Introduction and Basics to Zebrafish
Omkar Kumar Kuwar1, Kousik Maparu1, Shamsher Singh1, * 1 Neuropharmacology Division, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab 142001, India
* Corresponding author Shamsher Singh: Neuropharmacology Division, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab 142001, India; Tel: +91-9779980588; E-mail: shamshersinghbajwa@gmail.com INTRODUCTION
Zebrafish: Zebrafish (Danio rerio), a freshwater teleost formerly used as an experimental model from the past few decades, is found in lakes and rivers [1]. They belong to the Minnow family Cyprindiae of Cypriniformes, native to South Asia [2]. Zebrafish derived its name from the five prominent, uniformly pigmented horizontal stripes that adorn its body, evoking the pattern of zebra-like stripes that extend longitudinally from the caudal fin and are distinctive characteristics of the species [3]. Zebrafish exhibit a fusiform (spindle-shaped) body plane, with lateral compression and a superiorly directed mouth, adapted for surface feeding and optimal maneuverability in its aquatic environment. Often referred to as tropical fish, this well-known aquarium fish is regularly offered for sale under the trade name Zebra Danio, also located in ponds. Female Zebrafish have silver stripes instead of gold and a wider white belly than males, a blue stripe separated by gold stripes in a torpedo design [4]. It takes three months to reach sexual maturity at 28.5°C and are considered adults in an ideal environment, Zebrafish commonly survive for 3-5 years [5, 6].
Nowadays, Zebrafish have evolved as a novel research model in recent decades as the species has many benefits such as being inexpensive to purchase, easy to maintain, handle, and breeding in the common laboratory. They are incredibly robust creatures. A single Zebrafish often yields between 20 and 200 offspring per breeding event. Other key advantages include: they are transparent during early developmental stages and have rapid development ability. Specifically, Zebrafish embryos develop from a single cell to a larva with a formed head, tail, and beating heart within 24 hours. In between 72 hours, the central nervous system is fully developed and becomes functional, and the fins become motile. By 5 days post-fertilization (dpf), they achieve full viability and are capable of independent swimming and foraging [7]. This rapid development and high reproductive output
make Zebrafish an ideal model organism for both genetic and developmental biology research, particularly in high-throughput genetic studies. The basic organ system of Zebrafish is represented in Fig. (1.1).
Fig. (1.1)) Vital organs of Zebrafish.
ZEBRAFISH MAINTENANCE
As they are considered aquatic animals, they require good maintenance and care as per CPCSEA guidelines [8]. Various types of rotating systems constantly filter and oxygenate the water in which Zebrafish are kept to provide a marine environment that is favourable for their well-being. Fish waste products and extra foods are also filtered by the drainage system. Typically, the water and room temperature are kept between 26-28.5 °C and 12 hr light: 12 hr dark cycle. Un-purified water is passed through another tank after passing through a UV disinfection filter followed by a biological and active carbon absorption filter, a 50-micron canister filter, and finally 120-micron filter pad. To make dechlorinated water, it should be kept for at least 48 hours in a chamber and water should be circulated by a pump to keep it warm and speed up the dechlorination process [9].
The water pH-controller system should be monitored every day and pH should be kept around 6.8 to 7.5. NaHCO3 can be used to raise pH levels while required. It is important to clean fish tanks regularly. While cleaning the tank, stop the water supply and tilt the tank back to release the remaining water, and then carefully remove the tank from the system. Dirt accumulation and the growth of algae/fungus may be observed at the side walls and bottom of the tank. After filling the tank with dechlorinated water, place the baffle inside a clean tank. Using a fish net, carefully transfer fish into the tank and then shut the cover. Slide the cleaned tank into the system gently, then turn on the water supply. Before reusing the net, spray it with seventy percent ethanol, again rinse it with water, and allow it to dry. Take off the baffle from the unclean tank then spray with the same on both sides. Before reusing, give the tank and baffles a thorough rinse under tap water and let them completely dry. The filters need to be routinely checked and replaced with the circulation system to operate properly. Ensure that the water supply in each fish tank is neat and clean and that the filters are replaced regularly. Before replacing the 120-micron filter pad, make sure to reposition the filter toward the direction of water flow and utilize it completely before replacing it with a new one. Every week, the canister filter needs to be replaced. Use a wrench or your hands to spin the filter unit anticlockwise to remove it and then replace the canister filter and place a towel underneath to avoid or absorb water spills. Following the replacement of the old canister filter with a new one, carefully reinstall the filter into the aquatic system, manually adjusting its position as necessary. Twice every week or once every two weeks, the carbon filter needs to be replaced [10].
Use a wrench to carefully remove the carbon filter unit to replace the carbon filter. Replace the used activated carbon with fresh carbon by discarding it. Place the carbon holder back into the filter unit after re-fitting it. Reinstall the filtration unit inside the system. Before putting the filter into the system, flush some water through the pipes to clear all extra particles. Every six months, the biological filter needs to be washed. In a circulating system, a biological filter is often positioned between the carbon filter and the canister. Press the button for pressure release to reduce the filter pressure. For this phase, two people are usually required. After removing the filter's lid, extract the spore from the water and empty the contents of the filter into a container with a sieve. Nitrification and de-nitrification are two processes where a fine-pore bio-filter medium is used to separate spores [10].
Restore the spores in a fresh tank if the previous one is not clear. Replace the filter unit's lid, add de-chlorinated water, put it back into the system, and then turn on the water supply. Several nitrifying bacteria will reside in the spore in an acclimated aquarium. Because the nitrogen cycle in the aquarium depends on these microorganisms, remove the dirty spore which could cause a significant rise in ammonia (NH3) and nitrite (N=C-) as compared to the new biological filter, which contains the clean spores. If it is not handled properly, either of these nitrogen cycle intermediate stages may be hazardous to aquatic life and even fatal to Zebrafish. Consequently, the Zebrafish housing system needs another biological filter somewhere to facilitate the quick repopulation of crucial microorganisms on the new spores. UV filters should be changed every nine to ten months to control biological impurities (like bacteria) in the system. It should be mentioned that even though the globe seems to be in good working order, it is important to replace it since the UV filter disinfection dosage rate is 110 mill joules per square centimeter at the start of the bulb life then drops with time [9, 10].
FEEDING
Zebrafish should be provide a balanced diet by combining dry and live feeds. Food size varies from 100µ in the case of larvae and for adults 300-400µ in diameter, brine shrimp. The preferred salinity range for culturing brine shrimp is 35-40 ppt (specific gravity 1.024-1.028). The following procedures are outlined below that can be used to produce brine shrimp (Artemia sp.) eggs:
- Red sea salt is dissolved in water in a beaker and set on a magnetic stirrer to increase dispersion. Alternatively, you might oxygenate the water and mix the mineral salt in it using the airflow hose pipe. If red sea salt is unavailable, then use instant marine salt as a hatching medium. We hatch the brine shrimp (12-15g) in red sea salt water with 30-35 g/L of salt, even though brine shrimps can cope with a wide range of salinities (<1%) as well in brine shrimp eggs are difficult to obtain at local pet stores, enclosed brine shrimp may be used instead, provided they are "decapped" in smaller batches before being introduced into the shrimp hatchery [10].
- Before putting shrimp eggs into the brine shrimp hatcher, add 1-2 teaspoons of saltwater per liter. Use a pump to provide the hatcher a good ventilation and enable shrimp to hatch eggs in about 48 hours. It is important to note that...
