Evaluation of Tricaine Methanesulfonate Concentrations for Flow Anesthesia in Zebrafish (Danio rerio)

Zebrafish (Danio rerio) are often anesthetized by immersion in buffered tricaine methanesulfonate (MS-222). Although commonly utilized, immersion anesthesia presents a shortcoming of lethal asphyxiation with increased duration of exposures. A newer technique that circumvents this issue, known as flow anesthesia, has been adapted from larger aquatic species to zebrafish. Flow anesthesia improves safety by delivering oxygen-rich water along with the anesthetic across gill epithelium and allowing fish to be manipulated outside of water. Information on the construction of flow anesthesia apparatuses and parameters are sparse. The goal of this study was to create a flow anesthesia apparatus with materials commonly found within a research facility and to evaluate variable concentrations of MS-222 for anesthesia in zebrafish. Depth of anesthesia was monitored by quantifying respiratory rate and recording responses to physical stimulation. All concentrations of MS-222 evaluated (30-100 ppm) were successful at maintaining surgical anesthesia for up to 30 min. The anesthetic events were demonstrated to be safe, with an observed 97% survival rate. This work demonstrates refinements in zebrafish anesthesia and encourages future studies to evaluate MS-222 flow anesthesia for longer duration and evaluation of other commercially available anesthetics for efficacy in a flow anesthesia setup.