Zebrafish最新文献

The zebrafish is a valuable model organism that is widely used in studies of vertebrate development. In the laboratory, zebrafish embryonic development is normally carried out at 28.5°C. In this study, we sought to determine whether it was possible to modify the speed of embryonic development through the use of short- and long-term variations in incubation temperature. After incubation at 20°C-32°C, most early-stage embryos survived to the epiboly stage, whereas more than half of the embryos died at <20°C or >32°C. The rate of development differed between embryos incubated at the lowest (18°C) and highest (34°C) temperatures: a difference of 60 min was observed at the 2-cell stage and 290 min at the 1k-cell stage. When blastulae that had developed at 28°C were transferred to a temperature lower than 18°C for one or more hours, they developed normally after being returned to the original 28°C. Analyses using green fluorescent protein-buckyball mRNA and in situ hybridization against vasa mRNA showed that primordial germ cells increase under low-temperature culture; this response may be of use for studies involving heterochronic germ cell transplantation. Our study shows that embryonic developmental speed can be slowed, which will be of value for performing time-consuming, complicated, and delicate microsurgical operations.

The zebrafish is becoming an increasingly popular research animal around the world. Its welfare is affected by an array of environmental factors, such as food access and water quality. Holding density is an important welfare aspect, not least due to its interaction with other housing conditions. Despite the extensive use of zebrafish in research, little is known of how densities affect its welfare. In this systematic review, we have performed a large literature search, compiled, and evaluated all publications regarding zebrafish holding density. We have analyzed how density effects growth, reproduction, and stress response, including behavior, water quality, and pathogenic outbreaks in young and adult fish. Our review shows that the holding densities tested vary largely depending on the research focus, for example, body growth or behavior. In fact, research indicates that future recommendations on holding density could depend on which welfare aspects are considered. Overall, there is a need for more studies investigating the interactive effects of density on welfare indicators, such as reproduction coupled with stress response. We stress the necessity of including holding density in universal housing guidelines and reporting information on holding conditions of larvae and adults when publishing zebrafish work.

Cytogenetic studies in marine fish are scarce, and elemental cytogenetic information is available for not >2% of the species. Traditional cytogenetic methods require living individuals for their application, making the analysis of marine ichthyofauna very difficult. In this study, we present a detailed new protocol to obtain cytogenetic preparations from marine fish, through access to specimens in postmortem condition. The application of this protocol made it possible to access elemental cytogenetic information (diploid number) in six native species of the South Pacific Ocean, representative of five orders. In this way, we provide a new low-cost methodological tool for focused or large-scale cytogenetic analysis, both in economically important, native, or threatened species.

Hyperglycemia, a primary symptom in diabetes mellitus, is associated with difficulties in wound healing and regeneration. This condition is due to the length of the inflammatory phase and free radicals. Furthermore, there is evidence that molecular pathogenesis is involved in impaired wound healing in diabetics. As an animal model, zebrafish have many shared orthologous genes with human that are involved in protein regulation of wound healing and regeneration. Little is known about natural drugs that may be used to treat complications of wound healing in diabetes. Propolis, however, is known to consist of various organic compounds such as phenols and flavonoids with antioxidant and anti-inflammatory activities. This research aims to study propolis' effect on caudal fin regeneration and relative expression of several genes belonging to Hedgehog, bone morphogenetic protein (BMP), and Wnt signaling hyperglycemic (HG) zebrafish. GC-MS analysis and antioxidant activity testing were performed on ethanolic extract of propolis (EEP). Caudal fin regeneration was analyzed using ImageJ; blood glucose levels were measured; and relative gene expression analysis of shha, igf2a, bmp2b, and col1a2 was performed by the real-time polymerase chain reaction method with the β-actin housekeeping gene. Impairment of caudal fin regeneration in zebrafish hyperglycemia was characterized by a low percentage of regeneration and decreased relative gene expression. EEP at 15 ppm could increase the percentage of caudal fin regeneration and the expression of shha, igf2a, bmp2b, and col1a2. Based on the results, it appears that phenols and flavonoids from the EEP can improve the caudal fin regeneration of HG zebrafish.

