The African turquoise killifish, Nothobranchius furzeri, has emerged as an important vertebrate model for aging research due to its naturally short lifespan and hallmarks of aging. However, these characteristics create challenges for colony management, such as high breeder turnover, rapid generational change, and the risk of inbreeding or unintended selection in closed laboratory populations. Since new individuals cannot be introduced from the wild or purchased from commercial laboratory animal breeders, maintaining genetically stable laboratory stocks requires carefully controlled breeding strategies. Here, we describe an innovative colony management approach termed dynamic population breeding (DPB) and evaluated its performance in two commonly used laboratory lines with different lifespans, GRZ-D and MZCS-08/122. DPB integrates several key principles: the maintenance of overlapping breeding age cohorts, harem-based breeding groups, continuous monitoring of clutch quality and quantity, and the controlled use of embryonic diapause for flexible embryo storage and synchronized hatching. Embryos from multiple breeding groups were pooled, stored in diapause stage II, and hatched at defined time points to generate new cohorts while avoiding sibling-only populations. Using this approach, we systematically analyzed reproductive performance, embryo quality, hatching success, and survival in laboratory populations. Fertilization rates and clutch sizes remained within stable ranges across the reproductive period, although an age-dependent decline in fertilization efficiency was observed in both lines. Quality control at the clutch level allowed early identification and exclusion of low-performing breeding groups. Importantly, implementation of DPB reduced variability between cohorts and improved early-life survival of offspring, particularly during the first weeks after hatching. Overall, DPB provides a practical framework for maintaining stable and robust killifish colonies while minimizing unintended selection and inbreeding. By integrating diapause biology with structured breeding management, this strategy enhances reproducibility and sustainability of N. furzeri populations used in aging research.
This study presents a comprehensive bibliometric analysis of zebrafish (Danio rerio) research published between 2000 and 2025, based on data retrieved from the Web of Science Core Collection and analyzed using Clarivate's InCites platform. A total of 74,675 records were examined to uncover trends in publication volume, geographical and institutional distribution, international collaborations, disciplinary coverage, and thematic evolution over time. The results indicate a steady growth in zebrafish-related publications, particularly between 2000 and 2021, followed by a relative plateau. The United States and China lead in research output, with China showing rapid growth over the last decade. Collaboration networks remain dominated by a limited number of high-capacity countries, while many others, particularly those with limited infrastructure, remain underrepresented. The Cooperation in Science and Technology Member Countries also showed a noticeable decline in publication numbers following a 2021 peak. Thematic keyword analysis revealed a clear shift from early developmental biology themes-such as hindbrain and retinal development-toward emerging topics such as regeneration, oxidative stress, and toxicology. However, the findings suggest that this thematic diversification has not yet translated into widespread interdisciplinary integration. Zebrafish research remains largely anchored within classical biological disciplines, despite its increasing relevance to fields such as neuroscience, environmental health, pharmacology, and biomedical engineering. This mismatch between thematic scope and interdisciplinary adoption represents a potentially missed opportunity-especially in addressing complex global challenges. Strengthening cross-disciplinary collaborations and promoting the adoption of zebrafish in innovative, technology-driven research contexts may help unlock the full strategic potential of this versatile model organism.
OBJECTIVE:The skin barrier function is essential for maintaining skin health. Consequently, it is crucial to evaluate the protective and restorative effects of topical medications and cosmetics on the skin barrier. However, methods that can directly assess the skin barrier function in zebrafish beyond their regenerative capabilities are limited. Therefore, we aimed to develop an in vivo, in situ, high-throughput screening model for assessing skin barrier function in zebrafish embryos. METHODS:Lactic acid was used to induce skin barrier damage and crystal violet was used for staining to observe skin barrier damage. Histopathology was assessed using H&E, Masson, and Victoria blue staining. Cellular apoptosis was evaluated using in situ acridine orange (AO) staining and flow cytometry with propidium iodide (PI) staining. RESULTS:A significant increase was noted in fluorescence in the trunk and tail regions of zebrafish embryos following lactic acid stimulation compared with those in the control group, whereas exposure to the positive control for 4 h significantly reduced the fluorescence area, showing 35%-45% inhibition. Histological sections revealed that lactic acid caused noticeable damage to the epidermis of zebrafish embryos, characterized by thinning of the epidermal layer and the presence of inflammatory cell infiltrates. An increase in apoptotic cells was observed through in situ AO and PI staining using flow cytometry, and RT-qPCR revealed elevated expression levels of apoptosis-related genes, including fas, bax, and caspase 3. Furthermore, six additional samples from various sources were screened using this model, which yielded reasonable outcomes for both positive and negative samples, indicating its potential application value in evaluating the skin barrier protective efficacy of drugs or compounds. CONCLUSION:Our study supports the use of the lactic-acid-induced zebrafish skin injury model as a reliable in vivo and in situ method for evaluating skin barrier function.
