The Stability and Efficacy of Tricaine Methanesulfonate (MS222) Solution After Long-Term Storage

Abstract

Tricaine methanesulfonate (MS222) is widely used for the anesthesia and euthanasia of laboratory zebrafish. Fresh solutions have been recommended for each use; however, researchers often mix and store concentrated stock solutions for convenience and to reduce occupational exposure and environmental waste. While this is common practice, published guidelines are often inconsistent. Thus, the objective of this study was to evaluate the stability and anesthetic efficacy of MS222 after long-term storage and to develop specific storage parameters. Stock solutions (100 mg/mL MS222) were mixed and stored in amber jars at 4 °C and -20 °C for 2- and 6-mo. Stability of the solutions was analyzed using liquid chromatography-ion trap mass spectrometry and compared with fresh MS222. Fifty adult (30 male, 20 female) wildtype AB zebrafish (Danio rerio) were randomly anesthetized with 150 mg/L of one of the following MS222 solutions to evaluate anesthetic efficacy: 1) freshly prepared (0m); 2) 2 mo at 4 °C (2m4); 3) 2 mo at -20 °C (2m-20); 4) 6 mo at 4 °C (6m4); 5) 6 mo at -20 °C (6m-20). Time to cessation of swimming, loss of equilibrium, lack of response to von Frey (VF) stimulation, return of equilibrium, and resumption of swimming were compared between groups. Two fish from each group were euthanized at 24-h and 2-wk after anesthesia, and histopathology was performed. All solutions were determined to be stable under all storage conditions. No clinically significant differences were observed between the fresh and stored stock groups during anesthetic testing. No evidence of anesthetic-related histologic changes were noted in the gills, skin, kidneys, muscle, and central nervous system. Hepatic megalocytosis and a reduction in hepatic vacuolation were seen to varying degrees across all groups, but did not follow a treatment-related trend. Therefore, 100 mg/mL solutions of MS222 can be stored in amber jars at 4 °C or -20 °C for 6 mo and still used to effectively anesthetize zebrafish.

Figure 1.

Histologic scoring criteria for zebrafish exposed to MS222.

Figure 2.

Histopathology of select MS222 target-organs from zebrafish. Hematoxylin and eosin, scale bar = 20 µm. Gills (A through C), kidneys (D through F), liver (G through I), and ventral coelomic skin (J through L) of naïve zebrafish (A, D, G, J) were histologically indistinguishable from zebrafish exposed to fresh MS222 (B, E, H, K) and MS222 stored at -20 °C for 6 mo (C, F, I, L). Hepatic megalocytosis (arrows) was seen across all treatment groups and did not follow a treatment-related trend.