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

Abstract

Immersion in tricaine methanesulfonate (i.e. TMS) has been used for euthanasia of Xenopus laevis (African Clawed frogs). However, the time for preparation and potential human health hazards may pose as a barrier for large group culls. Here, we aimed to investigate whether immersion in bupivacaine is an effective means to euthanize this species. In experiment one, frogs (n = 10/group) were randomly assigned to 1-h immersion in 1 of 3 treatment groups: 1) TMS-5 (MS-222, 5g/L); 2) TMS-10 (MS-222, 10 g/L); or 3) Bupi-1.5 (0.5% Bupivacaine, 1.5 g/L). Frogs were then removed from solutions, rinsed with system water, and placed into a recovery cage. Heart rate was evaluated audibly via doppler ultrasound flow over 1 min at immediate removal (T1h), at 2 (T2h), and 3 (T3h) h in the recovery cage. In experiment two, frogs (n = 7/group) underwent 5-h & 19-h immersion in either TMS-5 or Bupi-1.5, with heart rate assessment at 5 and 19 hrs. Righting reflex and withdrawal reflex of the hindlimb were tested during the experiments. Experiment one—after the 1-h immersion, Bupi-1.5 treated animals had decreased heart rates compared to TMS-5 and TMS-10 treated animals by T2h. Neither TMS-5, TMS-10, nor Bupi-1.5 ceased heart rate after the 1-h immersion. Experiment two—after the 5-h immersion, Bupi-1.5 and TMS-5 treated animals were comparable in heart rates. 43% of TMS-5 animals and 14% of the Bupi-1.5 animals had completely ceased heart rates at T5h. At 19 h all remaining animals exhibited rigor mortis and had ceased heart rate. We recommend 19-h of immersion using either TMS-5 or Bupi-1.5 for cessation of heart rate in African Clawed frogs. These data are strong support for the use of secondary physical methods for euthanasia in African Clawed frogs when euthanasia by immersion is performed.

Gut microbiome diversity and function during hibernation and spring emergence in an aquatic frog

“…For euthanization, we employed a bath solution of tricaine methane sulfonate (MS-222) combined with sodium bicarbonate at a pH of 7.0. Given the recommended concentration range of 0.2 g/L to 5.0 g/L for amphibian anesthesia [24,25], we utilized 5 g/L for adults and 3 g/L for juveniles to ensure a humane and stress-free procedure [26][27][28]. A frog was deemed euthanized upon observing the loss of reflexes and respiratory and cardiac arrest [24,25].…”

Effective Rapid Blood Perfusion inXenopus

“…(IS 00001365_3). Though the primary method of euthanasia described is deemed an acceptable technique for euthanasia by the AVMA [11] it has not been found to lead to the cessation of heartbeat [12]. Even the frequently used secondary method of damaging the brain and spine through double pithing does not prevent the Xenopus heart from beating, nor does removing the heart from the animal.…”