Comparison of Tricaine Methanesulfonate (MS-222) and Alfaxalone Anesthesia in Zebrafish (Danio rerio)

Abstract

The research use of zebrafish has risen exponentially over the past decade while anesthetic options have remained largely unchanged.⁶ ricaine methanesulfonate (MS-222) is widely accepted as an anesthetic for routine husbandry procedures, however it has limitations and safety concerns. ¹¹ A greater variety of effective anesthetic options for surgical procedures would be advantageous for the research community. Adult zebrafish were randomly assigned to one of the following groups (n = 10, 5 males and 5 females): 200 mg/L MS-222; 6-, 10-, 13-, and 16-mg/L alfaxalone, and control. All zebrafish in the MS-222 group reached a surgical plane of anesthesia within 95 ± 32 s. By contrast, only 2 of 10, 1 of 10, 0 of 10, and 0 of 4 of the 6, 10, 13, and 16 mg/L alfaxalone groups, respectively, reached a surgical plane of anesthesia within the allotted 10-min period. Recovery time was also significantly slower in the alfaxalone groups as compared with MS-222, with some fish taking greater than 10 min to recover. In addition, 33 of 34 zebrafish (the 16 mg/L group was not completed due to safety concerns) in the alfaxalone groups lost opercular movements for greater than one minute during their anesthetic event and had to be removed to the recovery tank. The results demonstrated that alfaxalone was unable to provide a reliable and safe surgical plane of anesthesia at any of the drug doses tested. Therefore, we recommend alfaxalone not be used as an anesthetic for painful procedures on zebrafish and conclude that MS-222 remains a more viable anesthetic for immersion anesthesia in zebrafish.

Figure 1.

Experimental set-up. (A) Overview showing recovery tank on the left and anesthesia tank on the right. The pictured fish has lost his righting reflex and is anesthetized. (B) Image capture computer for recovery tank running Ethovision XT (Noldus) software. The fish is in the recovery tank.

Figure 2.

Plane of anesthesia and opercular movement. (A) All MS-222 exposed fish reached a surgical plane of anesthesia, while only 2 of 10, 1 of 10, and 0 of 10 alfaxalone exposed fish (6, 10, and 13 mg/L alfaxalone, respectively) met the criteria. (B) Among alfaxalone-exposed fish, 29 of 30 lost opercular movement (< 1 breaths per minute [bpm]), while none of the MS-222 treated fish exhibited opercular movements at less than 1 bpm.

Figure 3.

Mean time of return of the righting reflex. The time for the righting reflex to return was assessed as a measure of recovery. The MS-222 group had a significantly quicker return of the righting reflex than did any of the alfaxalone groups, ***P ≤ 0.001, ****P ≤ 0.0001. Error bars, 1 SD. n = 10 fish per treatment.

Figure 4.

Movement and bottom-dwelling time of fish exposed to alfaxalone or MS-222. (A) Mean distance moved by the fish during recovery. The MS-222 and control groups have similar mean distances traveled, while the alfaxalone groups moved significantly less. (B) Cumulative fish duration in the top or bottom tank areas during recovery. The control and MS-222 groups spent about equal time in the top and bottom area of the tank while the alfaxalone groups spent significantly more time on the bottom. The similarity between MS-222 and the control demonstrated a much faster return to normal swimming than each of the alfaxalone groups. (A, B) **P ≤ 0.01, ***P ≤ 0.001 compared with controls. ****P ≤ 0.0001. Error bars, 1 SD.

Figure 5.

Representative heat maps demonstrating recovery movement. Representative heat maps of 3 different zebrafish in each group during the 10-min recovery period. Time spent swimming in different areas of the tank during the recovery period for control (A-C), alfaxalone (10 mg/L; D-F), and MS-222 (G-I). Heat maps were generated by the Ethovision XT (Noldus) software. The colored area (blue to red) represents where the fish were swimming and the time spent in those regions (red indicates a longer time). All zebrafish in all 3 alfaxalone groups had similar heat map images, as seen in D-F. The similarity between MS-222 and the control shows a much faster return to normal swimming than do the alfaxalone groups.

Figure 6.

Mean distance moved during each minute of the recovery period. The MS-222 group showed rapid recovery from anesthesia and potentially some hyperactivity as compared with unanesthetized controls. Alfaxalone groups showed gradual increases in movement but did not travel as much as the control or MS-222 exposed groups during the 10-min recording period. n = 10 fish per treatment.