Two male specimens of the Cuban crocodile (Crocodylus rhombifer) weighing 250 gm were anesthetized using Ketamine 5 mg and Medetomidine 0.05 mg and euthanized using an intracardial injection of T-61 0.4 mL (Merck Animal Health. 200 mg embutramide for narcotic action and 50 mg mebezonium iodide for curariform action and 5 mg tetracaine hydrochloride, in aqueous solution). The skull was separated, and the temporal bones were removed using an oscillating saw. The eardrum and the columella were removed and the ears immersed in 2.5% glutaraldehyde and 1% PFA in 2.5% phosphate buffer. The temporal bones were placed in fixative for 48 h and in 0.1 M Na-EDTA for 3 weeks. Thereafter, the surrounding bone was further removed and the ears placed in 1% osmium tetroxide. The specimens were dehydrated in graded ethanol and embedded in Epon. The embedded specimens were divided into different pieces and mounted for semi-sectioning (1 µm thick). Sections were stained in toluidine blue and photographed. Areas of interest were thin-sectioned, and the sections were stained in lead citrate and uranyl acetate and examined at 80 kV in a Tecnai G2 Spirit TEM (Thermo Fisher/FEI Company, Eindhoven, Netherlands). Images were acquired with an ORIUS™ SC200 CCD camera (Gatan Inc. Pleasanton, CA, United States), using the Gatan Digital Micrograph software. A human SV from a cochlea taken out for an earlier study was used for analysis and comparison (Rask-Andersen et al., 2000 (link)).
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