SOP 10.5.6 - Humane Killing of Adult Rodents with CO2 Asphyxiation under Isoflurane Anesthesia (Rats)

1. Introduction and Definitions:

As per the UACC policy on humane killing of animals used in science, CO₂ humane killing is a conditionally acceptable method. Anesthetizing rodents with isoflurane prior to exposure to CO₂ will minimize pain and distress, this method of humane killing is considered an acceptable method. This method is for adult rodents only. Neonatal rats (up to 10 days of age) are resistant to the hypoxia induced by CO₂; therefore, alternative methods are recommended (e.g. decapitation using sharp blades. See SOP 10.5.5. Euthanasia Method for Rodent Neonates and Fetuses.

2. Materials:

• Isoflurane anesthetic machine (serviced within the last 12 months)
• Oxygen
• CO₂ Source with flow meter (facility outlet, or tank and regulator)
• Appropriately sized rodent cage lid with integrated connections for Isoflurane and CO₂
• Instruments (for secondary method of humane killing). See SOP 10.5.4 Humane Killing of Rodents via Physical Methods
• Body bags

3. Procedures:

• To minimize stress, animals should be humanely killed in their home cage with a maximum of two adult rats or one litter per cage (do not pool rats from different cages).
• Remove the lid and place the appropriately sized custom lid on the rodent cage.
• Disconnect the isoflurane anesthetic supply line from the induction chamber and connect it to the white quick connect on the lid.
• Connect the grey CO₂ supply line to the beige quick connect on the lid.
• Disconnect the scavenger hose from the induction chamber.
• Connect the scavenger hose to the adapter on the lid.
• Turn on the active scavenge to 15L/m.
• Turn on the oxygen flowmeter to 1L/min.
• Set the isoflurane vaporizer to 5%.
• Once the animal has lost consciousness (in lateral recumbency, breathing slower) while leaving the active scavenge on, turn off the isoflurane vaporizer and the oxygen flowmeter and promptly switch over to the CO₂ adaptor, setting the flowmeter to the maximum flow rate of 12 L/min.
• Maintain the CO₂ flow until the animal has stopped breathing, this process can take several minutes.
• Turn off the CO₂ flow meter and active scavenge and leave the animals in contact with loaded CO₂ cage for a minimum of two additional minutes.
• To confirm death, monitor animals for the following signs: no chest movement, no palpable heartbeat, poor mucous membrane colour, no response to toe pinch, colour change or opacity of the eyes.
• Once death is confirmed, increase the active scavenge to full flow
• Leave active scavenge on while removing dead rodent from cage
• Once the age is empty and the session is complete, ensure the active scavenge is off.

4. Secondary Means of Humane Killing:

• To ensure death, a physical method of humane killing is required unless justified in the Animal Use Protocol. See SOP 10.5.4 Humane Killing of Rodents via Physical Methods.

5. Disposal of Animal Carcass:

• Place carcass in body bag and then in fridge for disposal.