Quoting from guidelines to CARBON DIOXIDE EUTHANASIA OF RODENTS from University of Pennsylvania >>
Do not “pre‐fill” the euthanasia chamber with CO2. Start with room air then slowly fill the chamber with CO2 over several minutes
(OLAW). CO2 is heavier than room air, thus the chamber may need to be
“purged” between groups of cages.
“Because inspiration of high
concentrations of CO2 is both aversive and painful, a recommended
procedure is to place animals into a chamber that contains room air
and then to gradually introduce CO2.” (ACLAM)
CO2 first renders the animal anesthetized and then, with adequate exposure time, will result in death by CO2 narcosis. Animals should
be left for additional time within the euthanasia chamber, after
spontaneous movements have ceased, with CO2 continuing to flow.
“Animals should be left in the container until clinical death has been
Also, according to an article in The Scientist by Kerry Grens >>
It's been realized that at high concentrations, CO2 causes creatures torment. After reaching mucosal surfaces, it transforms into carbonic acid, which stings. Also, in people, even low fixations can be upsetting, bringing about shortness of breath.Therefore, explaining why high levels of CO2 is actually more painful for the rodents.