Deformities in aquacultured fish pose production challenges, particularly in marine aquarium aquaculture. H ead, spine, gill, and swim bladder deformities repeatably occur impacting the marketability of fish . To study the extent of the abnormality, a method of preserving and imaging must be developed. We have noticed that previous methods of euthanasia can cause stress, leading to exaggerations in features such as flared operculum, open mouths, and body curvature due to rigor mortis . When investigating the nature of craniofacial deformity, it is vital that the deformed features are not exaggerated during the euthanasia and fixing procedure .
Fine scale imaging via computerized tomography (CT) requires specimens to be positioned well, and ideally with each specimen in the same position for comparison. The easiest way to scan most preserved samples is through embedding in agars or resins. A gar e mbedding techniques are traditionally utilized on larval fish, embryos, and histological tissue sections to aid in image capture . This work, however, focuses on model species, that differ in size and application to marine aquarium fishes. There has been little to no research or protocols available for emending full fish for craniofacial analysis.
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During this study multiple methods of euthanasia, fixing, and agar embedding will be compared to find the best method for image analysis of specimens. Species aquacultured including clownfish (Amphiprion ocellaris), royal gramma ( Gramma lorteo), smallmouth grunt (Haemulon chrysargyreum), glassy sweeper (Pempheris schomburgkii), and the Atlantic lookdown (Selene vomer) will be utilized .
Fish representatives with mild (Figure 1A), moderate (Figure 1B ), and extreme (Figure 1C ) craniofacial deformities will be euthanized in Tricaine methanesulfonate (MS-222) in increments of 50 ppm buffered with 100 ppm sodium bicarbonate and fixed in 10% neutral buffered formalin . This procedure will result in natural, relaxed fish for imaging. The fixed fish will then be embedded in 1-2% agar at temperatures ranging from 30ºC to 50º C. M ethod development will determine the best viscosity and temperature to allow ample time to manipulate the specimen and remove bubbles before setting. Consistent, clear, bubble-free embedded specimens can then be scanned to examine the craniofacial features of the fish with increased accuracy.