ERA-NET: BlueBio - Indentification of broodstock performance indicators and markers to boost the aquaculture of emerging fish species.

The BESTBROOD project builds upon the current state-of-the-art and advanced biotechnologies to significantly improve broodstock management methods and enhance the reproductive performance of four emerging fish species (Senegalese sole, greater amberjack, spotted wolffish, and lumpfish) with strong commercial potential in European aquaculture. BESTBROOD has developed genetic and immune markers, bioindicators, and gained insights into optimal rearing conditions. It has improved control over maturation cycles and tested hormonal therapies and artificial fertilization methods to reduce reliance on wild fish for egg production. To achieve these goals, BESTBROOD has formed a multidisciplinary consortium of seven partners with established expertise in fish breeding, immunology, nutrition, and reproductive biology. Despite challenges posed by the COVID-19 pandemic, the project partners have successfully initiated investigations as outlined in the original proposal and met the established milestones. Significant advances have been made in Senegalese Sole by IFAPA. This includes revealing the genetic structure of the genome and identifying sex-associated markers for the chromosomic sex determination region. Genetic estimates for heritability and correlations related to growth and shape traits have been provided. These findings are crucial for selecting families with desirable breeding characteristics and identifying molecular markers associated with specific features. Additionally, an evaluation of sperm production and quality parameters was conducted by CUPIMAR using sole breeders fed different diets. Hormonal treatments associated with thermocyclers and dopamine inhibitors were identified as critical modulators of the gonadotropin-releasing hormone-mediated response. The study also examined sperm-related parameters that could affect in vitro fertilization protocols. For Greater Amberjack, HCMR developed an F1 broodstock preselected and maintained in a sea cage at the ARGOSARONIKOS fish farm. These fish were sexed, tagged, and subjected to various hormonal preparations and combinations administered by injection or slow-released implants to study the enhancement of spermiation. Sperm and blood samples were collected, and ongoing research includes histological analysis of germ cell proliferation and apoptosis, along with the immune and biochemical profile of lumpfish MMCs at Aldo Moro University, Italy. NORD has optimized sperm cryopreservation protocols for captive Spotted Wolffish breeders. They've tracked individual males regularly for spermiation at the onset of the spawning season. Identification of immune-reproductive markers from monthly collected blood samples is currently under intensive investigation. Seasonal variation in the expression profiles of reproductive-related biomarkers is also being studied using the same samples. Additionally, nucleic acids have been extracted from total blood and barcoded for transcriptomics studies. Juveniles and early maturing fish have been identified and conditioned for sampling as part of a comparative study. On the nutritional front, NORD and Akvaplan-niva have designed a new artificial diet tailored to the specific requirements of spotted wolffish broodstock. Comparative studies among wolffish have demonstrated significantly higher egg survival in broodfish fed the experimental diet, although little effect was observed in sperm quality parameters. In the case of lumpfish, a collaborative effort involving NORD, Akvaplan-niva, and the Italian partner collected gametes from mature farmed and wild breeders to assess reproductive state and identify any dysfunctions in captivity. The study did not reveal apparent oogenesis impairment in hatchery-produced lumpfish under the current rearing conditions, as hatchery-produced females exhibited similar gonad reproductive maturation, oocyte size frequency distribution, and fecundity compared to wild fish. However, an altered lipid profile observed in blood plasma, liver, and gonads suggests the need for an optimized diet to enhance oocyte quality. The BESTBROOD consortium has produced two PhD, and 3 MSc students, four broodstock management manuals, one for each species, consolidating existing knowledge and project results. Workshops held in Spain, Greece, and Norway, participation in major conferences, and the publication of several scientific and popular science articles have further disseminated the project's findings. Dissemination efforts continue to ensure the broad reach of knowledge generated through BESTBROOD.

Significant efforts to implement the principles and objectives of RRI were conducted. BESTBROOD-project was designed to meet the needs of the aquaculture industry, with a focus on improving knowledge exchange, innovation, communication, and dissemination to maximize the project's impact. The project bridged the gap between research and commercial operations to attract new investors to aquaculture, particularly in rural areas where fish farms are typically located. The project involved close collaboration with commercial farms to ensure swift implementation of up-to-date results in practical operations. Findings were shared openly on a website, at trade shows, at public events, and through open-access scientific publications and social media. Mobility between project partners was encouraged, and guest researchers from various institutes were hosted during the project. M.Sc. and Ph.D. students were also involved in the project. The consortium complied with ethics codes expressed in local, national, and European legislation and regulations throughout the project. •Stakeholder engagement was a primary focus, with industry partners and various stakeholders actively involved in developing best practices for sustainable aquaculture. This included NGOs, government agencies, and civil society organizations. Manuals providing insights into broodstock maintenance and handling were made freely accessible to stakeholders under a Creative Commons License. •All research was authorized by respective institutions' Bioethics and Animal Welfare Committees, following ethical guidelines under European regulations. Personnel managing live animals obtained FELASA certification, and principal investigators had the required functions to design and supervise the research. •Gender equality plans and laws were followed in both public and private organizations. Gender considerations were incorporated into experimental designs, personnel hiring, and student selection. •Open access and sustainability were key aspects of the research. Publications were made accessible under Creative Commons or gold open access, and manuals were assigned DOIs and deposited on technology platforms. Genomic data were also made publicly available on dedicated servers. •Sustainability efforts included promoting recirculation systems to reduce water usage and environmental impact and supporting emerging species to reduce the impact on fisheries. Overall, the BESTBROOD project embedded the principles of Responsible Research and Innovation into its stakeholder engagement strategy to foster a collaborative and inclusive approach toward sustainable aquaculture. Next steps •Bring research findings into using existing tools available in the market. •Find financial support to develop outputs further. •Explore opportunities to scale up in different settings. •Stakeholder engagement (farmers) to adopt technology developed. •Engage with different stakeholders for impact.

Appropriate broodstock management is essential for supporting reproductive function and reliable and consistent production of gametes and high-quality eggs, critical for the rapid increase in juveniles supply and turn aquaculture into a profitable industry. In all species commercialized to date, this has been the first significant challenge to overcome and it is still so for many emerging species. The BESTBROOD project aims to build upon state-of-the-art from previous EU and National projects and, evolve biotechnologies that significantly improve broodstock management methods and enhance reproductive performance of selected emerging species (Senegalese sole, greater amberjack, spotted wolffish and lumpfish). All these species are in an early commercial stage, but all have showed suitability for farming, high economic value and market demand. To create the BEST BROODstocks the project plans to: 1) develop genetic markers and bioindicators; 2) create knowledge on the best rearing conditions; 3) improve the control of the maturation cycles; 4) test hormonal therapies and artificial fertilization methods and; 5) reduce dependence on wild fish for egg production. As a result, the BESTBROOD will lead to a more stable and predictable supply of high volumes of high-quality eggs for the production of juveniles to meet market demands for sustainability, and increase Europe’s aquaculture competitiveness. The BESTBROOD brings together a multi-disciplinary consortium of partners with established expertise in fish breeding and reproductive biology to develop, validate and up-scale to the industry new tools and technologies and ensure research efforts are targeted to overcome the main reproductive bottlenecks affecting the project’s focus species. The BESTBROOD Multi-Actor Approach is oriented to the needs and demands of the aquaculture industry, improving knowledge exchange, innovation, communication and dissemination and thus increasing the final impact of the projects.