Zebrafish最新文献

The zebrafish (Danio rerio) has become an essential model in fields such as developmental biology, toxicology, genetics, and regenerative medicine due to its low cost, small size, transparent embryos, and genetic similarity to humans. Nowadays, this model is increasingly recognized as a valuable tool in other fields, including veterinary medicine and animal production research, particularly aquaculture, due to its unique characteristics that make it suitable for studying economically significant diseases affecting production species. However, unlike established models such as mice, zebrafish lack standardized protocols for housing, feeding, anesthesia, and sample collection, which affects study reproducibility. In addition, it is a common practice to use whole zebrafish larvae or juveniles for metagenomic studies rather than analyzing individual organs, despite the fact that gene expression can vary between organs. This approach complicates the attribution of findings to specific biological processes. To address this, various authors proposed protocols for sample collection in larvae, juveniles, and adult zebrafish; however, comprehensive studies encompassing nearly all fish organs are scarce. Understanding zebrafish anatomy and the technical requirements of the study is essential for accurate sample collection. Some challenges present during zebrafish dissection include the small size of the fish, the fragility of their organs, and the faster onset of autolysis and heterolysis after the death of the animal. Using magnifying lenses, microdissection tools, and conducting dissections on cold surfaces can help mitigate these issues. This article aims to improve sample collection for histopathological and genetic studies in adult zebrafish by establishing a comprehensive, organized, and systematic dissection protocol that accounts for the anatomical specifics of this experimental model.

Zebrafish (Danio rerio) is an important developmental biology and genetic animal model. Its educational use provides students with the opportunity for research-based learning, leading to the high-quality integration of theoretical knowledge with practical skills. In this study, we explored a didactic approach to teaching developmental biology using zebrafish. We describe the curriculum design and course workflow based on several pedagogical strategies and didactic tools that facilitate teaching effectiveness. Students' opinions confirm that knowledge is accessible during laboratories and lectures. Moreover, we observed an increasing number of students willing to perform research projects and conduct master's theses on topics related to developmental biology and model organisms in our department. Issues surrounding the use of animals in research and education are currently a subject of much discussion. Considerable attention is being paid to future animal-free alternatives. The course described here addresses this issue.

Zebrafish (Danio rerio) is a valuable model organism for studying developmental processes due to its external development and the optical clarity of its embryos and larvae. However, as development proceeds, zebrafish form increasingly opaque tissues that impede visualization of deep tissues and structures. Although tissue clearing methods have been used to facilitate imaging at these later stages, most of these methods have limited ability to clear dense tissues such as bone and cartilage, cause significant morphological distortion, and/or result in loss of fluorescent signal when used for imaging of fluorescent transgenes, dye-stained animals, or specimens generated using immunofluorescence or fluorescence in situ hybridization methods. Here, we report a novel imaging technique using a recently developed clearing reagent called LUCID that makes it possible to capture the complete cellular-resolution 3D structures of larval and juvenile zebrafish. We show that LUCID clears dense tissues such as pharyngeal cartilage in juvenile animals and even tooth bone in adults without causing either significant morphological distortion or significant loss of signal from transgene-driven fluorescent proteins, fluorescent nuclear DNA or actin staining dyes, or whole-mount in situ hybridization chain reaction fluorescence. Using this new approach, it is possible to perform complete high-resolution 3D imaging of whole fluorescently stained animals, even deep internal regions, providing a novel tool for elucidating the complex internal structures of developing zebrafish.

Zebrafish (Danio rerio) are often anesthetized by immersion in buffered tricaine methanesulfonate (MS-222). Although commonly utilized, immersion anesthesia presents a shortcoming of lethal asphyxiation with increased duration of exposures. A newer technique that circumvents this issue, known as flow anesthesia, has been adapted from larger aquatic species to zebrafish. Flow anesthesia improves safety by delivering oxygen-rich water along with the anesthetic across gill epithelium and allowing fish to be manipulated outside of water. Information on the construction of flow anesthesia apparatuses and parameters are sparse. The goal of this study was to create a flow anesthesia apparatus with materials commonly found within a research facility and to evaluate variable concentrations of MS-222 for anesthesia in zebrafish. Depth of anesthesia was monitored by quantifying respiratory rate and recording responses to physical stimulation. All concentrations of MS-222 evaluated (30-100 ppm) were successful at maintaining surgical anesthesia for up to 30 min. The anesthetic events were demonstrated to be safe, with an observed 97% survival rate. This work demonstrates refinements in zebrafish anesthesia and encourages future studies to evaluate MS-222 flow anesthesia for longer duration and evaluation of other commercially available anesthetics for efficacy in a flow anesthesia setup.

