Calcium and Phosphorus Contents, and Microstructure of Vertebrae in Rainbow Trout (Oncorhynchus mykiss) at Different Developmental Stages

The skeletal system of fish consists of the axial skeleton (skull, vertebral column, ribs, and intermuscular bones) and the appendicular skeleton, which are essential for behavioral and physiological functions such as locomotion, feeding, predator avoidance, and load-bearing. As for the vertebral column of teleosts, it is composed of many vertebrae connected from the head to the caudal base. The morphological characteristics of the vertebrae (such as the number and structure) vary among different fish species. These characters (especially the vertebrae number) provide an important basis for species identification. For instance, the number of vertebrae in Salmo salar is 57–60 (30 trunk vertebrae, 27–30 caudal vertebrae), while rainbow trout (Oncorhynchus mykiss) has a total of 63 vertebrae (including 33 trunk vertebrae and 30 caudal vertebrae), which can be used for species identification. Fish with a similar number of vertebrae require further skeletal morphological features to distinguish them. For instance, the three-dimensional structure of the same vertebra segment from 32 different teleost species (belonging to 10 different orders) were compared and analyzed using Micro-computed tomography (Micro-CT) scanning technology. The results showed that the lamellar trabeculae and its internal cavity structure on the spine differed between the species, suggesting that these structural characteristics can serve as additional evidence to classify and identify fish species. In addition, calcium (Ca) and phosphorus (P) are the most important mineral elements in a fish skeleton, and their contents vary among different fish. Therefore, there is potential to use the skeletal Ca and P contents in classifying and identifying fish and their life history characteristics.To examine the vertebrae number in O. mykiss, specimens of juvenile O. mykiss of body weight (1.27±0.21) g were cleaned and double-stained to obtain the whole skeletal image. A total of 63 vertebrae were identified, and all were completely ossified at this developmental stage. X-ray scanner technology scanned and photographed the entire skeletal structure of adult O. mykiss. The adult results were similar to the juvenile results of 63 vertebrae with both ends connected with the head or tail, and the ribs were attached to the trunk vertebrae. The ventral sides of the 1–33 trunk vertebrae were arranged in an arc, which was downward and separate. No intermuscular spine was evident. On the dorsal side of the vertebrae, the neural arches surrounding the neural canal were fused with the neural spines. Unlike the ribs, the caudal vertebrae had vascular arches, which formed passages for blood vessels and nerves and were fused with the vascular spines on the ventral side.The calcium and phosphorus content, and microstructure of the vertebrae in O. mykiss at different developmental stages were assessed. Vertebrae samples were collected at four developmental stages (young stage Ⅰ, young stage Ⅱ, adult stage Ⅰ, and adult stage Ⅱ; with an average body weight of 4, 35, 644 and 2 129 g, respectively). The calcium and phosphorus contents in the 1–6th vertebrae were assessed by inductively coupled plasma mass spectrometry (ICP-MS). The 4–6th vertebrae were scanned using Micro-CT technology. The results revealed the calcium and phosphorus contents of the vertebrae initially increased and then decreased during development. The highest levels of calcium and phosphorus in the vertebrae was at young stage Ⅱ, (4 711.121±567.948) and (3 649.488±446.961) μmol/g, respectively. The Ca/P molar mass ratio increased significantly with the growth of O. mykiss (P < 0.05). These results indicated that the degree of mineralization in the vertebrae increased with growth and development. Micro-CT scanning results indicated that the bone volume and surface of the vertebrae increased significantly with the growth of O. mykiss. The vertebrae segments became more obvious, and the structure became more complete. The vertebral microstructure indexes in O. mykiss at the different developmental stages suggested the trabecular number (Tb.N) significantly decreased with the growth of O. mykiss (P < 0.05). The highest levels occurred at young stage Ⅰ with (19.915±0.758) ind./mm, the lowest levels occurred at adult stage Ⅱ with (1.960±0.043) ind./mm. The trabecular thickness (Tb.Th) and trabecular separation/spacing (Tb.Sp), both significantly increased with O. mykiss growth (P < 0.05). Tb.Th and Tb.Sp of the vertebrae in O. mykiss were the lowest, (0.060±0.001) mm and (0.068±0.004) mm, respectively at young stage Ⅰ, and the highest levels, (0.718±0.026) mm and (0.402±0.029) mm, respectively) were at adult stage Ⅱ. In addition, the bone volume fraction (BV/TV), tissue mineral density (TMD), and bone mineral density (BMD) showed a trend of initially decreasing and then increasing. The lowest levels were at adult stage Ⅰ, (62.620±13.223)%, (460.300±102.825) mg/mL and (678.052± 4.417) mg/mL, respectively, and the highest BV/TV and TMD, (86.473±1.029)% and (654.797± 7.031) mg/mL were at adult stage Ⅱ. Conversely, the highest BMD, (820.527±5.003) mg/mL, was at young stage Ⅰ. The evaluation indexes of the bone spatial morphological structures (such as TV, BV, BV/TV, BS, Tb.Th, and Tb.Sp) increased significantly during the growth and development of rainbow trout, while Tb.N decreased significantly. The bone strength evaluation index, BMD initially decreased and then increased. The significant variation in the vertebra microstructure at the different developmental stages might be closely related to its function. These results indicate that the microstructure and element contents of vertebrae in O. mykiss changes significantly during development and the relative results could provide more reliable data for age, group, and taxonomic identification of fish