Macroscopic Examination of Skeletal Tissues in Phosphorus‐Deficient Rainbow Trout <i>Oncorhynchus mykiss</i>

Phosphorus (P) deficiency in aquacultured fish has become increasingly prevalent due to stricter environmental regulations and reduced dietary P inputs. P-deficient fish have been reported to exhibit morphometric alterations, notably in the ribs and neural/hemal spines; however, whether these deformities represent true pathological manifestations remains uncertain. This study re-examined skeletal tissues of juvenile rainbow trout Oncorhynchus mykiss using alternative protocols. Fish were fed either a P-deficient or a control diet for 1.5 months. Following formalin fixation, muscular tissues were enzymatically digested, and the skeletons were stained with alizarin red, alcian blue and aniline blue for detailed examination. P-deficient fish exhibited only minor rib deformities but showed extensive non-calcified regions across the skeleton, teeth, fins and scales. These findings were further confirmed by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS). In contrast, heating the skeletons (70°C or 100°C) markedly exacerbated skeletal deformities in P-deficient fish but not in control fish. These results indicate that bending deformities previously reported as clinical signs of P deficiency are not evident in intact fish but become conspicuous only after heating during analytical procedures. The expansion of non-calcified skeletal regions may serve as a sensitive diagnostic indicator of P deficiency in farmed fish.