GILTHEAD SEABREAMS (Sparus aurata)

3rd International Congress on Applied Ichthyology & Aquatic Environment

8-11 November 2018 Volos, Greece www.hydromedit.gr

COLLAGENULTRASTRUCTURE ULTRASTRUCTURE AND AND X-RAY X-RAY ANALYSIS COLLAGEN ANALYSIS OF OF VERTEBRAE VERTEBRAEOF OF GILTHEAD SEABREAMS (Sparus aurata) WITH THE SCOLIOSIS DEFORMITY Boursiaki V.1, Theochari C.1, Zaoutsos S.P.2, Mente E.1, Apostologamvrou C.1, Vafidis D.1 and Berillis P. 1* 1

Department of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, Fytoko Street, GR-38446, N. Ionia Magnisias, Greece. 2 Laboratory of Advanced Materials and Constructions Department of Mechanical Engineering Technological Educational Institute of Thessaly, 41110 Larissa, Greece Abstract The development of skeletal deformities in seabream (Sparus aurata) farming affects their growth, survival and production costs. Research has shown that the distribution of collagen in different fish tissues might be correlated to their swimming behavior. This study investigates whether gilthead seabream with scoliosis deformity showed collagen morphology abnormalities in their vertebras in comparison with those that did not show any skeletal deformities. Samples for decalcified vertebras of both groups were examined with transmission electron microscope and collagen micrographs were taken and analyzed. The results indicated that the fishes with scoliosis had significant smaller mean vertebras’ collagen fibrils diameter than the controls. Vertebrae in abdominal and caudal regions of the scoliotic S. aurata appeared to be smaller than the respective vertebrae of S. aurata without any skeletal deformity. Key words: Sparus aurata, scoliosis, vertebra column, collagen. *Corresponding author: Dr. Berillis Panagiotis ([email protected]). 1.

Introduction

Collagen is a group of naturally occurring proteins. It is abundant in most invertebrates and vertebrates (Gallop and Paz 1975, Adams 1978). It is the main protein of connective tissue and represents about one-fourth of the total body protein content (Bailey 1968). Its molecule is formed by three polypeptide strands, called alpha chains, each possessing the conformation of a left-handed helix. Collagen is one of the long, fibrous structural proteins whose functions are different from those of globular proteins such as enzymes. Tough bundles of collagen (collagen fibers) are a major component of the extracellular matrix that supports most tissues. In fishes, collagen fibrils form a delicate network structure with varying complexity in the different connective tissues in a pattern similar to that found in mammals. The collagen in fish is much more thermolabile and contains fewer, but more labile cross-links compared to the collagen from warm-blooded vertebrates. In general, contains fewer hydroxyprolin than in mammals, although a total variation between 4.7 and 10% of the collagen is observed (Sato et al. 1989). Different fish species contain varying amounts of collagen in their body tissues. This has led to a theory that the distribution of collagen may reflect the swimming behavior of the species (Yoshinaka et al. 1988). Gilthead seabream represents the 13% (548 million €) of the total 2014 EU aquaculture production (Annual Economic Report, 2016). Seabream is commercialized as whole fish at 350-500 g and any malformation is a limiting factor in their purchase by consumers. Deformities are a complex mixture of different bone disorders including vertebral and spinal malformations such as kyphosis, lordosis, scoliosis, platyspondyly and vertebrae fusion. The skeletal deformities are induced during the embryonic and post-embryonic periods of life and their development is not well understood. They are related with nutritional, environmental and genetic factors (Fernadez et al., 2008). Fish bones consist of calcium-phosphor hydroxyapatite salts (inorganic part, about 65% of bone‘s dry mass) embedded in a matrix of type I collagen fibers (organic part) (Mahamid et al. 2008). The relationship between collagen and hydroxyapatite is crucial for bone toughness and stiffness. The aim of this study was to determine if the skeletal deformity of scoliosis is associated with changes to the collagen fibrils morphology of the vertebras. 2.

