Animal protocols
Zebrafish breeding and embryo care
Zebrafish females may release eggs into the home tank water every morning, however these eggs need to be captured quickly to prevent the adult fish from eating them. We use a static tank and breeding insert to catch the eggs and protect them for collection.
- On the afternoon before eggs are to be produced, insert the breeding platform into the home tank of the fish to be bred. Adjust the water depth over the platform to be shallow (~30 mm).
- Ensure that the lights are controlled by the room timer and will turn on early the next morning. Leave the fish undisturbed overnight.
- When the lights come on, spawning will occur.
- 1-2 hours after lights on, visually inspect the breeding platform for the presence of eggs. Harvest the eggs and place them into a petri dish with fish water and a drop of methylene blue (anti-fungal).
- Count the eggs and record it on the log in room 9-329.
- Place the labelled, counted dish of fertilized eggs into the 29C incubator for 4 days. The fertilized eggs should hatch within 48 hours and the embryos will cling to the walls of the dish, displaying little spontaneous movement for the first 36 hours. During the first 96 hours, larvae do not need to be fed and will survive on their yolksacs.
- Each morning, visually inspect the dish of embryos for mortality, attachment, and spontaneous activity. Use a small pipette to apply small water streams to embryos that seem letheragic or malformed. Confirm the development of normal swimming (a burst-and-rest pattern).
- On the fifth day (5dpf), when >90% of embryos are spontaneously free-swimming, add them to the larval polyculture tank.
Making a static larval zebrafish and rotifer polyculture
At 5 days post-fertilization (5dpf), the zebrafish embryos have grown into larvae and have digested all the nutrients from their yolk. This requires the introduction of an external source of food. Zebrafish prefer live prey and consume them to thrive during their early growth stage (5-9dpf).The static polyculture tank maintains a balanced environment between the parameters necessary for rotifer survival and robust larval zebrafish growth. However, problems with waste buildup may quickly cause problems with water quality that can compromise the health of rotifers, larvae or both. Do not add pellet or flake food to the polyculture tank, add rotifers only.
- In Room 5-228A, calculate the density (XXX rotifers/mL) of the rotifer culture by counting them using a microscope and a Sedgewick-Rafter slide.
- Using the calculated density, calculate the volume that needs to be harvested from the culture bucket to obtain 500,000 rotifers, the typical number used for the 0.5 L polyculture tank. That formula is: Liters to be harvested = 500,000 rotifers / XXX rotifers/mL X 1,000 mL/L.
- Ensure that ammonia in the rotifer culture is less than 1.0 ppm. Add Chlor-Am-X if necessary.
- Harvest the calculated volume of rotifer culture into a flask and dilute the harvested rotifers to 333 mL with unfiltered culture water.
- Transport the harvested rotifers to the Fish Room (9-329).
- Set up a polyculture tank on the table: a small shallow breeding tank (Typically Dr. Burney will set aside a labeled tank for us).
- Fill the polyculture tank with ~200 mL of system water and ~100 mL of the harvested rotifers, allowing proper mixing. Fish water should be ~0.25 ppt salinity and harvested rotifers are 15 ppt salinity.
- Use the refractometer to confirm that the salinity of the polyculture water is now between 4 and 6 ppt. Do this before adding any larvae to the polyculture tank. The rotifers may settle to the bottom as they are temporarily shocked by salinity changes but within a few hours they will begin swimming again.
- Pipette any dead or malformed larvae from the incubation dish and discard them to avoid adding them to the polyculture tank. I also like to remove the empty or unfertilized eggs. Any dead or decaying tissue that gets into the polyculture tank may contribute to problems with water quality.
- Pour the larvae from the incubation dish into the polyculture, rinsing them off the dish with fish water. We have set a limit of 50 larvae per polyculture tank.
- Place the tank on the table in the Fish Room, covered and undisturbed.
Maintaining a larval polyculture
The larval polyculture should allow the survival of the rotifers for 5+ days during the early growth of the zebrafish larvae. During this time, the tank needs to be inspected daily.
- Observe the larval behavior. The fish larvae will hunt and eat the rotifers.
- Observe the water color and rotifer population. The polyculture water should appear light yellow-green with great clouds of rotifers swimming throughout. i) If the water looks too light, Algae may be added to the water drop-wise and distributed with gentle mixing. Do not add more than a few drops or the polyculture water may foul prematurely. ii) If there are fewer rotifers swimming, add more concentrated rotifers as supplemental feedings to ensure larval growth. Adding rotifer culture water will increase the salinity of the polyculture, however. Check salinity frequently and dilute with fish water to keep it below 6.5 ppt. Zebrafish larvae are adversely affected by anything much saltier than 7 ppt.
- Look for dead larvae. Some larvae will die in the polyculture tank (~5% seems typical). The dead bodies will fall to the bottom and can be best visualized with a flashlight using oblique lighting. These bodies should be siphoned out of the tank to prevent problems with water quality.
- After 5 days, the larvae can be transferred from the polyculture tank to a regular 1.8 L housing tank with all the baffles inserted. This tank should be placed on the housing rack and started on a very slow drip of fish water from the rack nozzle.
Zebrafish housing parameters
Our zebrafish are bred inhouse and derived from two batches (arrival dates?) of 3 pairs of adult wildtype (AB strain) zebrafish. These two batches of founder fish were ordered and introduced after the housing racks were disinfected. Every zebrafish used for our experiments have been derived from these twelve fish and their progeny. After polyculture, zebrafish are housed in a 2.8 L tank on a recirculating aquaculture rack (ZTEDU235 until 10/XX/2017; ZS760 since). These racks incorporate all that is necessary to maintain optimal water quality for zebrafish health and development. The recirculating system incorporates several filter processes to control microorganisms and waste including an ultraviolet sterilizer, a particulate filter pad, a fluidized bed biological filter, and carbon filters. The optimal water quality parameters for our system are:
- pH
- total dissolved solids
- NH3
- light/dark cycle
- housing density: fish/liter
Feeding schedule for developing zebrafish: days post-fertilization (dpf)
Zebrafish are fed by animal care staff for the majority of their needs. We provide them with rotifers to supplement their feeding.
- 0 - 5 dpf: None, petri dish in 29C incubator and eating their own yolk
- 5 - 9 dpf: Rotifers in larval polyculture.
- 10 - adult: Pellets and rotifers. (different size pellets)
Anesthesia in zebrafish using a MS-222 solution
If you need to anesthetize zebrafish, the safest way is to use MS-222 (tricaine mesylate).
- Make 1M TRIS base solution by mixing 24.23 g of TRIS base with 200 mL of RO water
- Make a stock (25x) MS-222 solution by combining 400 mg of MS-222, 97.9 mL of RO water, and 2.1 mL of 1M TRIS solution. Adjust to pH = 7.0 and store in -20C freezer.
- Make a working MS-222 solution by combining 4.2 mL of 25x stock MS-222 solution with 100 mL of fish water.
- Net one fish to be anesthetized and deposit it into the working MS-222 solution for 2-3 minutes.
- Once locomotion stops and operculum movements slow, the anesthetized fish can be removed from the MS-222 and manipulated for 3 minutes on a wet sponge. If longer times of anesthesia are necessary, the gills should be irrigated frequently with anesthetic solution.
- Fish need to be revived for 10-15 minutes in regular fish water before returning them to the housing tanks on the rack.