The zebrafish (Danio rerio) is a commonly used model organism for human diseases due to its genetic similarities with humans since over 80% of genes associated with human diseases are also found in the zebrafish. For most genetic experiments, it is essential to gather tissue for genotyping in a nonlethal procedure. A common method is removing a part of the tail fin from individual adult zebrafish with a scalpel. The fin usually regenerates within 14 days, leaving the fish without permanent damage. However, larval genotyping might offer significant advantages, such as saving resources and reducing the number of research animals and enabling experiments on zebrafish embryos with a specific genotype. Here, we show a cost-efficient method for fin-clipping zebrafish embryos as early as 48 h postfertilization (hpf). When done correctly, the fin will regrow quickly, being nearly completely restored at 120 hpf. While the amount of extracted DNA is low, it is still sufficient for commonly used methods of genotyping. Embryos with the desired genotype can then be used for phenotypic or functional analyses or be raised to adulthood.
Conducting longitudinal experiments on zebrafish disease models has the potential to deepen understanding of disease progression. Longitudinal experiments require rearing of zebrafish embryos/larvae in the laboratory for extended time periods to enable use of laboratory equipment for serial observation. We observed that in our laboratory, wild-type larvae did not survive past 14 days. As we were unable to identify a study that compared different factors that could affect survival of larvae raised in the laboratory, we aimed to define the most relevant parameters that impact larval survival and growth. We found that prolonged access to food had the greatest beneficial effect on survival, regardless of rearing density or rearing media. However, rearing density did impact the activity of 2.5-week-old larvae. Methylene blue is a frequent additive to zebrafish embryo-rearing medium. Methylene blue can impact cellular health, and methylene blue mitigated the decrease in motility of densely grown larvae. Methylene blue also promoted growth: larvae raised in methylene blue were significantly larger at 1 month than their counterparts raised without methylene blue. These data highlight factors that promote survival and growth of larval zebrafish raised in the laboratory for longitudinal studies.
Hawai'i and other island communities around the globe are experiencing high exposures to micro- and nanoplastics (MNPs). Widespread use of various types of plastics and the action of wave physics coupled with abundant silica creates a constant production of MNPs. The community health effects of exposure to MNPs are not yet fully understood. The aquatic vertebrate model zebrafish, Danio rerio, was utilized to evaluate the developmental and behavioral effects of nanoplastic (NP) exposure as well as visual confirmation of plastic accumulation after embryonic and larval exposure. Eye and yolk size, heart rate, angle of development, and early larval locomotion behavior were quantified after early NP exposure lasting 3 or 5 days. In this work, a digital laboratory environment is used as the context for research trainees spanning high school to graduate and medical school in Hawai'i to explore health outcomes of developmental NP exposures. Presence of MNPs in the early developmental environment led to changes in growth and behavior of exposed fish. Data analysis showed significant effects of exposures on changes in the rate of development and yolk consumption on day 2 and reduced body length and increased locomotion on day 6. Nanoplastic exposure also affected overall body length, but this effect was not significant. These data support the potential dangers of MNP exposure and demonstrate their persistent detrimental effects on development and behavior in young zebrafish. Accordingly, our work contributes toward shifting focus toward understanding longer impacts on health, growth, and behaviors and potentially the reduction of harm in developing vertebrates.
