Compatibility of eugenol anesthesia with classroom physiology experiments on nerve and muscle of frogs

Abstract

Frogs serve as valuable model organisms for studying physiological responses of nerve, skeletal muscle and the heart in undergraduate biology labs. Heretofore, induced hypothermia has been the preferred technique for pain and stress reduction prior to euthanasia by pithing. However, AVMA guidelines discourage hypothermia for restraint or euthanasia of amphibians and advocate anesthetic overdose as preferable to pithing alone. Unfortunately, the primary anesthetic used in fish and other aquatic invertebrates, MS-222, blocks normal nerve and muscle function in leopard frogs and renders them useless for classroom physiology experiments, as demonstrated by Medler (2019). Eugenol is a safe and affordable anesthetic that is effective in amphibians. We sought to test the hypothesis that eugenol anesthesia is compatible with classroom nerve and muscle experiments on frogs. Bullfrogs and leopard frogs were immersed in a bath of eugenol suspended in tap water at 275 to 350 mg/L. At various time points, a four step reflex test was performed including tests of righting response, superficial pain reflex, deep pain reflex, and corneal blink reflex. Once all reflexes were absent, the frog was euthanized by pithing and the gastrocnemius muscle was prepared for force recordings. The sciatic nerve was stimulated with a bipolar stimulating electrode while gastrocnemius force was measured using an isometric force transducer. The threshold voltage eliciting a muscle contraction and the peak isometric twitch force were recorded at 15 minute intervals for 2 hours. Control frogs were sedated by immersion in ice water for 20 to 30 minutes, pithed when reflexes were substantially weakened, and then subjected to the same stimulation protocol and force measurements. At these doses, eugenol reliably induces loss of reflexes in 20 to 35 minutes and neuromuscular preparations maintain the ability to respond to electrical stimulation. Preliminary results suggest that eugenol may produce a modest elevation of threshold voltage and a modest reduction in maximum twitch force compared to control frogs euthanized without chemical anesthesia. Eugenol may be an effective anesthetic to prepare bullfrogs and leopard frogs for classroom experiments on nerve and muscle. Funding was provided by the Bill and Linda Frost Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.