Introduction

Euthanasia is an act of causing a humane death in an animal by triggering speedy loss of consciousness, which leads to death with the least amount of pain, discomfort, or distress possible. An acceptable method of euthanasia, as per the American Veterinary Medical Association, makes an animal unconscious and dismissive to pain and distress as rapidly as possible, followed by the dysfunctioning of all respiratory and circulatory functions, as well as brain activity. The Euthanasia chamber euthanizes laboratory rodents through CO2 inhalation. It contains barbed fittings that can fit standard CO2 gas tubes

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Method of Euthanasia

There are several methods for euthanizing animals; however, an ideal euthanasia procedure fits certain characteristics for both the animal, employer, and the chemical used. Ideally, it should ensure the detention of an animal with minimal distress and the induction of sudden and permanent insensibility with minimal distress to the animal. Moreover, this technique should be acceptable to both the public and the person performing the treatment. It should also be cost-effective and easily performed by non-veterinary staff without substantial training. Finally, the chemical used for euthanizing is also critical. It should pose no or minimal hazard to the person doing the procedure, and leave no damage or residues that would hinder investigation or future use of the bodies. It should also not produce any residues that threaten other animals after the dead bodies are disposed of.

Considering all the parameters mentioned above for the best method of euthanasia, inhalation of CO2 is the closest to that ideal. It reliably and quickly produces unconsciousness with minimal distress to the animal. Moreover, it has no significant safety risks (Boivin et. al, 2017)

Principle

The CO2 gas is introduced into the chamber and slowly makes the animal lose consciousness, leading to a slow and humane death. 

Protocol

The most common form of euthanasia utilized for small animals is carbon dioxide (CO2) inhalation. Although CO2 is typically considered an appropriate euthanasia agent for small animals when provided properly, its acceptability is contingent on several essential parameters. The animals must be put to sleep by skilled workers. During euthanasia, different species should not be intermingled. When possible, animals should be euthanized individually. However, grouping animals for euthanasia is another approved and regular practice. Make sure the chamber is not too crowded. Euthanasia should be avoided in the animal room wherever possible. 

1. Place the animal(s) in the chamber without pre-charging or filling it with 100% CO2. To produce a balanced gas mixture and meet the goal of quick unconsciousness with little distress to the animals, a fill rate of 30-70 percent of the chamber capacity per minute, injected into the existing air in the chamber, is appropriate.
2. The time it takes to fall unconscious is usually between 2 and 3 minutes. Maintain CO2 flow for at least 1 minute after breathing stops. Remove the mice from the chamber if signs of dehydration and faded eye color are present; otherwise, continue to expose them to CO2. 
3. Death must be established by an appropriate means after the process is completed, such as determining cardiac and respiratory arrest or noting an animal’s fixed and dilated pupils. 

Technical Considerations for CO2 Euthanasia

The rate at which CO2 is displaced is crucial for humane CO2 application. Hence, a pressure-reducing regulator, flow meter, or restriction valve must be employed. A two-stage regulator provides the most control overflow rates. An initial regulator reduces the pressure in the tank to a predefined level, and then a flowmeter, flow gauge, or restriction valve delivers a precise CO2 flow to the euthanasia chamber. The flow rate can be increased after the animals are unconscious to reduce the time to death. An unconscious animal is defined as the loss of the righting reflex or achievement of lateral recumbency in the animal (Shomer et al., 2020).

The following equation determines the volume of the euthanasia chamber:

Euthanasia chamber volume in liters = (Height in cm) × (Width in cm) × (Length in cm) /1000 

Assessment Criteria for Death Confirmation

The following assessment criteria apply to all species and euthanasia procedures. 

Heart rate

The heart rate must be monitored for at least five minutes. Direct palpation of the carotid or femoral artery pulses and direct heart palpation are the most accurate methods of diagnosis. ECG can also be used to confirm death if there is no electrical activity in the heart.

Light-induced pupillary response

This should be done by shining a bright light into the animal’s eyes. A neurological response is shown by pupil constriction. The pupils will become dilated and insensitive to light after death. Some medicines and experimental substances, e.g., anticholinergics like atropine, also can inhibit pupillary responsiveness or modify it in other ways.

Deeply sedated animals can have shallow and erratic breathing patterns, which might be mistaken for a lack of spontaneous respiration. As a result, the absence of spontaneous breathing should not be utilized as the sole criterion for euthanasia confirmation.