The EWSR1 (EWS RNA binding protein 1) was originally discovered in Ewing sarcoma, a pediatric bone cancer. In a previous study, we identified zebrafish ewsr1a, the homologue of human EWSR1. Using a zebrafish mutant for ewsr1a, we demonstrated that Ewsr1a promotes differentiation of chondrogenesis in Meckel's cartilage, a component of the facial skeletal elements. Furthermore, we demonstrated that Ewsr1a interacts with the transcription factor SRY-BOX transcription factor 9 (SOX9) and modulates the transcription of its target genes. In this study, the role of Ewsr1a was further investigated in axial skeletogenesis. The maternal zygotic (MZ) ewsr1a/ewsr1a zebrafish display curved spines derived from irregularly spaced intervertebral discs (IVDs) and aberrant differentiation of nucleus pulposus cells. Using an in vitro cell culture system, we further show that the N-terminus of Ewsr1a is required for the interaction with Sox9. In zebrafish, the Ewsr1a also interacts with a Sox9 target gene, specifically the first intron of col2a1a. Zebrafish with the MZ ewsr1a/ewsr1a genotype display an increased level of collagen type II protein in the notochord starting at 36 h post fertilization. We propose that Ewsr1a contributes to IVD formation by regulating the expression of col2a1a.
Live-bearing fishes (Poeciliidae) are increasingly used as laboratory models, but their utility is hampered by high rates of filial cannibalism, which reduces fry recovery and increases husbandry demands. Traditional strategies, such as mesh tank inserts (TIs), rely on fry actively seeking refuge and are often ineffective. Here, we present a novel tank design called Baby Catchers (BCs) that use water flow to passively and automatically separate newborn fry from their mothers, fitting seamlessly into standard recirculating rack systems. We tested the efficacy of BC tanks compared to open tanks (OT) and traditional mesh TIs using Poecilia parae , a live-bearing species that is notorious for intense filial cannibalism. In short-term trials, BC tanks yielded significantly higher initial fry counts on day 0 and day 2, while mesh inserts performed no better than OT. Over 10 days, negative binomial generalized linear models predicted substantial progressive fry loss in OT (∼60% decline) and inserts (∼68% decline), but no significant change in BC, consistent with high fry survival. These results provide the first quantitative evidence that automated water-flow separation of fry dramatically reduces filial cannibalism in live-bearing fishes. The BC design offers a scalable, low-cost, and humane method for fry recovery, reducing technician effort, minimizing animal stress, and lowering barriers for research and breeding programs involving live-bearing fishes (e.g., swordtails, mollies, Poeciliopsis , mosquitofish, halfbeaks, etc.).
Zebrafish (Danio rerio) have become a prominent model organism in behavioral neuroscience and pharmacological studies due to their genetic homology with humans and transparent physiology. Despite their increasing use, many laboratories still rely on manual or semi-automated tracking systems to assess behavior. There are programs commercially available, which are expensive and may not be possible to purchase for those researchers with a limited budget. Here, we introduce AquaMaze, a standalone, open-source desktop application built with PyQt5 and OpenCV, designed for real-time and post-hoc video-based behavioral tracking of zebrafish. AquaMaze computes locomotion metrics, including swim distance, speed, quadrant occupancy, and rest/activity patterns, from user-defined video inputs. The software provides intuitive visualization with heatmaps and exports detailed CSV reports, offering a scalable solution for high-throughput behavioral assays. We demonstrate the capabilities of AquaMaze using standard zebrafish locomotor activity paradigms such as the novel tank test, swim behavior test, novel object recognition, and Y-maze paradigms. In all of these tests, it is shown that AquaMaze gives reliable and consistent results. Hereby we provide a fast and cheap tool for the zebrafish behavioral experiments.
Oral gavage is ideal for studies requiring controlled dose delivery and timing, such as repeated dosing and longitudinal analysis, as shown in this study. An anesthesia-free gavage technique was used to administer daily estradiol doses to adult zebrafish for 40 days to evaluate reproductive toxicity (developmental and reproductive toxicity one stage). Results showed that neither estradiol administration nor the gavage method caused stress or injury, but both impacted reproductive capacity in a dose-dependent manner. Females exposed to the drug exhibited a reduction in gonadosomatic index (GSI) and changes in follicle maturation, while in males, only the number of cells in the testis was reduced. The authors have no interests to disclose.
