Tenth Aquatic Models of Human Disease Conference 2022 Workshop Report: Aquatics Nutrition and Reference Diet Development.

Progress in biomedical research requires rigorous studies and reproducible outcomes. However, despite recent achievements, standard reference diets (SRDs) for aquatic model organisms, vital for supporting scientific rigor and reproducibility, are yet to be adopted. At this workshop, we presented findings from a 7-month diet test study, tightly coordinated and conducted across three aquatic research facilities: Zebrafish International Resource Center (ZIRC), Kent and Sharpton laboratories (Oregon State University), and Xiphophorus Genetic Stock Center (XGSC, Texas State University). We compared the impact of two commercial diets and a suggested zebrafish SRD on general fish husbandry, microbiome composition, and health in three fish species (zebrafish, Xiphophorus, and Medaka), and three zebrafish wild-type strains. We reported outcomes, gathered community feedback, and addressed the aquatic research community's need for SRD development. Discussions underscored the influence of diet on aquatic research variability, emphasizing the need for SRDs to control cross-experiment and cross-laboratory reproducibility. Species-specific reference diets are essential for model organism health and consistent research outcomes.

Modeling of cryopreservation pathway operation at an aquatic biomedical stock center for zebrafish.

Aquatic biomedical model organisms play a substantial role in advancing our understanding of human health, however, comparably little work has been directed towards developing dependable, high-throughput storage programs for valuable genetic resources. The Zebrafish International Resource Center (ZIRC) has developed a standardized cryopreservation pathway and stored thousands of genetic lines in their repository for use by the biomedical research community. This has yet to be replicated in other facilities, and an overall repository-level pathway has never been analyzed for aquatic species. To encourage repository development for other biomedical models and to improve the ZIRC storage process and system, this study used discrete-event simulation modeling to systematically analyze the cryopreservation pathway for efficiency, and to identify improvements. The models reflected "real-world" working conditions and were used to simulate key outputs, such as production capacity over time (throughput) and steps in the process that limit production (bottlenecks). With these models, recommendations were identified to eliminate waiting times and increase efficiency. These included following proper husbandry protocols because male quality significantly affected production time, and the use of part-time operators to assist with steps that had longer Waiting Times (i.e., time samples spent in a queue) to increase production capacity. Simulation process modeling is a powerful tool that can improve the operations of existing repositories. It can also support repository development at other biomedical stock centers, and at other facilities devoted to aquatic species such as research, conservation, and aquaculture production hatcheries.

Assessment of various standard fish diets on gut microbiome of platyfish Xiphophorus maculatus.

Diet is an external factor that affects the physiological baseline of research animals. It can shape gut microbiome, which can impact the host. As a result, dietary variation can challenge experimental reproducibility and data integration across studies when not appropriately considered. To control for diet-induced variation, reference diets have been developed for common biomedical models. However, such reference diets have not yet been developed for nontraditional model organisms, such as Xiphophorus species. In this study, we compared two diets designed for zebrafish, a commercial zebrafish diet (Gemma and GEM), and a proposed zebrafish reference diet developed by the Watts laboratory at the University of Alabama at Birmingham (WAT) to the Xiphophorus Genetic Stock Center custom diet (CON) to evaluate the influence of diet on the Xiphophorus gut microbiome. Xiphophorus maculatus were fed the three diets from 2 to 6 months of age. Feces were collected and the gut microbiome was assessed using 16S rRNA sequencing every month. We observed substantial diet-driven variation in the gut microbiome. Our results indicate that diets developed specifically for zebrafish can affect the gut microbiome composition and may not be optimal for Xiphophorus.

Disentangling the link between zebrafish diet, gut microbiome succession, and Mycobacterium chelonae infection.

BACKGROUND: Despite the long-established importance of zebrafish (Danio rerio) as a model organism and their increasing use in microbiome-targeted studies, relatively little is known about how husbandry practices involving diet impact the zebrafish gut microbiome. Given the microbiome's important role in mediating host physiology and the potential for diet to drive variation in microbiome composition, we sought to clarify how three different dietary formulations that are commonly used in zebrafish facilities impact the gut microbiome. We compared the composition of gut microbiomes in approximately 60 AB line adult (129- and 214-day-old) zebrafish fed each diet throughout their lifespan. RESULTS: Our analysis finds that diet has a substantial impact on the composition of the gut microbiome in adult fish, and that diet also impacts the developmental variation in the gut microbiome. We further evaluated how 214-day-old fish microbiome compositions respond to exposure of a common laboratory pathogen, Mycobacterium chelonae, and whether these responses differ as a function of diet. Our analysis finds that diet determines the manner in which the zebrafish gut microbiome responds to M. chelonae exposure, especially for moderate and low abundance taxa. Moreover, histopathological analysis finds that male fish fed different diets are differentially infected by M. chelonae. CONCLUSIONS: Overall, our results indicate that diet drives the successional development of the gut microbiome as well as its sensitivity to exogenous exposure. Consequently, investigators should carefully consider the role of diet in their microbiome zebrafish investigations, especially when integrating results across studies that vary by diet.

