Beyond Single-Agent Euthanasia, Gas Combination for Welfare-Refined Rodent Euthanasia: A Narrative Review

Abstract

Carbon dioxide (CO2) remains the most widely used euthanasia method for laboratory rodents because of its practicality and cost-effectiveness. Nevertheless, substantial evidence has raised welfare concerns related to hypercapnia-associated distress, nociceptor activation, and aversive behavioral responses prior to loss of consciousness. Inert gases such as nitrogen and argon have been proposed as alternatives, but their welfare implications remain variable and strongly dependent on delivery protocols. This narrative review synthesizes current evidence on gas-based euthanasia methods in laboratory rodents, with particular emphasis on emerging gas combination strategies, including carbon dioxide– nitrous oxide (CO2/N2O) mixtures. The review focuses on reported welfare-related outcomes, physiological mechanisms, and practical implementation considerations. A targeted and iterative literature search was conducted using PubMed/MEDLINE, Scopus, and Google Scholar to identify relevant studies published between 2000 and 2025. Experimental and comparative studies, reviews, and relevant regulatory guidelines involving mice and rats were included, and evidence was synthesized narratively due to substantial methodological heterogeneity. Across studies, single-agent gases exhibited distinct but not universally superior welfare profiles. Inert gases avoided hypercapnic acidosis but were associated with hypoxia-related behavioral responses under certain conditions. Selected CO2/N2O combinations were reported to achieve shorter induction times and fewer observable indicators of distress compared with CO2 alone, although findings were protocolspecific and based on limited evidence. Overall, gas combination approaches represent a context-dependent avenue for refinement rather than a universal replacement, underscoring the need for careful protocol selection and further research.