Consistent with the broad approach taken by the project, the training objectives were to:
1. Deliver integrated, multidisciplinary training to 15 ESRs in skeleton anomalies in aquaculture, including research skills at anatomical, histological, and molecular levels.
2. Deliver an integrated, multidisciplinary training in skeletal Biology research using innovative biotechnological approaches including the use of model fish.
3. Understanding of the socio-economic impact of their research, notably on new venture creation.
The ITN has delivered 5 Summer Schools dealing with fish breeding, manipulation, animal experimentation ethics, stock maintenance, recognition, recording and analysis of skeletal deformities in fish, statistical analysis, data mining, cell culture and generation of skeletal fish cell lines.
Conditions affecting skeletal health are of great concern in aquaculture, both for product quality (value) and of animal welfare. To make fish farming more sustainable by avoiding fish oils and fish meal in their diets, nutritional factors as well as overfeeding are of particular interest. Trials have been conducted to investigate the effects of dietary microminerals and vitamins in gilthead seabream (Sparus Aurata) larvae and juveniles. Meagre (Argyrosomus regius) larvae were fed diets supplemented with long-chain polyunsaturated fatty acids. The requirement of phosphorus in the diets for Atlantic salmon (Salmo salar) was investigated focusing on the vertebral column), as well as levels of carbon dioxide. Anticipating the risks due to climate change, different temperatures were used during rearing of meagre larvae. Gilthead seabream were grown at different densities or in different volumes. All these analyses have been conducted from a multidisciplinary approach, including anatomical, biochemical, genetic and immunohistochemical analysis of the skeleton.
Small model fish like medaka (Oryzias latipes) or zebrafish (Danio rerio), increasingly used in developmental biology, physiology and pathology in vertebrates, are pivotal in this project by linking the biomedical researchers to the aquaculture experts. First, drawing from information on human pathologies, the function of genes involved in osteoarthritis patients and early-onset osteoporotic was investigated by generating mutant cell lines for these genes or zebrafish lines deficient in the corresponding homologous genes to understand their role in skeletal development and homeostasis, identifying the WNT signaling pathway crucial for bone development and phenotype. In addition, dietary components such as oxidants and antioxidants were studied, with implications to both farmed and model fish and even humans. Micronutrients and probiotics were also investigated using bone-derived cell cultures and transgenic zebrafish, while the effect of dietary phosphate levels on vertebral body fusions were also investigated in wild type and mutant zebrafish. Transgenic and mutant zebrafish lines were generated and analyzed to study the mechanisms involved in aberrant notochordal sheath formation in zebrafish.
Extracts from marine organisms (invertebrates and microalgae) were identified that present beneficial osteoactive properties using transgenic zebrafish, selected extracts were also tested in gilthead seabream, potentially leading to the identification of new leads for prevention and treatment. Medaka and zebrafish larvae were also reared at different densities or different volumes, pointing at rearing densities that determine skeletal phenotypes. Mechanical stimulation was tested using zebrafish in a swim tunnel to understand the link between mechanical stimuli and bone homeostasis.
Innovative methods and applications were implemented throughout the entire project, such as micro-CT scans, Synchrotron X-ray tomographic microscopy, histology methods, genetic mutagenesis and transgenesis methods. Furthermore, an artificial intelligence (AI)-based software was developed to automatically quantify bone formation and mineralization in developing zebrafish larvae, medaka and the aquaculture species gilthead seabream. Finally, sophisticated economic analysis methods were applied for the first time to evaluate the socio-economic impact of skeletal deformities in farmed fish. Consumer and producer preferences, behaviors and responses within the European aquaculture marketplace, as well as cost and risk estimation.
Taken together, the work of the 15 ESRs that were hired on the project resulted in 39 scientific publications already published (24 still submitted or in preparation) and 129 presentations at international Conferences, while a wider audience was targeted by organizing 12 local workshops and several media presentations.
