DL-methionyl-DL-methionine as an efficient methionine source for promoting zootechnical performance and methionine-related pathways in the whiteleg shrimp (Penaeus vannamei).

Methionine (MET) supplementation is a current strategy to achieve shrimp requirement. Notwithstanding, the efficiency of the precisely formulated feeds can be diminished since shrimps are slow eaters and masticate feed externally that results in nutrient leaching. In this regard, a methionine dipeptide (DL-methionyl DL-methionine) benefits the feed industry by reducing MET water solubility while increasing its bioavailability. Therefore, the effects of feeding whiteleg shrimp (Penaeus vannamei) with increasing levels of methionine dipeptide were evaluated on zootechnical performance and methionine-, immune- and antioxidant-related pathways. A 74 d growth trial was conducted by feeding a control diet and four diets supplemented with AQUAVI® Met-Met at 0·08, 0·12, 0·24 and 0·32% of DM. Diet digestibility, body amino acids (AA) composition and nitrogen metabolites, metabolic enzymes, oxidative status and gene expression were evaluated. It can be concluded that graded dietary increase of methionine dipeptide up to 0·24 % for 74 d translated in significant gains on the growth performance, feed efficiency, nutrient and nitrogen gain and shrimp survival. Moreover, it was showed that Met-Met dietary spare leads to an improvement of free-AA pool and nitrogen metabolites concentration and reduces the signs of oxidative stress. Finally, in a closer look to the MET-related pathways passive to be altered by Met-Met spare, a clear modulation of the described antioxidant and cell proliferation routes was detected.

Effect of Dietary Inorganic and Chelated Trace Mineral Supplementation on the Growth Performance and Skeletal Deformities of European Seabass and Senegalese Sole Post-larvae.

Trace elements such as Cu, Fe, Mn and Zn are essential minerals in fish diets, especially important at early larval stages. The chemical speciation of these elements directly influences their uptake efficiency and metabolic utilization. In order to optimize the form of trace elements incorporated into larval feed, two experiments were conducted using two commercial fish species, European seabass (Dicentrarchus labrax) and Senegalese sole (Solea senegalensis), and two chemical forms (inorganic and glycinate chelates). Several fish performance parameters were measured, as well as bone status parameters to assess which form of mineral results in optimal fish biological performance. European seabass and Senegalese sole post-larvae were unresponsive (P > 0.05) to dietary treatments in terms of dry weight (DW), standard length (SL), relative growth rate (RGR) or feed conversion rates (FCR) when fed diets supplemented with chelated over inorganic trace minerals. This study suggests that replacing dietary inorganic mineral supplementation by their organic glycinate-chelated forms brings no beneficial effects on somatic growth and bone development in Senegalese sole and European seabass post-larvae fed high-quality commercial microdiets. Additionally, we show that mineral leaching from diets can be significant, but the use of chelated minerals can potentially mitigate this leaching phenomenon. Therefore, the selection of the dietary mineral form should take into account not only their economic value, but also their biological effect and environmental impact. Data generated in this trial provides new knowledge in trace mineral nutrition of early-stage marine fish.

Effect of Dietary Manganese and Zinc Levels on Growth and Bone Status of Senegalese Sole (Solea senegalensis) Post-Larvae.

Essential dietary trace elements, such as zinc (Zn) and manganese (Mn), critically influence a wide range of physiological, metabolic, and hormonal processes in fish larvae and post-larvae. Despite their importance for normal fish growth and skeletal development, trace mineral nutrition has not been extensively studied in the early stages of development of fish. Post-larvae of an emergent aquaculture species, Senegalese sole (Solea senegalensis), were the subject of this study in order to better understand the effects of diet supplementation of trace minerals upon fish larval development and performance. Sole post-larvae were fed a combination of organic Mn (45 and 90 mg kg-1 feed) and organic Zn (100 and 130 mg kg-1 feed) and survival, growth, mineral deposition rates, and vertebral bone status were assessed. Our results showed that although no significant effect was found on the growth performance of Senegalese sole post-larvae, Mn and Zn supplementation to a commercial microdiet for marine fish larvae at higher dietary levels (Mn at 90 mg kg-1 and Zn at 130 mg kg-1) improved larval survival, decreased the severity of vertebral malformations, and increased the deposition of Mn in bone.

