Evaluation of Effective and Practical Euthanasia Methods for Larval African Clawed Frogs (Xenopus laevis)

Larval, or tadpole-stage Xenopus laevis frogs are a popular research model for developmental biology and disease studies. Existing euthanasia guidance documents offer recommendations for both eggs and adult stages, yet do not specifically address the larval stage. Data evaluating effective euthanasia methods for groups of X. laevis tadpoles would therefore be useful. The goal of the current study was to evaluate the efficacy of various immersion euthanasia procedures on tadpoles: tricaine methanesulfonate (MS222) at 6 g/L, eugenol at 800 μL/L and rapid chilling (2 to 4 °C). We also evaluated tadpoles at various developmental stages (NF stages 46, 47 and 49). Tadpoles (n = 70) were exposed to euthanasia solution for 15 min, and controls (n = 40) were placed in housing tank water for 15 min. All animals were then placed in recovery tanks containing housing tank water for 4 h to confirm irreversibility of each agent. Cessation of the heartbeat was assessed at the end of euthanasia solution exposure and at each hour thereafter. We found that immersion in a 6 g/L solution of MS222 resulted in 100% euthanasia of all larval stages tested. Conversely, eugenol produced variable euthanasia rates that were affected by both age group and batches of stock solutions. Rapid chilling was completely ineffective as a euthanasia method in our study. Based on our findings, we recommend MS222 as an effective and practical means of euthanizing large numbers of X. laevis tadpoles.