This study aimed to evaluate the viability of gametes in zebrafish (Danio rerio), at different rigor mortis stages. Viability assessments were conducted on oocytes at various developmental stages using LIVE/DEAD and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. For sperm evaluation, both kinetic (computer-assisted sperm analysis) and morphological assessments (Rose Bengal staining) were performed. Results demonstrated that rigor mortis progression significantly impacted oocyte viability during post-rigor stages, with the following viability rates: pre-rigor (70.43 ± 12.31%), fresh/control (46.43 ± 12.54%), post-rigor (27.62 ± 22.29%), and rigor mortis (comparable to fresh/control). Conversely, sperm kinetics exhibited nuanced responses to the rigor mortis stages, with specific parameters showing sensitivity, whereas the others remained relatively stable. Sperm motility was higher in the fresh/control (63.23 ± 19.03%) and pre-rigor (58.96 ± 14.38%) compared to the post-rigor group (3.34 ± 4.65%). This study highlights the significance of the pre-rigor for successful gamete collection and preservation. These findings provide valuable insights for conservation efforts and optimization of genetic resource management for endangered fish species. This study aimed to develop effective assistive reproductive techniques by elucidating the interplay between rigor mortis and gamete quality, contributing to the broader goals of species conservation and maintenance of genetic diversity in fish populations.

Zebrafish are widely used and increasingly adopted in developmental biology, disease modeling and tissue regeneration research. However, a freely available and user-friendly zebrafish husbandry management application has been lacking. Here, we developed an open-source fish management application, referred to as VMS-Fish (https://shinypath.wustl.edu/vmsfishdb/), to accurately track and manage zebrafish stocks. The VMS-Fish application includes default user logging and exclusive administrative privileges, making it a robust and error-free option for individual users, small laboratories and large consortia managers. With easy online and offline installation options, VMS-Fish provides an intuitive and freely available resource for zebrafish researchers worldwide.

Zebrafish is an ideal system to study the effect(s) of chemical, genetic, and environmental perturbations on development due to their high fecundity and fast growth. Recently, single-cell sequencing has emerged as a powerful tool to measure the effect of these perturbations at a whole-embryo scale. These types of experiments rely on the ability to isolate nuclei from a large number of individually barcoded zebrafish embryos in parallel. Here, we report a method for efficiently isolating high-quality nuclei from zebrafish embryos in a 96-well plate format by bead homogenization in a lysis buffer. Through head-to-head single-cell combinatorial indexing RNAseq experiments, we demonstrate that this method represents a substantial improvement over enzymatic dissociation and that it is compatible with a wide range of developmental stages.

The small characins represent a systematic puzzle in the Neotropical ichthyofauna as a result of independent miniaturization processes, adaptive convergence and lack of diagnostic characters for several genera. In order to diminish the taxonomic uncertainties and the evolutionary pathways in Hemigrammus, we carried out an integrative genetic analysis in the putatively widespread Hemigrammus marginatus Ellis, 1958 by combining cytogenetic and molecular data based on the mitochondrial Cytochrome C Oxidase subunit I (COI). Specimens of H. marginatus from the type locality in Itapicuru River basin and other two populations from coastal rivers in northeastern Brazil were analyzed and compared with the available data from other regions in South America. Conspicuous macro and microkaryotypic differences were detected between the samples from northeastern and southern Brazil (Upper Paraná River basin). Likewise, the DNA barcoding and species delimitation analyses recovered distinct Molecular Operational Taxonomical Units within H. marginatus. Therefore, the population from the type locality should be referred to as H. marginatus stricto sensu, representing a restricted characin taxon from coastal drainages (including the São Francisco River basin) along northeastern Brazil, while other populations of this small characin fish need to be taxonomically revised and managed as unique lineages.

Despite decades of research with laboratory rodents, the mechanisms underlying learning and memory, and their impairment, are still not fully understood. The zebrafish is a newcomer in this research area, but it has shown great promise. Food is often employed as a reinforcer in learning tasks with rodents. However, for zebrafish, food has been a problematic reinforcer. Controlling timing and localization of its delivery is difficult. What food types zebrafish prefer is also rarely studied? Here, we describe a novel food delivery hardware and procedure. The apparatus is simple, cheap to manufacture, and easy to employ. Using this new method, we compare how zebrafish respond to three food types, artemia nauplii, crushed tropical fish flakes, and small zebrafish pellets. In binary choice tasks, we show that zebrafish spend significantly more time near the artemia delivery cylinder, swim closer to, and visit this cylinder more frequently compared to food cylinders delivering flakes or pellets, while responses to these latter two cylinders do not differ from each other. We conclude that the newly developed method allows the quantification of food preference in zebrafish, and that it will lead to the identification of highly rewarding food types for learning studies in this species.

Photoperiod and temperature are two of the most powerful environmental cues that entrain circadian clocks. Being ectothermic, fish must keep their body temperature within a physiological range to optimize biological processes mainly applying behavioral strategies. Here, we developed a low-cost, automated system that allows to create a horizontal multiple-step thermal gradient and video record fish behavior for long-term periods. To validate the system, we assessed daily thermal preference and locomotor activity in the teleost Danio rerio. Our apparatus provides the opportunity to investigate the behavioral thermoregulation of captive fish, and our results highlight the importance of considering thermal preferences when designing husbandry protocols.