Advancing Zebrafish Husbandry: Takeaways From the 2024 Husbandry Workshop and Husbandry Summit

Introduction

A Zebrafish Husbandry Workshop and Summit took place on February 19–20, during the World Aquaculture Society Meeting 2024 in San Antonio, TX. The 1-day workshop encompassed session topics ranging from zebrafish disease detection and control, various husbandry methods, and technologies. The workshop was followed by the first Zebrafish Husbandry Summit, a panel discussion between several workshop speakers and the audience. Approximately 50 participants attended the event (Fig. 1). Looking back on 2 decades of impactful workshops, the discussion highlighted anticipated areas of focus for future husbandry efforts.

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FIG. 1.

2024 Husbandry Summit Panel Discussion. February 19–20, World Aquaculture Society Meeting 2024, San Antonio, TX. Approximately 50 attendants participated in the event discussing zebrafish husbandry conventions, best practices, and health monitoring issues for biomedical research facilities.

Currently, the available literature on husbandry data remains insufficient. Many published studies focus on singular parameters, failing to address real-world scenarios adequately, where countless husbandry parameters act in concert on the animal population. Frequently, changes in one environmental parameter can have consequential impacts on several others that zebrafish are exposed to. Hence, a major topic of discussions was how husbandry information could be better obtained, integrated and curated, and disseminated in the zebrafish research community. There was consensus that consistent husbandry among laboratories supports research reproducibility and scientific rigor.

Summit Participants

Kathleen Hartmann kicked off the session by sharing that the aquaculture industry has a key concern in the US, namely that new regulations are becoming more and more “European Centric”, which is something the zebrafish community also needs to be aware of. It was discussed that this is particularly urgent for the exchange of scientific materials and fish lines domestically and across international borders. A key problem encountered by many researchers in the US is the extremely diverse regulatory landscape for animal exports to all the different destination countries. The U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS), Veterinary Services (VS) (USDA/APHIS/VS) provides several helpful tools online that support research animal exchanges, for example the International Regulations (iRegs) for the export of animals, which includes aquatic species (https://www.aphis.usda.gov/animal-product-export). ¹ This updated repository of country-by-country information helps both senders and recipients to better understand relevant regulations and the needed paperwork. Where the system falls short unfortunately, is that zebrafish and other biomedical aquatic models are grouped under aquaculture, rather than purpose-bred animals from closed research facilities with health monitoring programs. Because laboratory animal exports are controlled through APHIS, rather than for example DHHS or the Department of the Interior, destination countries typically require inspection standards that have been developed for aquaculture or agricultural facilities but pose significant impediments for the exchange of laboratory animals. Indeed, exchange of laboratory rodents and Drosophila was facilitated in the past only after these species were categorized as pests, relieving them from significant regulatory burden for international shipping. Thus, communication and agreement are urgently needed across international agencies and countries that regulate laboratory animal welfare and disease control. The zebrafish community urgently needs to engage and lobby with USDA/APHIS to ask for help developing different standards for scientific animal exchanges with other countries.”

George Sanders and Kristen Kwan suggested that for facility line importations, both better communication of health status information about incoming fish, as well as more harmonized pathogen monitoring guidelines are needed across domestic and international facilities in the research community. Facilitating communication and collaboration between facilities, implementing standard health reports or summaries, and prioritizing adequate quarantine procedures can streamline, and indeed accelerate and facilitate, the sharing of fish across facilities while minimizing pathogen risks.

Steve Watts and his team have developed and tested an open-source, laboratory-formulated and purified standard reference diet (SRD) for adult and juvenile zebrafish. ² Even in cases where minor adjustments to ingredients may be necessary, it offers significant qualitative advantages over commercial, closed-formula diets, which may undergo undisclosed ingredient modifications. The rodent scientific community achieved a standard reference diet in less than 20 years and a first draft for a zebrafish SRD was achieved in about 10 years. As a next milestone, it is now urgent to develop community acceptance and demand. Several laboratories have already successfully tested this proposed standard reference diet.^3–6 To achieve community acceptance, Dr. Watts emphasized the importance of setting up a consortium with one or more centers to oversee testing, diet development progress, and achieve the goal of a widely adopted SRD for zebrafish and, eventually, other aquatic model organisms. ⁷ The consortium will invite and encourage more laboratories to use and test the SRD and publish their findings. Lastly, an educational approach to informing the community and associated partners needs to be developed and defined (journals, organizations, granting agencies, etc.) and the support from the American Society of Nutrition, World Aquaculture Society, National Institutes of Health (NIH), International Zebrafish Society (IZFS, izfs.org), and/or National Animal Nutrition Program needs to be enlisted to support these efforts. To this end Dr. Watts suggested that the Zebrafish Husbandry Association (ZHA) distributes information on the SRD, which can be promoted through ZHA’s monthly webinars, Danio Zoom Friday chats, and conference flyers.

