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

Abstract

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.

Prevalence of Ranavirus Infection in Three Anuran Species across South Korea

“…To prevent potential transmission of ranavirus between sampling locations, we sanitized all collecting equipment using the following protocol: brushing off mud and vegetation, spraying 10% bleach (Yuhan Clorox, Seoul, South Korea) and cleaning with tap water, spraying 70% ETOH, and completely air-drying all field equipment after each collection [ 32 ]. We individually preserved sampled tadpoles (Voucher number G01621DJ-G03995LC) in 99% ETOH after euthanasia by submerging them for more than 15 min in 0.5% MS222 [ 33 ]. In the laboratory, we randomly subsampled a maximum of eight tadpoles of each species from each location, determined their Gosner developmental stage [ 34 ], extracted liver tissues, and preserved the tissues at −80 °C.…”

Bupivacaine as a euthanasia agent for African Clawed Frogs (Xenopus laevis)

“…Although the definitive reason for this disparity between this current study and the 2009 study is unclear, the differences in age of the frogs may have contributed to the difference in outcome. Concentration and duration of TMS to effectively euthanize African Clawed frogs has also been shown to vary by life stage [ 23 ]. Whereas all frogs utilized in the 2009 study were aged 2–3 years old, we included retired breeders from various colonies whose age was not specified.…”

“…described, but may be logistically challenging and time consuming if larger groups must be culled [17]. In fish and amphibians, TMS acts centrally on the central nervous system to produce general anesthesia when the body is immersed in solution, and has also been investigated & utilized off-label for euthanasia [5,6,[18][19][20][21][22][23][24]. As discussed in the introduction, there are logistical drawbacks to utilizing TMS for this cause, prompting investigation for an alternative.…”

First Report of Endemic Frog Virus 3 (FV3)-like Ranaviruses in the Korean Clawed Salamander (Onychodactylus koreanus) in Asia

“…Two diseased larvae of the Korean clawed salamander (O. koreanus) were collected from a mountain stream in Chuncheon-si (City), Republic of Korea (37.840727 • N, 127.798621 • E) between December 2022 and March 2023. We first described external pathological symptoms and euthanized them by cooling them down in 500 mL of ice and water (5:1, 2-4 • C) for 15-30 min [21] followed by decapitation using a guillotine [22]. Onychodactylus koreanus was collected with permission granted by the Mayor of Chuncheon-si (#4180 00085202 200004).…”