Effect of flow rate on aversion to gradual-fill carbon dioxide exposure in rats

Laboratory rats are commonly killed by a gradually increasing concentration of carbon dioxide (CO2), but an optimal flow rate for minimizing distress during this procedure has not yet been identified. While fast flow rates minimize time to unconsciousness, slow flow rates may allow an animal to lose consciousness before CO2 concentrations become sufficient to cause pain and dyspnoea. We used an approach-avoidance test, where access to a food reward was contingent on entering a chamber that was gradually filling with CO2, in order to determine whether rats' aversion to gradual-fill CO2 exposure varies with flow rate. During testing, the exposure chamber was filled with either air or CO2, and we examined CO2 flow rates ranging from 3% to 27% of the chamber volume/min. The rats left the chamber before loss of consciousness at all flow rates, and at higher flow rates they left the chamber more quickly. The maximum CO2 concentrations tolerated were at intermediate flow rates, but even at the optimal flow rate of 14%/min rats left the chamber once the CO2 concentration reached just 15.9%, and no rats remained in the chamber until they lost consciousness. Rats tolerated even lower concentrations when flow rates were higher and lower, but this response function was not steep. For example, at the highest flow rate of 27%/min rats left the chamber at a CO2 concentration of 13%. In conclusion rats are sensitive to CO2 flow rate and are most tolerant of CO2 when introduced at a flow rate of around 15%. Despite this modest effect of flow rate, gradual-fill CO2 exposure is still aversive to rats over all flow rates tested and research into alternative methods of euthanasia is now urgently required.