Laboratory rodent euthanasia

Euthanasia is defined as “…the act of humanely killing animals by methods that induce rapid unconsciousness and death without pain or distress” (The Guide for the Care and Use of Laboratory Animals (2013) National Academies Press, p. 123). The American Veterinary Medical Association (AVMA) evaluates techniques used for euthanasia and offers species-specific guidelines that provide the most humane conditions possible. The AVMA may update its guidelines as new information becomes available, but the panel provides a comprehensive review and update at least every 10 years. A new report was issued in 2013 which provides substantive updates to the methods used for rodent euthanasia.

While the USDA does not require usage numbers to be reported for mice and rats, it is estimated that over 90% of all animals used in research belong to the genera Mus and Rattus. Very often at the completion of a research study, euthanasia is required. Even if a planned experiment does not include terminal euthanasia, there may be a need to humanely euthanize subjects for unanticipated reasons – namely, if an animal has reached a point where it is experiencing pain or distress that cannot be relieved. Grant applications to the NIH for projects involving animals are required to propose methods that conform to most recent AVMA guidelines (or a scientific justification for non-compliance must be given).

It is important that scientists choose a method that is compatible with their research objectives. For example, if they are studying neurochemistry, they need to avoid anesthetic agents that modify brain chemistry. There are other factors that should also be taken into consideration such as: worker safety, species suitability, time requirements, pain and distress potential, reliability and legal requirements.

Inhaled Anesthetic Overdose or Preceding Exsanguination

This method uses an anesthetic gas, typically isoflurane, to euthanize the animal. The inhalant is delivered via anesthetic vaporizer or by an open drop method in a sealed chamber. Ether is acceptable according to the AVMA guidelines but not recommended due to risk of explosion.

Advantages

Inhaled anesthetics are generally easy to apply as simple equipment may be used.
Inhaled agents are good for use on small animals where venipuncture is difficult.

Disadvantages

The majority of inhaled anesthetics carry a risk to personnel safety.
Certain inhaled agents are explosive.
Often a secondary method is required to verify euthanasia.
Inhaled agents are aversive to mice and rats thereby causing distress.

CO₂ Induction

Carbon dioxide (CO₂) is commonly used as a rodent euthanasia method because it is generally safe, inexpensive and readily available in most animal facilities. Animals lose consciousness more quickly if they are exposed to a high concentration of gas – however this may be painful and distressing to the animal and is now deemed unacceptable by the AVMA. Gradual exposure has recently been shown to be a more humane approach due to decreased aversion. A flow rate of 10-30% cage volume/min is now recommended by the AVMA.

CO₂ euthanasia is usually carried out using a chamber filled by a compressed gas cylinder or a plumbed manifold system. Whereas pre-charged chambers were commonly used historically to euthanize rodents, a device that assures compliance with the AVMA flowrate guidelines, such as a Quietek™, is now required. CO₂ from dry ice sublimation is not acceptable for use because the flow cannot be regulated. Euthanasia in the home cage can be achieved with CO₂ in most circumstances, and this is recommended when possible because it reduces distress related to unfamiliar environments.

Advantages

CO₂ can be administered in the home cage – this reduces stress.
CO₂ is inexpensive, readily available, nonflammable, and nonexplosive.
Good for smaller animals where venipuncture may be difficult.
This method is preferable for high volume euthanasia, since animals can be grouped together (note that they should not be crowded because this condition causes distress).
This method poses minimal hazard to personnel when proper ventilation is available.
CO₂ induction does not distort cellular architecture.

Disadvantages

Can cause distress when administered at a high rate.
It is difficult to ensure optimal flow rate without a device such as the Quietek.
Rodent Neonates (up to 10 days of age) are more resistant to this method than older animals.

Pharmacological Agents (injection)

Using this method, the animal is injected with a barbiturate, typically sodium pentobarbital, at a concentration high enough to cause an overdose. While these drugs should be administered intravenously, it is considered acceptable to administer intraperitoneally. Sodium pentobarbital is a Class II drug which is regulated by the DEA. Pentobarbital combinations are also available which have the advantage of less strict regulations.

Advantages

Barbiturates euthanize animals very quickly, with minimal discomfort to the animal.
Barbiturates are relatively inexpensive.

Disadvantages

Intravenous administration is recommended for best results. This requires trained personnel.
The animal must be restrained.
Barbiturates have strict DEA and State regulations so they are difficult to obtain and require extensive accounting for use and storage.

Cervical dislocation

Cervical dislocation is a technique the animal’s spinal column is manually dislocated from the skull, causing immediate death. Use of this technique is limited to mice and immature rats (<200g).

Advantages

Cervical dislocation is fast and inexpensive.
No special equipment is required.
This method does not chemically contaminate tissue.

Disadvantages

This technique can be aesthetically unpleasant for personnel.
Extensive personnel training is required for cervical dislocation to be humane.
Not suitable for rats over 200g.
Prior sedation may be recommended or required by some institutions.
May be difficult to perform on rodents younger than 10 days.

Decapitation

Decapitation is the process by which the head is quickly removed using a special guillotine with a sharp blade. This technique is typically employed when studying brain chemistry or blood analytes that are sensitive to modification or degradation once euthanasia is complete. The effect of this technique on personnel should be evaluated with care.

Advantages

Fast and humane if administered correctly.
Does not chemically contaminate tissue or blood.

Disadvantages

Significant potential danger for personnel because of sharp blade.
Proper training and monitoring required.
Prior sedation may be required by some institutions.
Handling and restraint may distress animal prior to euthanasia.
Smell may be distressing to other animals that are nearby.
Can be aesthetically unpleasant for personnel.
Improperly maintained equipment will produce undesirable results.

Other

The preceding list identifies the most common forms of rodent euthanasia employed in biological research. Many other techniques are used on a less frequent basis. Typically use of other methods is driven by a specific goal of the project and requires case-by-case approval with scientific justification.