An enzymatic procedure for dechorionating the fish embryo, Oryzias latipes.

Abstract

AbstractThe tough, fibrous resilient, acellular capsule (chorion) of fish embryos has been characterized as protein‐keratin‐like in composition. It has resisted digestion by common proteinases, such as trypsin, chymotrypsin, pepsin, and papain. We have been able to remove the chorion with pronase, a commercially available (Calbiochem) proteolytic enzyme of bacterial origin with broad substrate specificity.The enzyme at concentrations of between 0.5 and 0.00625% was tested at room temperatures in several Ca++ and Mg++‐free solutions including a weak salt solution (0.1% NaCl), Yamamoto solution (0.7% NaCl), and distilled water with either phosphate or Tris buffer at pH 6.0–11.5. The embryos were in their first day of development at the start of the treatment.It was found that the enzyme was most effective in Tris‐buffered water, less so in weak salt solution and least effective in Yamamoto solution. Phosphate buffer caused some precipitation of the enzyme. The chorion was thinned over a wide range of pH (7.0–11.0) and in concentrations of enzyme between 0.5–0.0125% within 21–36 hours. Although the embryos frequently escaped from the thinned chorion by rhythmical movements, many of the embryos could be removed easily with the aid of common forceps. Once removed from the chorion the embryos were placed in phosphate buffered weak salt solution (Rugh), where, with few exceptions, they developed normally.Considering least mortality and highest yield of embryos with maximally thinned chorions, Tris‐buffered distilled water at pH 9.5 or 10.0 in an enzyme concentration of 0.025% was found to be optimal. Under these conditions 78% of the total number of embryos treated had completely thinned chorions within 24 hours.