Mike Kent stressed that there is a lack of training for veterinarians that are involved in aquatic animal care. Over the course of 2 decades, a number of pathogens have been identified that infect zebrafish populations. The diagnosis of these infections is often difficult, and histochemical approaches are limiting in their sensitivity and timing. Advancements in health screening, particularly molecular tools using environmental samples, offer promising opportunities for better understanding and managing fish colonies. ⁸ This approach is more sensitive, proactive, and representative of colony prevalence, while being less invasive on animals, requiring fewer animals to be sacrificed for diagnostic purposes.

To this end it is also urgently necessary to have more veterinarians trained in aquatic pathogen diagnostics. There is an urgent need for buy-in from funding providers, institutional administrations, and PIs to support the increase of the pool of veterinarians that can oversee aquatic facilities in their roles as institutional attending veterinarians. Importantly, funding is needed to support candidates and Institutions that provide such training.

Zoltan Varga pointed out that the zebrafish research community has always been known for its spirit of collaboration and mutual support among laboratories. Collaborative opportunities at a broader scale can be pursued if organizations like the IZFS, Zebrafish Husbandry Association (ZHA, zhaonline.org), and the Zebrafish Information Network (ZFIN, zfin.org) come together, ensuring diverse perspectives are incorporated. This collaboration could involve joint efforts such as hosting webinars, maintaining a shared wiki platform, and extending invitations to speak at each other’s conferences. In addition, enhancing the quality of fish-related publications can be achieved by providing authors with standardized descriptive language or husbandry “ontologies” and comprehensive parameter lists that build on the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. ⁹ Data can be included in publications either through platforms like protocols.io, wiki-style databases such as at ZHA and ZFIN, or through the inclusion of more detailed husbandry materials, methods, and data in the supplemental section of publications.

The community must review the concepts of standardization and harmonization and commit to supporting the distinct definitions of engineering, performance, and experience standards. These are supported by “must,” “should,” and “may” guidance which expressly require a collaborative approach among Institutional Officials, Compliance Officers, Institutional Animal Care and Use Committees (IACUC)/Home Offices or equivalent, Veterinarians, Animal Facility Staff, and Researchers. This approach ensures that individual research goals are accomplished and at the same time optimal animal welfare is maintained, avoiding unnecessary distress, discomfort, or pain, thus, achieving rigorous experimentation and reproducible research outcomes.

Steve Ekker highlighted that good husbandry practices must be based in and be consistent with sound scientific practice. To this end, even more peer-reviewed and published husbandry papers are needed to establish a scientific community consensus and avoid single publications driving temporary trends. There is a big challenge; however, more reviewers are needed for peer reviewing manuscripts. This is volunteer work and a “culture of expectation of review” is needed. It is desirable that more experienced reviewers train and include graduate students and post-docs in this process to increase the pool of reviewers.

He suggested a community wide survey such as for example in the 2017 Health and Husbandry issue of the Zebrafish journal to help determine what the key husbandry issues and questions are in the zebrafish research community.

Petronella Kettunen called for a critical reassessment of existing publications and the production of comprehensive review articles to identify and address gaps in husbandry knowledge. She accentuated this problem by summarizing her review of housing density studies.

In her own study on housing density, Dr. Kettunen measured cortisol levels and observed that fish housed at a 1 fish per liter stocking density exhibited higher cortisol levels compared with those housed at higher densities, even up to 16 fish per liter.^10,11 However, she also pointed out that it is essential to note some limitations of the study, which lasted only nine weeks and was conducted in static fish tanks and not on recirculating water filtration systems. Thus, Dr. Kettunen’s conclusion that 16 fish per liter only necessitated increased feed inputs could not determine potential consequences of increased bio load on recirculating system dynamics, which may lead to water quality issues and necessitate more rigorous filtration system maintenance protocols. Moreover, fish husbandry requires adequate space for growth and development. Overcrowded tanks are associated with heightened risks of developmental deformities, pathogen transmission, and size disparities among fish populations requiring more research to find an upper limit.

