CO and N(2) are not humane alternatives to CO(2) euthanasia in rats.

Carbon dioxide (CO(2)) is commonly used for rat euthanasia, but its potential negative side effects call for alternatives causing less distress while providing a fast and reliable loss of consciousness (LOC). Euthanasia with CO(2) is currently performed in a highly non-standardized manner with different CO(2) volume displacement rates/minute (VDRM) ranging from replacing 30 to 70% of the chamber volume with 100% CO(2) per minute. Therefore, we evaluated euthanasia with 100% CO(2) at 30% and 70% VDRM as well as carbon monoxide (8.6% CO at 70% VDRM), nitrogen (100% N(2) at 70% VDRM) and isoflurane anesthesia (6% and 2.1% at 70% VDRM) in female and male rats. We used a multi-modal approach including telemetry for ECG, blood pressure (BP), heart rate measurements, EEG, and plethysmography. Video-recordings were used to track behavior during euthanasia. Stress hormones and lung pathology were assessed post-mortem. A VDRM of 70% CO(2) resulted in a rapid LOC compared to 30%, thus, minimizing the time in which distress and eventually pain can be experienced. Nevertheless, the higher amount of distress experienced until LOC potentially outweighs the benefits of a faster LOC. CO and N(2) resulted in longer times to LOC, seizures before LOC, increased stress hormone levels, and caused more lung damage. Therefore, CO and N(2) are not humane alternatives to CO(2) and should not be recommended for euthanasia in rats.  GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00424-026-03156-1.