Figure 3

Examples of double stained fish with alcian blue/alizarin red from different dietary treatments showing different levels of ossifications and typologies of skeletal deformities. (a) Larva fed with control diet (0.66*10⁸ total VA IU kg^-1 DW) exhibiting mostly red coloured skeletal structures (calcified bone) with the exception of pterygiophores and sclerotic elements still stained with alcian blue (cartilage). (b) Larva fed moderately increased levels of VA (1.5×VA group, 1.00*10⁸ total VA IU kg^-1 DW) showing a larger proportion of skeletal structures stained in blue (cartilage) in comparison to the control group; e.g. pectoral fin girdle, epurals, parahypural and specialized neural arch in the caudal fin complex, and frontal, pterotic and parietal bones in the cranium. (c) Larva fed with the highest levels of VA (10×VA group, 6.82*10⁸ total VA IU kg^-1 DW) showing intermediate values in blue colouration with regards to the control group. The skeletal structures stained in blue (cartilage) are those already reported in the larva from the 1.5×VA group. (d) Larva showing a severe deformity (double lordosis and kyphosis) affecting haemal vertebrae. (e) Deformed caudal fin complex showing the fusion of the hypurals 3-4 (HF3-4) and 1-2 (HF1-2), as well as the fusion (VF) and compression (VC) of different haemal vertebrae centra (23-24 and 22-23, respectively). (f) Fusion of vertebral bodies (VF) from haemal vertebrae number 22 and 23 and underdevelopment of vertebrae 24 (UV). (e) Head of a larva with a slight prognathism (asterisk) due to an underdevelopment of the premaxillar and maxillar bones, and vertebral compression (VC) between centra of prehaemal vertebrae number 2 and 3.