Angiosarcoma is a clinically aggressive tumor with a high rate of mortality. It can arise in vascular or lymphatic tissues, involve any part of the body, and aggressively spread locally or metastasize. Angiosarcomas spontaneously develop in the tp53 deleted (tp53^del/del) zebrafish mutant. However, established protocols for tumor dissection and transplantation of single cell suspensions of angiosarcoma tumors result in inferior implantation rates. To resolve these complications, we developed a new tumor grafting technique for engraftment of angiosarcoma and similar tumors in zebrafish, which maintains the tumor microenvironment and has superior rates of engraftment.

Short-lived turquoise killifish (Nothobranchius furzeri) becomes a popular model species for neuroscience. However, the effects of sex and rearing conditions on behavior and brain monoamines in N. furzeri are unknown. In this article, we study the body mass, behavior in the novel tank diving test, levels of noradrenaline (NA), dopamine (DA), serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) in the brain of 108 day-old N. furzeri males and females reared in small (one male and one or two females in 4-L tanks) and large (four males and four females in 25-L tanks) groups. Males were heavier and had a lower NA level in the brain compared with females. The behavior of males and females did not differ in the novel tank diving test. Their DA, 5-HT, DOPAC, and 5-HIAA levels in the brain did not differ too. Males from small groups spent more time near the tank's bottom. Rearing in small groups reduced the DA level in the female brain and the DOPAC level in female and male brains. However, group size did not affect body mass, 5-HT and 5-HIAA levels in the brain. Thus, group size is important for behavior and neuroscience studies of N. furzeri.

Over the past decade, the zebrafish has been increasingly employed in biomedical neuroscience research due to its numerous evolutionarily conserved features with mammals. Its simple brain and the several molecular tools available for this species make the zebrafish an appealing model to study mechanisms of complex brain functions, including learning and memory. Most learning paradigms developed for the zebrafish have employed visual stimuli as the associative cue. Spontaneous color preference is a potential confound in such studies. It has been analyzed in zebrafish using colored objects, but with conflicting results. It has rarely been explored with colored light, despite the increasing use of computer-generated visual stimuli. Here, we employ a light emitting diode (RGB-system) light-based color preference task in the plus-maze. In two independent experiments, zebrafish were tested in a four-choice or dual-choice condition by using four different-colored lights (red, green, blue and yellow). Our results suggest a light preference hierarchy that depends on context, since yellow was preferred over green in the four-choice condition whereas blue was preferred over all other colors in the two-choice condition. These results are useful for future color-light-based learning experiments in zebrafish.

Teleost fish such as Danio rerio (zebrafish) have been successfully used in biomedical research since decades. Genetically altered fish lines obtained by state-of-the-art genetic technologies are serving as well-known model organisms. In Europe, following Directive 2010/63/EU, generation, breeding, and husbandry of new genetically altered lines of laboratory animals require governmental state approval in case pain, suffering, distress, or long-lasting harm to the offspring derived by breeding of these lines cannot be excluded. The identification and assessment of pain, distress, or harm, according to a severity classification of mild, moderate, severe, or humane endpoint, became a new challenging task for all scientists, animal technicians, and veterinarians for daily work with laboratory zebrafish. In this study, we describe the performance of the assessment of welfare parameters of selected pathologic phenotypes and abnormalities frequently found in laboratory fish facilities based on veterinary, biological, and physiological aspects by using a dedicated score sheet. In a colony of zebrafish, we evaluated the frequency of genotype-independent abnormalities observed within 3 years. We give examples for severity classification and measures once an abnormality has been identified according to the 3Rs (Replacement, Reduction and Refinement).

The ability to carry out high-resolution, high-magnification optical imaging of living animals is one of the most attractive features of the zebrafish as a model organism. However, increasing amounts of pigmentation as development proceeds and difficulties in maintaining sustained immobilization of healthy, living animals remain challenges for live imaging. Chemical treatments can be used to suppress pigment formation and movement, but these treatments can lead to developmental defects. Genetic mutants can also be used to eliminate pigment formation and immobilize animals, but maintaining these mutants in lines carrying other combinations of transgenes and mutants is difficult and laborious. In this study, we show that CRISPR duplex guide ribonucleoproteins (dgRNPs) targeting the slc45a2 (albino) and chrna1 (nic1) genes can be used to efficiently suppress pigment formation in and immobilize F0 injected animals. CRISPR dgRNPs can be used to generate pigment-free, immobile zebrafish embryos and larvae in any transgenic and/or mutant-carrying background, greatly facilitating high-resolution imaging and analysis of the many transgenic and mutant lines available in the zebrafish.