A Simple Method for Inducing Masculinization of Zebrafish Stocks Using 17α-Methyltestosterone.

Severely skewed sex ratios in zebrafish stocks can pose significant hurdles for line propagation and sperm cryopreservation. To overcome female-biased sex ratios in stocks derived from imported sperm samples, the Zebrafish International Resource Center has implemented routine supplementation of larval food with 17α-methyltestosterone to skew gonadal sex differentiation toward masculinization. Resulting stocks averaged 80% males.

Is catalase an effective additive to alleviate oxidative stress during cryopreservation of zebrafish sperm at the repository level?

The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration for: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA or methanol, membrane integrity was significantly decreased (70-75%) compared to controls. With catalase, sperm cells maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.

A Systematic Analysis of Metal and Metalloid Concentrations in Eight Zebrafish Recirculating Water Systems.

Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism. Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study, eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating water systems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our results help to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metal concentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish.

Cryopreservation of Pooled Sperm Samples.

Cryopreservation of sperm cells is currently the most efficient tool for managing large and small collections of valuable genetic resources. Cryopreservation minimizes expenses for animal and facility maintenance such as personnel, water, power, and space. It extends the time offspring can be produced from individual organisms, reduces the need to maintain live populations, provides flexibility for planning future experiments and research projects, and can prevent catastrophic loss of irreplaceable research lines. In this chapter, we present the sperm collection, dilution, cryopreservation, thawing, and in vitro fertilization procedures used at the Zebrafish International Resource Center (ZIRC).

Temporal and Concentration Effects of Methanol on Cryopreservation of Zebrafish (Danio rerio) Sperm.

We evaluated the cryoprotective effects of methanol on zebrafish sperm at different concentrations, exposure times, and stages during cryopreservation. Samples were collected by crushing of dissected testes or abdominal stripping. After exposure to 0%, 2%, 5%, 8%, and 10% methanol for 0-11 min, fresh sperm (1 × 106 cells/mL) did not show changes in plasma membrane integrity (measured by flow cytometer), but cell size changes (light scatter) were observed after exposure to 8% or 10%. After exposure for 0-60 min, fresh sperm (1 × 108 cells/mL) did not show significant changes in survival or membrane integrity. Sperm cryopreserved in 5%, 8%, and 10% methanol showed high post-thaw survival, in 5% and 8% showed high post-thaw motility, and in 5% showed highest post-thaw membrane integrity compared to other concentrations between 0% and 10%. Within 0-60 min after thawing, no significant differences in cell survival and membrane integrity were found for any concentration (p ≥ 0.269). Comparison of 5% and 8% methanol for dissected testes (n = 20) revealed no difference in post-thaw motility, membrane integrity, cell survival, fertilization, or hatching, embryo viability; for stripped sperm (n = 10), no differences were observed in post-thaw membrane integrity, fertilization, and embryo viability, however, higher motility and survival were detected in 5% than in 8% methanol. Thus, a concentration of 5% methanol seems most suitable for cryopreserving zebrafish sperm based on post-thaw survival and motility.

Workshop Report: Zebrafish and Other Fish Models-Description of Extrinsic Environmental Factors for Rigorous Experiments and Reproducible Results.

Current standards for husbandry and maintenance of zebrafish and other aquatic species in the laboratory are diverse, and are subject to laboratory performance, engineering, and practice standards (the Guide), institutional interpretation, national animal welfare laws, and cultural differences. Consequently, it is difficult, and probably not advantageous, to establish a single standard in view of the hardy nature of zebrafish and the diversity of research requirements it is used to address. Based on their natural habitat, zebrafish can thrive in a variety of environmental conditions, which is a specific advantage for working with this laboratory organism. However, it also makes reporting and reproducibility difficult, because variations in the husbandry and environmental conditions, including the environmental conditions before and during experiments, are often underreported in the scientific literature. This lack of consistency presents a potential problem for research reproducibility. To begin addressing this emerging scientific gap, the National Institutes of Health's (NIH) Office of Research Infrastructure Programs (ORIP), Division of Construction and Instruments (DCI), hosted a workshop in late 2017, entitled "Zebrafish and Other Aquatic Models: Reporting of Environmental Husbandry Conditions for Rigorous Experiments and Reproducible Results," that was attended by ∼60 participants. The objectives of the workshop were to bring together a diverse group of stakeholders-researchers, facility managers, veterinarians, journal editors, commercial vendors, and others to (1) review current husbandry and environmental management practices for the care of zebrafish and other aquatic organisms in the laboratory and to (2) propose a process for the development of a minimal set of environmental parameters that should be reported in publications to ensure rigor and robustness of experiments and reproducible outcomes. The participants also discussed how these recommendations, as an initial step, might be collected, disseminated, implemented, and improved upon after future iteration.

Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation.

Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

10th European Zebrafish Meeting 2017, Budapest: Husbandry Workshop Summary.

A husbandry workshop on July 3, 2017, at the 10th European Zebrafish Meeting in Budapest, Hungary (July 3-July 7, 2017), focused on the standardization, optimization, and streamlining of fish facility procedures. Standardization can be achieved for example by developing novel software and hardware tools, such as a fish facility database for husbandry and environmental facility management (Zebrabase, Oltova), or a hand-held, air-pressurized fish feeder for consistent food distribution (Blowfish, Argenton). Streamlining is achieved when work hours are reduced, as with the standardized fish feeder, or by limiting the number and types of fish diets and observing the effect on animal welfare and performance (Barton). Testing the characteristics of new fish diets and observing whether they produce better experimental outcomes (Certal) optimizes diets and improves fish productivity. Collectively, the workshop presentations emphasized how consistency and harmonization of husbandry procedures within and across aquatic facilities yield reproducible scientific outcomes.

Fluorescent light exposure incites acute and prolonged immune responses in zebrafish (Danio rerio) skin.

Artificial light produces an emission spectrum that is considerably different than the solar spectrum. Artificial light has been shown to affect various behavior and physiological processes in vertebrates. However, there exists a paucity of data regarding the molecular genetic effects of artificial light exposure. Previous studies showed that one of the commonly used fluorescent light source (FL; 4100K or "cool white") can affect signaling pathways related to maintenance of circadian rhythm, cell cycle progression, chromosome segregation, and DNA repair/recombination in the skin of male Xiphophorus maculatus. These observations raise questions concerning the kinetics of the FL induced gene expression response, and which biological functions become modulated at various times after light exposure. To address these questions, we exposed zebrafish to 4100K FL and utilized RNA-Seq to assess gene expression changes in skin at various times (1 to 12h) after FL exposure. We found 4100K FL incites a robust early (1-2h) transcriptional response, followed by a more protracted late response (i.e., 4-12h). The early transcriptional response involves genes associated with cell migration/infiltration and cell proliferation as part of an overall increase in immune function and inflammation. The protracted late transcriptional response occurs within gene sets predicted to maintain and perpetuate the inflammatory response, as well as suppression of lipid, xenobiotic, and melatonin metabolism.

Report of Workshop on Euthanasia for Zebrafish-A Matter of Welfare and Science.

The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.

A Simple and Inexpensive Microinjection System for Zebrafish Embryos and Larvae.

Microinjection is a widely used technique to inject defined volumes and concentrations of substances and explore their physiological function in vivo. The technique has been particularly successful with zebrafish embryos; however, the injection equipment can be relatively expensive and therefore available only to well-funded laboratories. In this study, a simple, cheap, easy-to-assemble, and easy-to-use setup with a straightforward, accurate, and efficacious calibration method is introduced. The accuracy of this calibration method was tested by comparing with the results of calibration methods that are currently used in high-cost systems. Injection success with this low-cost system was verified based on the presence of injected dyes in zebrafish embryos, the absence of any significant morphological and behavioral differences between 3,4,-dichloroaniline-treated and untreated embryos, and larval viability.

An Affordable Intraperitoneal Injection Setup for Juvenile and Adult Zebrafish.

Intraperitoneal (IP) injections are an effective and reproducible route of drug administration. However, current IP equipment can be either costly, inaccurate, or unsafe for zebrafish. We describe a simple, low-cost IP setup, which can be easily assembled from inexpensive and readily available parts, and which provides a safe, reproducible, and accurate IP-injection method for experimenters.

Biosecurity and Health Monitoring at the Zebrafish International Resource Center.

The Zebrafish International Resource Center (ZIRC) is a repository and distribution center for mutant, transgenic, and wild-type zebrafish. In recent years annual imports of new zebrafish lines to ZIRC have increased tremendously. In addition, after 15 years of research, we have identified some of the most virulent pathogens affecting zebrafish that should be avoided in large production facilities, such as ZIRC. Therefore, while importing a high volume of new lines we prioritize safeguarding the health of our in-house fish colony. Here, we describe the biosecurity and health-monitoring program implemented at ZIRC. This strategy was designed to prevent introduction of new zebrafish pathogens, minimize pathogens already present in the facility, and ensure a healthy zebrafish colony for in-house uses and shipment to customers.