However, since nutritional needs change through the larval development, the precocious detection of skeletal deformities could aid to determine the influence nutrients on early larval development.In this sense, the establishment of the alcian blue-alizarin red
double staining technique for developing European sea bass
larvae becomes useful to describe skeletogenesis as well as to
evaluate any factor that could induce skeletal deformities.
Although the ontogeny of the cephalic (Gluckmann et al.,
1999) and appendicular (Marino et al., 1993) skeleton has been
investigated in this species, there is no information about the
characterization of the ossification process using a quantitative
methodology. Quantification of bone mineralization could also
serve to determine and localize possible disruptions during this
process that could constitute the origin of skeletal deformities.
In order to validate bone quantification analysis based on the
double staining approach, it was found appropriated to study
in parallel the expression pattern of the osteocalcin gene, which
serves as marker for the mineralization process. Osteocalcin
(Bone Gla protein) is indeed the most abundant non collagenous
protein in the extracellular matrix of bony tissues
(Nishimoto et al., 1992), it is synthesized by matures osteoblasts
and constitutes nowadays a marker for bone remodelling
in various vertebrates