissues for research or other purposes.

Using broiler chickens as a model species, this experiment demonstrated that T-61 is effective for use in birds. Despite there being great variation within avian species, the authors’ clinical experience has shown T-61 to be effective for euthanizing a wide range of birds including but not limited to neonate layer chicks, turkeys, waterfowl, psittacines, raptors, and passerines. Furthermore, due to the limited avian-specific research on medication, and the limited number of medications licensed for birds (much less that are species-specific), it is common practice for drug use to be extrapolated from mammals and among species of birds. As many medications and injectable euthanasia solutions, including pentobarbital, are extrapolated among avian species (1), the evidence of T-61 effectiveness from this study can be extrapolated to other avian species. The authors reiterate, that to overcome species variability, when extrapolating among species, and to provide effective euthanasia, higher doses of T-61 are recommended. The dosage must be sufficient to produce anesthesia followed by death. Therefore, users are also advised to monitor birds for muscle relaxation, loss of brainstem reflexes, and indications of anesthetic overdose; cardiac and respiratory dysrhythmia followed by cessation, and pale to cyanotic mucous membranes and skin. Further research investigating genera or order specific dosages is recommended. To conclude, T-61 is effective as well as efficient and is suitable as a sole euthanasia agent for avian species. When injected intravenously as the sole euthanasia agent, it rapidly induces insensibility and brain death, ensuring the bird is unable to experience pain, distress, and the occurrence of cardiac arrest and circulatory collapse, and reliably results in death.