Physiologic, Behavioral, and Histologic Responses to Various Euthanasia Methods in C57BL/6NTac Male Mice

Area of Science:

• Veterinary Science
• Animal Welfare
• Laboratory Animal Science

Background:

• Rodent euthanasia using carbon dioxide (CO2) is common but raises welfare concerns regarding potential pain and stress.
• Alternative methods like isoflurane and barbiturates are explored for more humane euthanasia.
• Evaluating physiological and behavioral stress indicators is crucial for assessing euthanasia techniques.

Purpose of the Study:

• To compare the stress and physiological responses of three common mouse euthanasia methods: pentobarbital-phenytoin, CO2 inhalation, and isoflurane followed by CO2.
• To determine if pentobarbital-phenytoin offers a less stressful alternative compared to CO2-based methods.
• To assess cardiovascular, behavioral, and adrenocorticotropic hormone (ACTH) responses to different euthanasia protocols.

Main Methods:

• Mice underwent euthanasia via intraperitoneal injection of pentobarbital-phenytoin, CO2 inhalation, or isoflurane anesthesia followed by CO2 inhalation.
• Cardiovascular parameters, behavioral indicators of pain/stress, and ACTH levels were measured.
• Euthanasia time and lung pathology were also assessed.

Main Results:

• Adrenocorticotropic hormone (ACTH) levels suggested pentobarbital-phenytoin may be less stressful than isoflurane or CO2 euthanasia.
• Cardiovascular and behavioral data did not consistently support isoflurane or pentobarbital-phenytoin as significantly less stressful than CO2.
• CO2 euthanasia was the fastest method; however, it resulted in the most severe lung pathology.

Conclusions:

• CO2 euthanasia, with or without isoflurane, is considered an acceptable method due to its speed and comparable stress levels based on most indicators.
• Pentobarbital-phenytoin may induce less ACTH elevation, indicating potentially lower stress.
• Alternative methods are preferable for studies requiring analysis of lung tissue due to CO2-induced pathology.