Summit Discussion

A discussion ensued about the future implications for publications. We discussed and proposed a more collaborative approach for the future that will also enhance the rigor and reproducibility of scientific studies: These efforts are particularly crucial given the upcoming development of a new edition of “The Guide for the Care and Use of Laboratory Animals” by the National Research Council (USA). ¹² It is imperative to ensure that the forthcoming guide is built upon a robust scientific foundation, free from the limitations of trendy, incomplete, or outdated references. For example, when papers only report mean standard length and weight, the extremes of fish size can be overlooked, which are essential for animal group dynamics and hierarchies, potentially leading to inconsistent and non-reproducible research results. Because many assumptions about stocking density from previous publications have not been held up by other studies, a more comprehensive list of environmental husbandry parameters that influence animal well-being should be also reported for example animal age. Principal investigators should include husbandry protocols and outcomes in manuscripts for publication. Therefore, they need a core competency in husbandry and need to include enough information in the methods section or in the Supplementary Data S1. Technicians and managers have the required competency based on their day-to-day work in the facility. They are in the best position to review, write up, and make available to PIs husbandry materials and methods, from the time studies were conducted, including the specific fish strains, and importantly if there had been any deviations from normal environmental parameters. Thus, PIs and animal facility technicians or managers should touch base at end of study and evaluate whether husbandry conditions were normal—according to the planned protocol. Disclosing significant deviations from normal or expected husbandry conditions will further enhance replicability of studies. In addition to this proposed practice, the zebrafish community could approach scientific journals to better prepare reviewers about zebrafish husbandry standards in the future. For example, a standard checklist for reviewers could be prepared for materials and methods sections that capture key husbandry parameters and permits inclusion of deviations such as suggested above.

Collaboration between researchers and facility managers is paramount for acquiring accurate husbandry data. Facility staff, often equipped with advanced degrees in aquaculture or marine science, possess the expertise necessary to maintain optimal fish health and growth. Recognizing facility staff as collaborators rather than impediments to research can significantly enhance publication quality.

A further possibility to enhance husbandry protocol availability is to establish or expand existing centralized protocol repositories. Possibilities include the Zebrafish Information Network (ZFIN, zfin.org) which can be directly updated by users, protocols.io, or a future wiki-style repository hosted by ZHA. However, it would be good to avoid redundancies and avoid loss of effort or focus by multiple parallel sites. Harmonization of fish husbandry can be further enhanced by improving the quality of zebrafish research publications by including thorough, yet concise descriptions of husbandry Standard Operating Procedures and extrinsic data monitoring, either by referring review of husbandry practices to platforms like protocols.io that can store husbandry protocols with associated DOI identifiers, or through the supplemental data sections. To this end, a goal was identified that helps eliminate jargon and unnecessary synonyms thus supporting FAIR (Findable, Accessible, Interoperable, and Reusable) publication standards by developing and providing authors with controlled vocabularies, descriptors, and comprehensive husbandry parameter lists that can be founded on the ARRIVE guidelines. Thus, a yet to be developed standard checklist for reviewers (materials and methods inclusions), veterinarians and facility managers should be developed. This list could be developed in parallel to a husbandry ontology or “HO,” comparable to already existing genetic (GO) or morphological ontologies in various databases (ZFIN.org). Standardizing language and information about husbandry terminology—not necessarily standardization of procedures—might be a highly desirable community tool for a harmonized husbandry approach in the future. Therefore, the zebrafish community should establish (or expand existing) protocol repositories and include model organism databases (e.g., ZFIN) that already have frameworks for dealing with nomenclature and ontologies. In addition, to foster community engagement, the ZHA plans to launch a 10-part monthly webinar series with the IZFS focusing on Reporting and Reproducibility. ZHA aims to invite experts to discuss essential reporting practices and the impact of various variables on research outcomes. An email has already been sent to the community. The goal is to propose a tiered list of essential husbandry parameters to this journal and Journal of the American Association for Laboratory Animal Science (JAALAS), along with a standardized form for documenting these parameters in aquatic research, prioritizing essential variables to ensure even partial data collection is beneficial.

In summary, addressing these key areas identified during the summit can substantially improve the quality and reliability of zebrafish husbandry research, fostering a more collaborative and informed scientific community. This includes the urgent need for more comprehensive and higher-quality literature on zebrafish husbandry, enhanced communication and collaboration between researchers, veterinarians, and facility staff, and widespread adoption of a standardized (open-source) reference diet.