EVALUATION OF HUMANE PROCEDURES FOR EUTHANASIA AND FIXATION SUITABLE FOR HISTOLOGICAL EXAMINATION OF BLADDERTIPPED ANEMONES (ENTACMAEA QUADRICOLOR)

Although bladdertipped anemones (Entacmea quadricolor) are commonly kept in private aquaria and in zoological collections, very limited information regarding the anesthesia and euthanasia of this species is available. We evaluated three anesthetic agents commonly used for aquatic invertebrate anesthesia (142 g/L magnesium chloride [MgCl2], 4 g/L tricaine methanesulfonate [MS-222] and 10% ethanol) as first step agents for inducing unresponsiveness in bladdertipped anemones during a 30-min immersion as well as their potential as a single method for euthanasia. Subsequently to anesthesia, one of three methods of euthanasia (freezing, immersion in 10% formalin or 96% ethanol) were adopted in order to assess their efficacy as a secondary euthanasia method for the anemones. To evaluate the efficacy of the anesthesia and euthanasia methods, animals were placed in untreated saltwater for 48 hours after the procedures to evaluate potential recovery. None of the anesthetic agents were effective as a reliable single-step euthanasia, but all induced unresponsiveness to the second step of euthanasia. Freezing, immersion in formalin, and 96% ethanol were all 100% effective as secondary euthanasia methods. Lastly, three fixative solutions (10% buffered formalin, Bouin´s fixative, 96% ethanol) were tested for suitability for histopathological examination. Based on our study, immersion of bladdertipped anemones in MgCl2 at 142 g/L followed by freezing, immersion in 10% formalin or 96% ethanol appear to be humane and practical euthanasia methods in bladdertipped anemones. Immersion in formalin is the superior method as the second step of euthanasia for histopathological analysis.