不同发育期虹鳟脊椎骨的显微结构及钙、磷元素含量分析

Calcium and phosphorus contents, and microstructure of vertebrae in rainbow trout (Oncorhynchus mykiss) at different developmental stages

中文关键词: 虹鳟  脊椎骨  生长发育  显微结构  元素含量

英文关键词: Oncorhynchus mykiss  Vertebrae  Growth and development  Microstructure  Element content

中文摘要:

      为揭示虹鳟(Oncorhynchus mykiss)脊椎骨的形态结构、元素组成等特征在生长发育过程中的变化情况，本研究分别采集4个发育期(分别为幼鱼Ⅰ期、幼鱼Ⅱ期、成鱼Ⅰ期和成鱼Ⅱ期，平均体质量分别为4、35、644和2 129 g)的虹鳟脊椎骨样品，利用电感耦合等离子体质谱(ICP-MS)检测其第1~6节脊椎骨中钙、磷元素含量，并运用显微CT (Micro-CT)技术对其第4~6节脊椎骨进行扫描与三维重建。结果显示，虹鳟第1~6节脊椎骨的钙、磷元素含量在不同发育期均呈先升高后降低的趋势，脊椎骨中钙、磷元素含量在幼鱼Ⅱ期最高；脊椎骨钙/磷摩尔质量比在生长发育过程中显著增加。第4~6节脊椎骨的显微结构扫描结果显示，脊椎骨的骨小梁数量(trabecular number, Tb.N)随虹鳟的生长呈显著降低趋势；骨小梁厚度(trabecular thickness, Tb.Th)和骨小梁分离度(trabecular separation/spacing, Tb.Sp)在生长发育过程中显著增加；脊椎骨的骨体积分数(bone volume fraction, BV/TV)、组织矿物质密度(tissue mineral density, TMD)和骨矿物质密度(bone mineral density, BMD)等指标均在成鱼Ⅰ期中最低，其次为幼鱼Ⅱ期；脊椎骨中BV/TV及TMD在成鱼Ⅱ期中最高，而BMD在幼鱼Ⅰ期中最大。上述结果不仅为虹鳟发育生物学研究提供了基础数据，还可为鱼类年龄鉴定和分类鉴定等研究提供理论依据。

英文摘要:

      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).猠⁔獨楥杳湥椠晲楥捳慵湬瑴汳礠⁩摮畤物楣湡杴⁥摤攠癴敨污潴瀠浴敨湥琠⁤慥湧摲⁥瑥栠敯⁦爠敭汩慮瑥楲癡敬⁩牺敡獴畩汯瑮猠⁩据漠畴汨摥†灶牥潲癴楥摢敲⁡浥漠物敮⁣牲敥污楳慥扤氠敷⁩摴慨琠慧⁲景潷牴⁨愠条敮Ɽ†杤牥潶略灬Ɐ⁰慭湥摮⁴琮愠硍潩湣潲浯椭捃⁔椠摳散湡瑮楮晩楮捧愠瑲楥潳湵潴晳†晩楮獤桩⹣੡ted 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 change