Whole-mount Bone Staining of Selected Cyprinidae Fishes of Punatsangchu, Bhutan

Abstract

Whole-mount bone staining was used to study skeletal system and reproductive toxicology for a long time. Alizarin red S and Alcian blue were used for a decade to stain specimens, and it is still widely used today. The study was conducted to standardize the rapid whole-mount bone staining protocol for larger specimens of selected cyprinidae and to compare meristic and morphometric data before and after staining, which further generates comparative information on the osteological characteristics of the fishes found in Punatsangchu River. The specimens were collected using purposive sampling and stored in 10% formalin. The protocol of the whole-mount bone staining method was modified by changing the concentration of reagent and the timing required based on the size of the specimens. Formalin, Triton X-100, and potassium hydroxide were used as fixative solutions. The enhancement solution consisted of ethylene glycol, Triton X-100, and potassium hydroxide. Alizarin red S and ethylene glycol were used as the bone staining solution and Alcian blue as the cartilage staining solution. Tween 20 and potassium hydroxide were used as clearing solutions. Details of the meristic counts were compared following both pre- and post-staining procedures. In all four species, the meristic counts revealed significant differences (p= .03, N=20). However, comparative morphometric measurements revealed no significant difference between and within species (p=1.00). The strong stout spines on the dorsal and anal fins remained the same, indicating that bone staining was useful in species identification.