Danio rerio

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Zebrafish

Common Name: Zebrafish

Scientific Name: Danio rerio

Domain: Eukaryotic

Biosafety Level: BSL1/BSL2, Varies depending on research context

Benefits

Zebrafish, a small and robust organism, presents numerous advantages as a model organism:

• Small size (2.5-4 cm long) and robust organism
• In its larval stages it is transparent and as it matures to an adult it develops stripes that
  run along the length of the body and look blue in color. Transparency provides optical
  clarity, in vivo study of molecular mechanisms by allowing for live imaging.
• Cheaper to maintain than mice.
• External fertilization, fast growth rate and production of multiple offsprings which allows
  study over multiple generations.
• Zebrafish have similar genetic structure to humans and share 70% of the genes. In
  addition to genomic similarity, the presence of conserved organs and organ systems
  between human and zebrafish contributes to development of a number of successful
  models of human diseases.
• Ability to create gene knockouts using CRISPR/Cas9 and TALEN techniques.
• Ability to create tissue specific transgenic animals. Recent improvement of the
  Tol2-based transgenic system in zebrafish has allowed the control of gene expression in
  a spatiotemporal manner by coupling with regulatory elements such as GAL4/UAS or
  Cre/LoxP.
• Fully sequenced genome. Its genome is 1,505,581,940 base pairs in length and contains
  26,247 protein-coding genes.

Applications

Zebrafish serve as a model for various research areas such as development, genetics, immunity, behavior, physiology, and nutrition research:

• Developmental biology research - Zebrafish is a key model organism for the study of
  vertebrate developmental biology. Cell division, migration, differentiation and cell fate,
  neurological development,etc is studied using zebrafish models. The strengths of this
  model system lie in its external, visually accessible development, ease of experimental
  manipulation, and common genetic underpinnings with other vertebrates including
  humans.
• Human disease modeling - Zebrafish is used as a successful model for metabolic
  diseases such as Duchenne muscular dystrophy, human melanoma, acute lymphoblastic
  leukemia, polycystic kidney disease, nephronophthisis, acute kidney injury, Parkinson’s
  disease, Huntington’s disease, Alzheimer disease, myocardial infarction.
• Cancer Research -Another cell transplant technique applied to zebrafish is
  xenotransplantation of tumor cells into embryonic or juvenile fish. Mammalian tumor cells
  can survive, divide, metastasize, and induce angiogenesis in the zebrafish embryo
  before the immune system develops or in immunosuppressed juvenile fish. This
  technique can be combined with transgenic cell labeling to visualize the interaction of
  tumor cells with their microenvironment in vivo. In addition, mutagenic or chemical
  screens can be devised to identify genes or chemicals that modify tumor metastasis or
  angiogenesis
• Testing Chemical Compounds in Zebrafish Embryos - The zebrafish embryo represents
  a convenient model for studying interactions between chemical compounds and animal
  physiology. The value of the zebrafish model is derived from its small size, chemical
  permeability, ease of observation, and physiologic similarity to other vertebrates
  including humans. The small size of zebrafish embryos allows them to be arrayed in 96-
  or 384-well plates. Chemical compounds can then be added to the wells and
  experimental endpoints measured after the desired incubation period. This process can
  be mechanized so that large libraries of compounds can be screened efficiently. The
  aqueous environment of zebrafish is advantageous, because chemical compounds can
  be directly added to the fish's water and freely diffuse into the developing embryo
• Random mutagenesis strategies in zebrafish have generated thousands of distinct
  mutant phenotypes, many of which are consistent with human genetic disorders.Along
  with study of these diseases, rescue by gene therapy and other pharmacology agents to
  alleviate symptoms are screened and tested.

