Change in CO2 Euthanasia Chamber Displacement Rate

The 2013 AVMA Guidelines for the Euthanasia of Animals recommend a displacement of 10 - 30% of the chamber volume per minute when performing rodent euthanasia by CO2 overdose. An internal study recently conducted by DLAR personnel and PITT investigators concluded that a 10 - 30% chamber displacement rate is not optimal for minimizing pain and distress when conducting CO2 euthanasia. The internal findings are supported by recent publications and historical strain dependent signs observed in mice and rats across University animal facilities that include a significantly prolonged time to unconsciousness, severe aversive behavior, and clinical signs of pain and distress associated with a 30% chamber displacement rate.

Based on the above, the DLAR submitted and the IACUC approved a program-wide deviation to the AVMA guidelines in November 2016. The program wide recommendation for rodent CO2 euthanasia has been changed from a 10 - 30% displacement rate to a 50 - 55% displacement rate. As a result, CO2 euthanasia equipment must be recalibrated to provide the appropriate flow of CO2 into the euthanasia chamber. Depending on the euthanasia equipment setup, either the flow meter setting or the flow gauge component of the pressure regulator must be adjusted to control the euthanasia chamber displacement rate between 50 - 55%.

First, the euthanasia chamber volume is determined by the following equation:

    (height in cm) X
Euthanasia chamber =  (width in cm) X (length in cm)
volume in liters                               1000

Flow Meter Setting

Flow meters control the delivery of gas in liters/minute (LPM). The flow meter setting is determined by multiplying the euthanasia chamber volume in liters by the desired chamber displacement rate. For example, the CO2 flow meter rate for a 50% chamber volume/minute displacement rate is determined by the following equation:

Flow meter setting (LPM) = (Euthanasia chamber volume in liters)
X 0.5 volume/min

Flow Gauge Setting

If a flow meter is not used to control CO2 gas flow, a two-step pressure regulator should be used on the CO2 supply tank to regulate gas flow. The flow gauge measures the gas flow in cubic feet/hour (CFH). This is different than a pressure gauge that measures gas pressure in pounds per square inch (PSI).

To calculate the flow gauge setting, determine the flow meter setting in LPM as described above then convert the value from LPM to CFH by the following equation:

LPM X 2.12 = CFH

All CO2 euthanasia equipment must be recalibrated no later than January 31, 2017 to provide a 50 - 55% chamber displacement rate. Research personnel should carefully observe rodents euthanized in the recalibrated equipment in outside use areas to assure that the euthanasia is humane and completed with minimal to no pain or distress. Report any concerns to your DLAR facility assigned veterinarian who will be happy to review your calculations and euthanasia equipment set up and work with you to resolve any issues.

The RCCO and IACUC monitor euthanasia practices and equipment during protocol audits, the semi-annual facility inspection process, and other post-approval monitoring activities. Investigators will be asked about the recalibration of CO2 euthanasia equipment during these activities to assure compliance with the 50 - 55% chamber displacement rate.

The PI is responsible for making the changes described above to PI-owned rodent CO2 euthanasia equipment. Division of Laboratory Animal Resources (DLAR) veterinary staff is responsible for making the changes described below to DLAR rodent euthanasia equipment provided inside a DLAR facility.

Any questions about this policy should be directed to your DLAR facility assigned veterinarian. If you are unsure how to contact your veterinarian, visit the DLAR website or call 412-648-8950.