Applications

Euthanasia is a critical research procedure. Several times scientists have to find out the effects of respiratory distress or hypoxia on the whole body in normal, diseased, or specific conditions such as injury, pregnancy, etc. In those cases, the euthanasia chamber helps establish hypoxic conditions for lab animals in a controlled environment with minimal pain. For example, Hallak et al. (2000) researched the damaging effects of maternal hypoxia on the fetal neurons and CNS. They utilized a euthanasia chamber as a hypoxia chamber and reported the significant neuronal damage with overall reduced brain and body size of the fetus. Similarly, Lauder et al. (2011) reported the reduced intraperitoneal adhesions with chitosan-dextran gel. For the observation of the results, they euthanized the rats using a euthanasia chamber. 

Precautions

Euthanasia is a sensitive process. It should be led with compassion and sympathy. On the other hand, it demands proper technique and training. Following are some of the suggested precautions:

1. The chamber fill rate should be checked if unconsciousness has not developed within 2 to 3 minutes. A malfunctioning flow meter, a lack of CO2 supply, and/or leakage should all be investigated. Unintended recovery can be avoided by using appropriate CO2 concentrations and exposure periods.
2. It is crucial to confirm death following CO2 exposure. In the case of CO2 narcosis, it must be followed by a secondary technique of euthanasia, such as decapitation, bilateral pneumothorax, or neck dislocation if the animal is not dead.
3. The CO2 euthanasia chamber should be cleaned after each usage and at the end of the day to eliminate any debris or pheromones that may have been released during the last euthanasia session. Alternatively, each group should be given a new/unused container.
4. On a brief examination, a profoundly sedated or seriously ill animal can appear dead; metrics such as lack of movement or evident lack of respiration are insufficient to proclaim a killed animal dead. Hence, all research employees must get adequate and suitable training in all laboratory euthanasia methods and for  evaluating vital signs to confirm animal death. 

Strengths and Limitations

Strengths:

The Euthanasia chamber is a specially designed instrument for CO2 euthanasia that allows euthanizing small lab animals with minimum pain and distress. It allows easy observation of animals with fully controllable CO2 flow rates. 

Limitations

Several studies have shown that CO2 causes pain and discomfort in animals, so its usage should be carefully examined (Conlee et al., 2005).

Summary

1. Euthanasia is the act of causing a humane death in an animal by triggering speedy loss of consciousness 
2. The Euthanasia chamber is specially designed for CO2 euthanasia. 
3. The chamber has clear, transparent walls that allow easy observation of the lab animal. It has a CO2 gas inlet and outlet with a stopcock and barbed fit for CO2 gas tubes.
4. Euthanizing lab animals require proper training and expertise.

References

1. Boivin, G. P., Hickman, D. L., Creamer-Hente, M. A., Pritchett-Corning, K. R., & Bratcher, N. A. (2017). Review of CO₂ as a Euthanasia Agent for Laboratory Rats and Mice. Journal of the American Association for Laboratory Animal Science : JAALAS, 56(5), 491-499. 
2. Euthanasia of Research Animals.   Retrieved from https://services-web.research.uci.edu/compliance/animalcare-use/research-policies-and-guidance/euthanasia.html
3. Guidelines for Euthanasia of Rodents Using Carbon Dioxide. (2020).   Retrieved from https://oacu.oir.nih.gov/system/files/media/file/2021-06/b5_euthanasia_of_rodents_using_carbon_dioxide.pdf
4. Hallak, M., Hotra, J. W., & Kupsky, W. J. (2000). Magnesium sulfate protection of fetal rat brain from severe maternal hypoxia. Obstetrics & Gynecology, 96(1), 124-128. doi:https://doi.org/10.1016/S0029-7844(00)00844-9
5. Conlee, K. M., Stephens, M. L., Rowan, A. N., & King, L. A. (2005). Carbon dioxide for euthanasia: concerns regarding pain and distress, with special reference to mice and rats. Laboratory animals, 39(2), 137–161. https://doi.org/10.1258/0023677053739747
6. Lauder, C. I. W., Garcea, G., Strickland, A., & Maddern, G. J. (2011). Use of a Modified Chitosan–Dextran Gel to Prevent Peritoneal Adhesions in a Rat Model. Journal of Surgical Research, 171(2), 877-882. doi:https://doi.org/10.1016/j.jss.2010.06.028
7. Shomer, N. H., Allen-Worthington, K. H., Hickman, D. L., Jonnalagadda, M., Newsome, J. T., Slate, A. R., . . . Wilkinson, M. (2020). Review of Rodent Euthanasia Methods. Journal of the American Association for Laboratory Animal Science : JAALAS, 59(3), 242-253. doi:10.30802/AALAS-JAALAS-19-000084