Materials and Methods

At the present study 20 adults individuals of Sparus aurata were collected from a commercial fish farm. Fishes were divided in to two groups. One with the presence of scoliosis (10 fishes, mean weight 336.69±26.48gr,

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mean length 25.11±0.91 cm), and one without any skeletal deformity (10 fishes, mean weight 352.64±39.18 gr, mean length 27.39±0.96 cm). Each fish was X-rayed at 50 kV (Figure 1). The vertebral column of S. aurata was divided into three regions: the cervical, with four vertebrae, the abdominal, with twelve vertebrae and the caudal, with eight vertebrae. The length of the vertebrae of these three regions was measured using the K-PACS V1.6.0 (Image Information Systems Ltd.). Vertebra samples were taken from the part of the vertebra column that the scoliosis occurred (caudal region), fixed with glutaraldehyde, decalcified, dehydrated and embedded with resin. Ultrathin sections (60-80nm) were taken, stained with uranyl acetate and PTA and examined under a Philips CM10 electron microscope equipped with a digital camera (Veleta, Olympus). Micrographs were taken and collagen fibrils diameter and period were measured using a special algorithm. Further computational details appear in Tzaphlidou and Berillis (2002). The same procedure was followed for gilthead seabreams without any skeletal deformity (vertebras were taken from the caudal region of the vertebra column).

Figure 1: Apical X-ray of S. aurata with scoliosis. The scoliosis area is highlighted with an arrow. 3.

Results

The collagen fibrils mean diameter and the period are presented in Table 1 and Figure 2, 3. There is a significant difference between the vertebras’ collagen fibrils diameter of the two groups (P0.05). Table 2. Mean lengths of S. aurata vertebrae in different regions. Cervical (mm)

Abdominal (mm)

Caudal (mm)

S. aurata without any skeletal deformity

4.73a ± 0.19 (40)

7.47b±0.10 (120)

6.43d ± 0.20 (80)

S. aurata with scoliosis

4.92a ± 0.16 (40)

6.95c±0.08 (120)

5.36e ± 0.18 (80)

Note: Results are median ±Interquartile Range. Numbers in brackets show the number of measurements. Means in a column followed by the same superscript are not significantly different (P > 0.05).


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Figure 2: A general collagen fibril diameter histogram of the vertebrae (nm) of both groups. S. aurata without any skeletal deformity and S. aurata with scoliosis.

Figure 3: Collagen fibrils of the vertebrae (A) of S. aurata without any skeletal deformity and (B) of S. aurata with scoliosis.


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Figure 4: Vertebrae collagen fibrils’ periodicity did not differ between S. aurata without any skeletal deformity and S. aurata with scoliosis. 4.

Discussion

Gilthead seabream is one of the most important farmed fish species. Fishes with skeletal abnormalities are not preferred by the consumers, have diminished survivance and increase the production cost. As it has already been reported the development of skeletal deformities is not well understood and could be related to nutritional, environmental and genetic factors (Fernadez et al. 2008). Rapidly growing animals are more likely to develop pathological lesions to the skeleton. Thus, the hypothesis that growth rate may be another risk factor in the occurrence of spinal deformities is acceptable (Halver et al. 1969, Weisbrode and Doige 2001). In bone, collagen represents more than 90% of the organic bone matrix. It confers resistance to the structure and establishes the biomechanical properties of the tissue (Moro et al. 2000). Lim and Lower (1978) showed that vitamin C deficiency in channel catfish (Ictalurus punctatus) leaded to vertebra column deformities (kyphosis, scoliosis, lordosis) and to a decrement in the collagen content of bone. Our results showed a correlation between the scoliosis deformity and the vertebras’ collagen fibril diameter in seabream. No similar correlation between the scoliosis deformity and the vertebras’ collagen fibril period detected. The result that the mean collagen fibril D-period of scoliotic seabreams and the ones without any skeletal deformities was similar (P > 0.05), implies that the arrangement of collagen molecules or the covalent crosslinking between them, is not affected in the same way as the diameter is, so the structure of collagen fibrils appears to be a more rigid marker than their diameter. To form fibrils the collagen molecules are assembled in parallel and are mutually staggered by D-period or integral multiples of D-periods. In our results, the normal axial relationship between collagen molecules in a collagen fibril from a vertebra of a scoliotic S. aurata fish seems not to be disturbed. In our results, the normal axial relationship between collagen molecules in a collagen fibril from a vertebra of a scoliotic S. aurata fish seems not to be disturbed. Our collagen fibrils’ periodicity measurements are smaller than 68 nm, but in electron microscopy, dehydrated specimens usually provide lower values, than those obtained with low-angle X-ray diffraction of hydrated specimens and atomic force microscopes in which D is near to 68 nm (Tzaphlidou, 2005).\ Acknowledgements Part of the research was financed by the Research Committee of the University of Thessaly.

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