Maintenance

• Zebrafish is an ectothermic, tropical species native to South Asia. Facilities utilizing
  zebrafish as a model should filter municipal water with sediment and carbon pre-filters,
  reverse osmosis, and deionization to create high-quality base water. Adding small
  amounts of high-quality sea salts produces "fish-ready" culture water. Monitoring and
  maintaining daily temperature, pH, and conductivity within ranges of 26–29°C, 7–8, and
  200–3000 micro-Siemens respectively is essential. Weekly measurements and
  maintenance should include alkalinity, hardness, carbon dioxide, and dissolved oxygen,
  aiming for 50–75 ppm, 100–200 ppm, 0–15 ppm, and 6–8 ppm respectively. Nitrogenous
  waste, harmful even at low concentrations, necessitates weekly monitoring of ammonia,
  nitrite, and nitrate. Ammonia and nitrite levels should be as close to zero ppm as
  possible, while nitrate levels below 50 ppm are acceptable. Biological filters employ
  ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria to transform toxic
  ammonia into less harmful nitrate, with nitrate reduced via routine water exchanges.
  Before making adjustments, cross-verification of water quality parameters using two
  methods is advised. Adjustments should be gradual to mitigate stress on research fish
  and biological filters. These conditions help maintain environment similar to natural
  habitat of zebrafish while providing ideal conditions for research.
• Zebrafish exhibit a well-documented tolerance for a wide temperature range, with lower
  and higher temperatures posing no conflict with animal welfare standards. Lower
  temperatures lead to slower embryo/larvae growth, while adults show no known
  behavioral differences within the proposed temperature range.Temperature influences
  both water chemistry and animal physiology. Oxygen solubility in water decreases as
  temperature rises, but recirculating systems negate the need for additional oxygenation
  due to constant water movement. For consistency and standardized husbandry, housing
  water temperature typically ranges from 24°C to 29°C. Emphasizing the avoidance of
  sudden temperature changes is crucial.Developmental stages are often referenced in
  hours post-fertilization (hpf) based on a temperature of 28.5°C.
• Modern laboratory conditions often provide zebrafish with a static dark-light (D-L) cycle,
  usually 10 hours of darkness and 14 hours of light. Gradual changes in light intensity
  mimicking dawn and dusk can be included optionally. Although different settings, such as
  a 12:12 D-L cycle, don't directly impact animal welfare, they may influence physiological
  processes like spawning frequency and breeding success.
• A combination of live and processed dry feeds generally enhances growth, generation
  time, and reproductive performance, indicative of well-being. Dry feed diets are
  considered nutritionally complete, while live feeds and associated fish prey-capture
  behavior offer enrichment. Feeding frequency should align with fish developmental
  stages. Larvae, which grow rapidly, benefit from two to three daily feedings of live feed if
  possible. Feeding can be manual or automated. Automated feeding robots can dispense
  both dry and live feeds in defined quantities, though adjusting for varying animal counts
  requires precise programming. When using automation, daily visual inspections of fish
  tanks by staff remain vital.
• Females can lay hundreds of eggs per mating, but allowing at least a week's recovery
  between spawnings is recommended for sufficient regeneration and maturation of new
  ova. Adults not designated for embryo production should be kept in mixed-sex groups to
  facilitate natural breeding behavior and prevent egg-associated oviduct inflammation
  ("egg-bound" females). Managing fish colony genetics is crucial to minimize inbreeding's
  negative effects and prevent loss of genetic diversity over time. Due to space and cost
  constraints, distinct fish strains and lines are often kept in smaller groups. To counter
  inbreeding depression, each new generation should result from an outcross, with sibling
  matings reserved for necessary situations.
• Rearing embryos and larvae holds significance in zebrafish facilities. Embryos are
  maintained in embryo medium, often containing 0.5 mg/l methylene blue to combat
  fungal infections. Stocking densities of up to 100 embryos per 35 ml are used in 9 cm
  diameter Petri dishes under a 28.5 ± 0.5℃ D-L cycle. Employing sterilized water is
  essential, with removal of extraneous materials like feces, scales, and unfertilized eggs
  necessary prior to transferring the eggs. Regular medium changes and removal of
  non-viable embryos, dead eggs, and chorion remains are advised.
• A clean environment is fundamental for upholding high animal health and welfare
  standards. To achieve this, careful consideration must be given to prevent
  cross-contamination during routine husbandry procedures. Equipment in contact with
  fish, such as nets and mating boxes, should be dedicated to specific systems and periodically sanitized. While chemical sanitization is viable, rinsing adequately to prevent
  water contamination is crucial. A simpler and safer cleaning method involves heating to
  at least 60℃ for a minimum of one hour. Isolating equipment used for quarantine from
  main facility equipment is essential.