Effect of Trace Minerals and B Vitamins on the Proliferation/Cytotoxicity and Mineralization of a Gilthead Seabream Bone-Derived Cell Line.

Trace minerals and vitamins are known modulators of bone metabolism, and dietary optimization of these components may improve skeletal development and reduce the occurrence of skeleton deformities in farmed fish. As for larval stages, mineral and water-soluble vitamin nutrition requirements are lacking in research efforts and knowledge is scarce. An in vitro cell system developed from gilthead seabream vertebra and capable of mineralization was used to assess the effect of B vitamins (thiamin and pyridoxine) and trace minerals (copper, manganese, and zinc in a sulfated and chelated form) on cell proliferation and extracellular matrix (ECM) mineralization. Dependent on dose, inhibition of cellular proliferation and/or cytotoxic effects was observed for all nutrients tested and LD50 values were determined: copper, 67.4-69.5 ppm; manganese, 20.9-29.8 ppm; zinc, 37.1-42.8 ppm in sulfated and chelated form respectively; thiamin, 6273 ppm; pyridoxine, 14226 ppm. ECM mineralization was enhanced by mineral (dose and form dependent) and vitamin (dose dependent) supplementation, at non-toxic concentrations below the determined LD50s. This in vitro work confirmed the mineralogenic action of trace minerals and water-soluble vitamins and provided valuable insights for subsequent in vivo nutritional trials.

Osteotoxicity of 3-methylcholanthrene in fish.

Many chemicals produced by human activities end up in the aquatic ecosystem causing adverse developmental and reproductive effects in aquatic organisms. There is evidence that some anthropogenic chemicals disturb bone formation and skeletal development but the lack of suitable in vitro and in vivo systems for testing has hindered the identification of underlying mechanisms of osteotoxicity. Several fish systems - an in vitro cell system to study extracellular matrix mineralization and in vivo systems to evaluate bone formation and skeletogenesis - were combined to collect data on the osteotoxic activity of 3-methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon. Anti-mineralogenic effects, increased incidence of skeletal deformities and reduced bone formation and regeneration were observed in zebrafish upon exposure to 3-MC. Pathway reporter array revealed the role of the aryl hydrocarbon receptor 2 (Ahr2) in the mechanisms underlying 3-MC osteotoxicity in mineralogenic cell lines. Analysis of gene expression in zebrafish larvae confirmed the role of Ahr2 in the signaling of 3-MC toxicity. It also indicated a possible complementary action of the pregnane X receptor (Pxr) in the regulation of genes involved in bone cell activity and differentiation but also in xenobiotic metabolism. Data reported here demonstrated the osteotoxicity of 3-MC but also confirmed the suitability of fish systems to gain insights into the toxic mechanisms of compounds affecting skeletal and bone formation.

Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectives.

BMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage- and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to down-regulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events.

Development and characterization of Xl1, a Xenopus laevis chondrocyte-like cell culture.

We describe the development and characterization of a new cell line, designated Xl1, derived from vertebra and long bones of Xenopus laevis. These cells can mineralize their extracellular matrix upon addition of an inorganic phosphate donor and vitamin C, as characterized by von Kossa staining. In addition they express genes such as matrix gla protein (mgp), alkaline phosphatase, type II collagen, and retinoic acid receptors, representing a valuable tool to analyze expression and regulation of Xenopus cartilage-associated genes. Continuous treatment with retinoic acid (RA) inhibited mineralization, alkaline phosphatase expression and its activity, suggesting that RA is a potential negative regulator of Xl1 cell differentiation. These cells are receptive to efficient transfer of DNA using conventional methods including calcium phosphate, liposome-mediated transfer or electroporation and were found to express basal levels of mgp at least 50-fold higher than the routinely used Xenopus A6 cell line, as seen by transcription assays with the distal X. laevis mgp promoter. Being the first amphibian cell line derived from bone tissue, the Xl1 culture provides an excellent in vitro tool for functional promoter studies, being suitable, among other uses, for identifying promoter elements mediating cartilage-expressed genes as shown here for mgp.