There are many factors to consider when choosing a euthanasia method. Let’s take a look at the common house mouse, Mus musculus, the most common laboratory animal model (discounting fruit flies, which are often euthanized using ethanol or isopropanol fumes, or by placing them in a freezer for 48 hours). Two common methods for euthanizing mice and other rodents are gas-induced hypoxia and cervical dislocation. Gas is far and away the most common due to the simplicity of the protocol.

In the gas method, mice are taken from their colony and placed in a sealed container. This can be a glass jar with a lid or simply a plastic bag clamped shut with a squeezed hand. A gas, most commonly CO2 due to its affordability and ease of regulating flow, is introduced by dropping a tube connected to a pressurized tank. This induces death by displacing air in the container. A small animal will fall unconscious within about 10 seconds, and signs of brain death come within about 30 seconds. 

“Agony” and “Suffering” as Alabama Experiments with Nitrogen Executions - Bolts
Alabama said Alan Miller’s execution by lethal gas would be “more humane.” He writhed and gasped for air in his final moments.

However, animals euthanized in this method often display signs of distress. Hyperventilation and clawing at the walls of the container are common. Mice and rats can be so averse to any exposure to CO2 that they will willingly forego rewards like food to avoid it, or even engage a different aversive stimulus. Inert gasses like argon and nitrogen have been used in some instances, but are considered inhumane as animals also show distress when euthanized this way. Nitrogen may be particularly problematic since it is lighter than air and might not as quickly sink to the bottom of the chamber to displace available air as other gasses might, so this might prolong the process. Research has shown that sedation before gas is introduced does not reduce neurological signs of distress.

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Witnesses watched through a window at an Alabama prison as Kenneth Eugene Smith became the nation’s first person to be put to death using nitrogen gas.

There are also other reasons to avoid gas euthanasia. If the tissues a researcher is interested in studying are prone to acidification then CO2 might not be the best choice, as CO2 readily combines with water to produce carbonic acid, which drops bodily pH. Incidentally, the chemical also creates a painful burning sensation in the lungs, nasal passages, and any exposed mucus membranes. 

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A federal judge is hearing testimony about the nation’s first two nitrogen gas executions, weighing whether to let Alabama carry out a third such execution next month

To avoid this complication, cervical dislocation should be considered. In this technique, a mouse is held firmly down on its stomach, with a researcher’s finger or some other material used to hold and brace the back of the animal’s skull. A sharp pull on the animal’s tail should dislocate the skull from the cervical vertebrae. If the chest cavity is opened immediately researchers will see that the animal’s heart may continue beating for upwards of 30 seconds post-mortem. This is normal.

These techniques for smaller animals like mice and rats are generally not acceptable for larger  animals like sheep, goats, pigs, and cattle. For these animals, overdose with barbiturates is acceptable, as are captive bolt pistols and firearms. If euthanizing at scale, barbiturates may become cost prohibitive.

When using firearms, an appropriate caliber and bullet should be used, and should be aimed at the head. Hollow point bullets fragment on impact and produce more brain damage but do not penetrate as far as a standard round nose bullet. For larger livestock like cattle, calibers between 0.32 and 0.45 are recommended. One study found that a 9mm handgun did not perform adequately when tested on cattle skulls in a controlled environment. In general, adequate results can be achieved when the combination of firearm, ammunition, distance from target and the target itself can be optimized.

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