Intraocular pressure (IOP) monitoring is a critical aspect of diagnosis, and management of ocular diseases. Therefore, a tractable vertebrate model capable of reliable IOP characterization is highly desirable. The present study aims to perform a preliminary investigation of a handheld rebound tonometer to stably measure IOP and further characterize baseline IOP values in the zebrafish model. The IOP in both eyes (oculus dexter, OD, and oculus sinister, OS) of wild-type zebrafish was measured using the iCare PRO rebound tonometer at the same time points on testing days for 18 months. The long-term IOP values of male zebrafish (3-21 months) were range from 21.96 ± 0.99 to 23.39 ± 0.32 mm Hg (OD) and 22.10 ± 0.93 to 23.38 ± 0.26 mm Hg (OS), while insignificant difference was noted among testing days (p > 0.05). Similarly, insignificant difference was observed in female zebrafish with IOP values range from 22.10 ± 1.12 to 23.19 ± 0.72 mm Hg (OD) and 22.30 ± 1.00 to 23.15 ± 0.36 mm Hg (OS) (p > 0.05). A comprehensive evaluation of IOP profiles confirmed no significant differences based on gender, time point, and between eyes. These findings indicated that our iCare tonometer-based experimental methods provide high measurement and time-point consistency, confirming their long-term reproducibility and reliability in measuring IOP in the zebrafish.
Zebrafish are a powerful, accessible vertebrate model for teaching core biology concepts. BioEYES is a well-established K-12 outreach program that uses inquiry-based classroom investigations to leverage these strengths. However, its traditional model relies on dedicated personnel and substantial infrastructure, limiting sustainability at individual institutions. Here, we describe UCI BioEYEs, a graduate student-led adaptation that maintains BioEYES' educational strengths while reducing logistical and financial barriers. In partnership with middle school science teachers at a dual-language (English-Spanish) campus, UCI BioEYEs delivers week-long modules built around live zebrafish embryos and larvae. Graduate students lead hands-on investigations in cell biology, development, environmental effects on embryogenesis, and Mendelian inheritance. Activities use standard classroom equipment (e.g., school-grade stereoscopes, smartphone-based imaging), enabling repeated use without specialized infrastructure. Over four years, UCI BioEYEs reached over 1,100 middle school students. Teachers cited curriculum integration, sustained engagement with live vertebrate models, graduate student role models, and inclusive bilingual design as key to long-term adoption. Graduate instructors reported major gains in teaching confidence and science communication skills. Thus, a graduate student-led structure sustains zebrafish-based outreach benefiting K-12 learners while supporting graduate training. UCI BioEYEs offers a transferable framework for scalable, inclusive science education. All curricular materials are provided here to support adoption by other institutions.
Zebrafish are an emerging vertebrate model for traumatic brain injury (TBI) research, but current larval injury paradigms, especially weight-drop methods, can be difficult to standardize. Here, we describe the Zebrafish Pneumatic Injury Device (ZePID), a compact pneumatic piston system that delivers controlled, reproducible pressure pulses to induce TBI in larval zebrafish. ZePID showed a strong linear relationship between input and delivered pressure and reduced variability relative to weight-drop approaches. In 6-days postfertilization larvae, a 150 psi injury increased maximum swimming speed and total distance traveled, consistent with seizure-like hyperactivity after TBI.
In many research studies, the handling and procedures performed can generate stress and require the use of anesthesia or sedation. In this sense, plant-based extracts named essential oils have emerged as potentially effective and environmentally safe anesthetic/sedative options. Here, we sought to evaluate the essential oil of Varronia curassavica (VCEO) as an anesthetic/sedative ingredient. Here, the anesthetic and sedative capabilities of the VCEO using adult zebrafish (Danio rerio). The anesthetic induction was performed under four different concentrations (100, 200, 400, and 600 mg L-1), followed by evaluation of recovery time; furthermore, a 3 h long exposure was performed for sedative testing, where fish were exposed for 3 h at three different concentrations (50, 80, and 100 mg L-1) under a superpopulation condition, such as those faced during transport. To assess stress, we collected whole-body cortisol from each individual after each exposure. The concentrations of 400 and 600 mg L-1 induced anesthesia (S4) but promoted excessive recovery times; on the contrary, they reduced the cortisol levels of the fish compared with the positive and negative controls. In the sedative testing (3 h long exposure), the animals exposed to 50 mg L-1 remained in S2 stage from the first 10 min, and those exposed to 80 mg L-1 after 20 min. The 100 mg L-1 induced stage S3a. There were no significant changes in cortisol levels observed at these concentrations. Limitations include measurement of whole-body cortisol at a single endpoint and the consistently slower induction and recovery with VCEO compared with a reference anesthetic MS-222, a property that constrains its practical use and warrants further dose-response, time-course, and recovery-optimization studies. Therefore, VCEO appears to be a promising anesthetic and sedative alternative, effective and plant-based, without increasing the physiological stress of the animals.
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.).
Brazil has emerged as a significant contributor to the global zebrafish (Danio rerio) research community, yet a comprehensive analysis of its national output, collaboration networks, and thematic focus has been lacking. This study provides a systematic bibliometric analysis of 801 Brazilian-corresponding articles published from 2020 to 2025, representing ∼2.7% of global production. Our findings reveal a marked concentration of scientific output, with the South and Southeast regions contributing ∼65% of national publications, led by the states of Rio Grande do Sul (24.8%) and São Paulo (20.4%). Despite this geographic disparity, a robust and integrative national collaboration network connects all regions, with an average 34% of publications involving interstate co-authorship. International partnerships are substantial (30.4% of output), led by the Southeast and South regions, and feature distinct geographic profiles, including the Central-West's links with South Asia. The field is characterized by a strong applied focus, with Toxicology (34% of studies), Pharmacology (18.3%), and Neuroscience (15%) dominating the research landscape, aligning with the predominant use of the adult zebrafish model (64%). Publication is heavily concentrated in environmental and toxicology journals, with nearly half of all output published by Elsevier (47%). These results map a dynamic, collaborative, and thematically focused national research community that is resilient yet faces persistent regional inequalities. The study establishes a critical baseline for understanding the structure and drivers of Brazilian science in a globally relevant model organism.
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.