Cryopreservation enables the long-term storage of viable biological material at ultra-low temperatures and forms the foundation for germplasm cryobanks that maintain valuable genetic lines of model organisms such as zebrafish (Danio rerio). However, reliable and reproducible cryopreservation protocols for fish germline stem cells remain difficult to develop, partly because key steps such as equilibration are often overlooked or assigned arbitrarily. Here, we optimized equilibration time for cryopreservation of zebrafish ovarian tissue. Ovarian fragments were equilibrated in 2 M methanol + 0.1 M glucose + 10% egg yolk for varying durations (15-120 min) before controlled slow cooling and storage in liquid nitrogen. Post-thaw viability was assessed using a Trypan Blue exclusion assay. A 60-min equilibration yielded the highest viability of ovarian cells in Experiment 1 (55.7 ± 1.7%), whereas a 30-min equilibration yielded the highest viability in Experiment 2 (75.9 ± 2.4%), but was not significantly different from the 60-min equilibration in Experiment 2 (75.6 ± 2.0%) (p = 0.998). Equilibration alone accounted for a 48.7% increase in post-thaw viability relative to controls. The framework presented here provides a reproducible method for determining species-specific equilibration optima and supports the development of effective germplasm cryobanks for both model and endangered fish species.
Chorions, also known as egg membranes, form quickly after fertilization and play a role in subsequent embryonic development in fish. While the enzymatic hardening of the chorion due to the release of cortical alveoli (CA) components by the exocytosis of CA has been well-demonstrated, the initiation mechanism of this process has remained unresolved. Knockout lines with the prss59.1 trypsin paralog gene exhibited abnormalities in chorion elevation. Prss59.1 has been shown to be expressed on the chorion's surface. Therefore, we hypothesized that a trypsin-like enzyme expressed on the chorion could trigger chorion elevation. In this study, we attempted to improve the effectiveness of Hank's solution at preventing chorion elevation. By adjusting the concentration of the solution's contents, we developed a modified Hank's solution that can stop chorion elevation almost completely. Using this solution, we demonstrated that trypsin can induce chorion elevation. These results support our hypothesis that a trypsin-like enzyme initiates chorion elevation. This assay method can be used for the further analysis of the chorion elevation mechanism.
The use of zebrafish (Danio rerio) larva as an experimental model has gained a lot of interest in epilepsy research due to its multiple advantages over mammalian models. The present study investigated the time-dependent expression of c-fos, an immediate early gene, and a marker of neuronal activation, following pentylenetetrazole (PTZ)-induced seizures in zebrafish larvae at 7-day post-fertilization . The larvae were exposed to 8 mM PTZ for a 15-min period, transferred to fish system water, and processed for c-Fos expression analysis at 15, 30, 45, 60, and 90 min of the start of the experiment. c-fos mRNA and c-Fos protein levels were quantified, and Pearson correlation analysis was conducted to assess their relationship. PTZ exposure induced seizure-like behavior and resulted in a dynamic temporal expression of c-Fos, with both mRNA and protein achieving peak levels at 45 min and declining by 90 min. This approach applied a fixed exposure duration and defined post-exposure time points, which allowed a more accurate temporal profiling. The observed peak expression at 45 min suggested an optimal window for evaluating c-Fos expression in the PTZ-induced seizures model of zebrafish larva. These findings provided a valuable reference for selecting experimental endpoints in zebrafish larva seizure studies and enhanced the reliability of c-Fos as a marker of neuronal activation.
The study investigates the role of cobalt chloride (CoCl2), a hypoxia-inducing agent, in promoting tissue regeneration using zebrafish as a model system. Caudal fins of adult zebrafish were amputated and transdermally exposed to 1% CoCl2. The extent of fin regeneration and the neovascularization process at the growth front were analyzed. CoCl2 exposure significantly enhanced regeneration compared with controls, with increased fin length and more prominent blood vessel sprouting and anastomosis. Molecular and proteomics analyses revealed an upregulation of angiogenic and pro-angiogenic factors, particularly Vascular Endothelial Growth Factor (VEGF). To verify the role of VEGF in CoCl2-mediated tissue regeneration, the amputated fins were exposed to inhibitors such as genistein and SU5416. These results suggest that CoCl2 promotes tissue regrowth and wound healing by stimulating angiogenesis. The findings highlight the therapeutic potential of CoCl2 in enhancing regeneration and wound repair through the HIF-1α/VEGF signaling pathway, with potential implications for treating ischemic wounds.
