Last updated: March 5, 2012
I. PURPOSE
Zebrafish adults, larvae and eggs and used extensively for certain physiological and developmental experiments. It is important that a steady supply of adults be available at all times for research purposes.
II. RESPONSIBILITY
It is the responsibility of the LAR staff to understand the handling and care of zebrafish and the Aquatics Habitat Stand-alone Re-circulating Fish Rack System (ZF0601)
III. PROCEDURE
- Daily:
- Morning
- Observe fish in the tank for signs of disease, injuries or death. Euthanize fish that are dying (see C below).
- Check that the filter and heater are working properly. Water temperature should be between 78 and 85F.
- Light timer should be set for a 14:10 light/dark cycle
- Record date, room temperature, tank water temperatureand pH from digital readout monitor and initial log book.
- Feed fish with a small pinch of Tetra flakes sprinkled over top of water.
- Feed fish fresh brine shrimp once daily. Use small cup net to add brine shrimp (washed) to tank so that salt water is not added at same time.
- Sweep and mop floor
- Fill out checklist and initial daily log
- Afternoon
- Feed fish with a small pinch of Tetra flakes sprinkled over top of water (add enough food so that all the fish get some and all the food is eaten within 5 minutes).
- Saturday and Sunday (fish are fed 1-2 times daily on weekends)
- Feed fish with a small pinch of Tetra flakes and fresh rinsed brine shrimp when first come in
- Record water pH and temperature
- Fill out checklist and initial daily log
- Monthly
- Change filter cartridge every 3 months
- Change charcoal filter according to manual
- Calibrate digital probe according to manual
- If and when necessary (on an as needed basis):
- Complete tank wash. Wash small individual tanks through cage washer without detergent
- Euthanasia
- MS 222 is commercially available as tricaine methane sulfonate (TMS), which can be used for the euthanasia of amphibians and fish.
- Tricaine is a benzoic acid derivative and, in water of low alkalinity (< 50 mg/L as CaCO3); the solution should be buffered with sodium bicarbonate. A 10 g/L stock solution can be made, and sodium bicarbonate added to saturation, resulting in a pH between 7.0 and 7.5 for the solution.
- The stock solution should be stored in a dark brown bottle, and refrigerated or frozen if possible.
- The solution should be replaced monthly and any time a brown color is observed.
- For euthanasia, a concentration ≥ 250 mg/L is recommended and fish should be left in this solution for at least 10 minutes following cessation of opercular movement.
- Water Tests for Fish Tanks
- Follow directions on each individual test package
- Daily , record pH and temperature from digital read out
- Weekly, test for ammonia, nitrite and salinity in all tanks
- Monthly, test for alkalinity, iron, dissolved oxygen and hardness in all tanks
- Preparing Brine Shrimp
- In brine shrimp incubator, mix 1-2/3 tablespoons solar salt in 1 L of tap water
- Add 1/2 level teaspoon brine shrimp eggs (stored in HSB 009B; excess eggs stored in freezer) to water solution
- Aerate with aerator system for 24 hours
- After 24 hours, transfer entire contents to glass holding container with aeration system
- Rinse brine shrimp incubator and begin a new solution daily as above
- Breeding
- Breeding will be performed at the specific request of the PI or animal user
- Number breeding tanks to correspond to the numbers on individual tanks
- At 4 PM on the day of request, LAR staff will setup breeding tanks and place dividers in individual breeding tanks
- Place males on one side of breeding tank divider and females on the other side
- Pull breeding tank dividers at 7 AM the following morning
- Growing zebrafish fry
- Record DOB, number of embryos and parental tank on log sheet
- Days 0-5
- Maintain embryos in 5 ml of Egg Water in a Petri dish
- Remove dead embroyos and debris daily using a one-piece disposable pipette with bulb (can be done under a microscope)
- Record activity on log sheet
- Egg Water recipe: mix 1.5 ml stock salts with 40 g "Instant Ocean" sea salts to 1 L distilled water
- Day 5 to 3 weeks
- Place embryos in crystallization dishes and add fresh Egg Water proportionately
- Remove 1/3 Egg Water daily from dish and replace with fresh Egg Water
- 5-11 day old fry
- Feed 2-3 drops (using disposable pipette) of 24-hour old brine shrimp hatch 2-3 times per day (rinse brine shrimp in strainer under tap water to remove salt and unhatched cysts at each feeding time)
- 12-17 day old fry
- Feed 1.5 ml (1 full pipette) of 24-hour old brine shrimp hatch 2 times per day (first rinse brine shrimp as noted above)
- 17-21 day old fry
- Feed 3 ml (2 full pipettes) of 24-hour old brine shrimp hatch 2 times per day (first rinse brine shrimp as noted above)
- 3 week old fry
- Place in a small cylindrical aquaria with regular zebra fish water (as prepared and tested for adult zebrafish)
- Remove and replace 1/3 to 1/2 volume of stock zebrafish water daily
- Add transition food - amount that will be consumed within 5 minutes to stocking density of fry (Reef Larval Fish Diet is stored in HSB 009B) in addition add 1.5 ml (1 full pipette) of 24-hour old brine shrimp hatch 2 times per day (washed as noted above)
- Feed adult volumes upon maturation (1.5 ml of dilute brine shrimp per 8 adult zebrafish)
- Zebrafish Book http://zfin.org/zf_info/zfbook/cont.html#cont1
- Maintenance
- Aquatics Habitat Tank System is supplied with reverse osmosis water system that is maintained by McGowan Water Company, Missoula, MT
- The tank system is maintained accourding to Aquatics Habitats "ZF0601" Stand-alone Unit manual
