Several nutritional studies have found a direct effect of several vitamins in chondrogenic and osteogenic development during early life stages of marine fish species. In the present study, the effect of vitamin A (VA) in gilthead sea bream skeletogenesis was evaluated by means of four different dietary regimes (enriched rotifers) containing increasing levels of total VA (75, 109, 188 and 723 ng total VA mg − 1 DW). Dietary treatments were offered to larvae during the rotifer-feeding phase (4-20 days after hatching), while later all groups were fed with Artemia nauplii and weaned onto the same inert diet. Different dietary doses of VA affected gilthead sea bream larval growth, survival, performance (maturation of the digestive system) and quality (incidence of skeletal deformities). Higher levels of dietary VA than those included in the commercial emulsion for rotifer enrichment led to different levels and typologies of skeletal deformities, indicating that gilthead sea bream larvae were very sensitive to small increases in dietary VA. The degree of ossification was affected by the level of VA in enriched rotifers: the higher amount of VA in the diet, the higher number of skeletal pieces ossified (R = 0.585, P = 0.04). Dietary VA affected the normal process of bone formation and skeletogenesis, the skeletal structures mostly affected by high amounts of dietary VA were those from the cranial skeleton (splanchnocranium), vertebral centrums and caudal fin complex. The premaxilla, maxilla and dentary bones were the cranial structures affected by dietary VA levels, resulting in a large incidence of animals with compressed snout. Dietary VA also affected the normal development of the opercular complex, and a dose-response dependant effect was observed in relation to the incidence of specimens with incomplete operculum. Body shape was also affected by the level of dietary VA, increasing the incidence of specimens with lordosis, kyphosis and/or scoliosis with the dose of VA, being the prehaemal and caudal vertebrae the most affected regions of the vertebral column with this kind of abnormalities. The caudal fin complex was the most affected region of the skeleton affected by dietary treatments as seen by the high incidence of skeletal deformities in fish fed different doses of dietary VA. Deformities affected all skeletal elements composing the caudal fin, although the most affected ones were, in order of importance, the epurals, hypurals, parahypural, neural arch and uroneurals. Differences in sensitivity to dietary VA amongst caudal fin skeletal elements might be due to their differential ontogenetic development and differences in the exposure time to VA. An excess of dietary VA also accelerated the intramembranous ossification process of vertebral centrums leading to one or two supranumerary vertebrae, and a high incidence of fused and compressed vertebral centrums. The sensibility of the developing skeletal structures to dietary VA levels should incline us to test lower doses of VA in live preys enrichments during ear...

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The effects of different levels of vitamin A (VA) in Senegalese sole larval performance and development were evaluated by means of a dietary dose-response experiment using enriched Artemia metanauplii as a carrier of this micronutrient. Larvae were fed from 6 to 27 days post hatch (dph) with enriched Artemia containing graded levels of total VA (1.3, 2.1, 4.5 and 12.9 µg VA mg − 1 DW). The content of VA in live prey directly affected its accumulation in larvae and early juveniles. Retinyl palmitate accumulated during larval ontogeny, whereas retinol showed the opposite trend, decreasing from hatching until 41 dph and then remaining constant until the end of the study.

During the last decades, the finfish larviculture industry has considerably improved its rearing methods under intensive conditions, but larval quality remains one of the main problems for the proper sustainable success of this productive sector (Kou moundouros 2010, Boglione et al. 2013a). The quality of farmed fish is directly related to the quality of the fry, and depends on both organoleptic and morphological characteristics that should be as similar as possible to that of wild fish, which is considered to be the ABSTRACT: This study provides a comprehensive description of the tissue organization of nondeformed and deformed opercula and vertebrae from gilthead sea bream Sparus aurata juveniles by means of histological, histochemical and immunohistochemical approaches. Two types of opercular anomalies are described: the folding of the opercle and subopercle into the gill chamber, starting at the upper corner of the branchial cleft and extending down to its lower third; and the partial lack of the operculum (opercle, subopercle, interopercle and preopercle underdeveloped) with a regression of the loose edge extending down to its lower third. Histological observations revealed a rare type of bone remodelling process in the opercular structure, which consisted of the coalescence of contacting bone tissues (presumably from the preopercle and opercle), resulting in skeletal tissue with a trabecular aspect filled by a single-cell epithelium of cubic osteoblastic-like cells. Differences in collagen fiber thickness and its 3-dimensional arrangement between normal and deformed opercula were also found. Lordotic vertebrae were characterized by the formation of fibrous cartilage in the haemal and/or neural sides, indicating that a metaplastic shift occurred during the process of lordosis. Another major histomorphological change found in lordotic vertebrae was the complete loss of notochordal sheath integrity. Histological alterations were coupled with an imbalance of cell death and cell proliferation processes in lordotic vertebrae as well as that of bone formation/resorption, and extracellular matrix deposition activity differences which might have resulted from the remodelling process occurring in lordotic vertebrae. Altogether, these results provide an increase in our basic knowledge of bone disorders that contribute to our understanding of the mechanisms by which these skeletal anomalies appear in this fish species and which hamper its production efficiency.

The ability of zebrafish to fully regenerate its caudal fin has been explored to better understand the mechanisms underlying de novo bone formation and to develop screening methods towards the discovery of compounds with therapeutic potential. Quantifying caudal fin regeneration largely depends on successfully measuring new tissue formation through methods that require optimization and standardization. Here, we present an improved methodology to characterize and analyse overall caudal fin and bone regeneration in adult zebrafish. First, regenerated and mineralized areas are evaluated through broad, rapid and specific chronological and morphometric analysis in alizarin red stained fins. Then, following a more refined strategy, the intensity of the staining within a 2D longitudinal plane is determined through pixel intensity analysis, as an indicator of density or thickness/volume. The applicability of this methodology on live specimens, to reduce animal experimentation and provide a tool for in vivo tracking of the regenerative process, was successfully demonstrated. Finally, the methodology was validated on retinoic acid- and warfarin-treated specimens, and further confirmed by micro-computed tomography. Because it is easily implementable, accurate and does not require sophisticated equipment, the present methodology will certainly provide valuable technical standardization for research in tissue engineering, regenerative medicine and skeletal biology.