Zebrafish (Danio rerio) is recognized as a versatile model for hematopoietic studies due to its transparency, genetic similarity to humans, and ease of manipulation. This study describes a protocol for the ex vivo extraction and differentiation of macrophages from the renal medulla of zebrafish using transgenic lines and L929 cell-conditioned medium (LCCM) as an alternative to recombinant M-CSF. Adult TgMpegmCherry zebrafish were maintained under controlled conditions. Following euthanasia, renal medullary cells were isolated, treated with antibiotics, and cultured in either LCCM or recombinant human M-CSF. Macrophage differentiation was assessed using confocal microscopy (mCherry), flow cytometry, and functional phagocytosis assays. The protocol enabled efficient differentiation of progenitor cells into macrophages, with an average of 49.1% mCherry+ cells after 7 days in LCCM, outperforming recombinant M-CSF. Differentiated cells demonstrated strong phagocytic activity, confirming macrophage functionality. This method provides an accessible approach to obtaining ex vivo zebrafish-derived macrophages, enabling immunological and hematopoietic studies and allowing for functional comparisons with traditional murine macrophage protocols.
The transcription factor oligodendrocyte transcription factor 2 (Olig2) plays a central role in specifying motor neurons and oligodendrocytes during vertebrate neural development. While transgenic reporter lines such as TgBAC(olig2:EGFP) have been instrumental in visualizing olig2 expression, they fall short in directly reporting endogenous protein levels and may not fully recapitulate native gene regulation. To address these limitations, we generated a TgKI(olig2-mNeonGreen) zebrafish line using CRISPR/Cas9-mediated knock-in at the endogenous olig2 locus. The resulting Olig2-mNeonGreen fusion protein localizes specifically to the nucleus, enabling direct live imaging and accurate quantification of Olig2-expressing cells. We confirmed that the knock-in preserves endogenous mRNA expression and protein function, and that homozygous fish develop normally. As proof of concept, modulation of Sonic Hedgehog signaling altered Olig2-mNeonGreen+ cell numbers as expected, confirming the reporter's responsiveness to known upstream inputs. This TgKI(olig2-mNeonGreen) line offers a robust tool for studying neural progenitor dynamics in vivo.
The optokinetic response (OKR) is a widely used measure of visual and neurological function in zebrafish (Danio rerio). In this study, we employed a simple and cost-effective OKR assay system for adult zebrafish utilizing a mobile phone and a stereomicroscope. The setup was validated by comparing control fish with those exposed to light-induced retinal degeneration (LIRD), demonstrating significantly impaired OKR responses. This custom-made setup offers valuable applications in visual neuroscience, disease modeling, and drug discovery.
DaniocellDesktop is a cross-platform interactive desktop application designed to facilitate reanalysis of a previously published 462,243-cell single-cell RNAseq dataset that profiled cell-type-specific gene expression across the first 5 days of wild-type zebrafish development. DaniocellDesktop enables custom redefinition of cell populations, identification of differentially expressed genes, and generation of several types of publication-ready plots to show gene expression patterns, gene co-expression patterns, and gene expression over time within these previously published data without requiring any specific programming knowledge. This software is available from https://daniocell.nichd.nih.gov/desktop/.
In zebrafish housing systems with water recirculation, a daily renewal of at least 10% of the total water volume is generally recommended to preserve optimal water quality. Such aquatic housing systems often rely on reverse osmosis (RO) water. RO production uses large amounts of running water, a high proportion of which is then discharged into waste. A key parameter for water quality control is nitrate, the final transformation product of the nitrogen cycle in water. Here, we used this parameter to test whether daily water renewal could be reduced in our zebrafish housing system without impact on water quality. For 18 months, divided into four periods, we progressively reduced the rate of water renewal from 10% to 6%. We show that nitrate levels are not impacted when renewal rates remain above 8%, making it possible to significantly save on water in zebrafish breeding systems.
