Sturgeon fishes optimizing of staining protocol for skeletal studies during developmental stages

Abstract

Alcian blue-Alizarin red staining protocol during different growth stages of Beluga (Huso huso), as a model of sturgeon fishes, for skeletal ontogeny studies was prepared successfully. For an optimal staining protocol design both fish size and their morphological characteristics at each developmental stage were considered, since such parameters notably influence the staining of tissues. Three developmental windows were determined for an optimal staining procedure: (i) 11-20 mm, (ii) 29-30 mm, and (iii) more than 30 mm total length (TL). Generally, for skeletal staining, alcian blue and alizarin Red are used for stain cartilage and bone respectively. The incubation times of the different solutions were adjusted to allow the stain penetration for revealing the integrity of cartilaginous and bony tissues without significant tissue degradation. Since it was possible to detect the first cartilaginous and mineralized structures in specimens as small as 11 and 20 mm TL, respectively, this procedure is a useful tool to study the sturgeon fishes skeletal ontogenesis, to precociously diagnose skeletal malformations in small larvae and eventually to better characterize the effect of different environmental and ⁄ or nutritional factors on the ossification status of specific skeletal components.