Zebrafish are very interesting and easy organisms to work with.  They can be used in many activities in a biology lab.  Some of these activities are found on the zebrafish web site.   The development of zebrafish, as with other fish, is related to the temperature that they are raised at.  The warmer the water that the embryos are in the faster they develop.  This increase in the rate of development can be seen quantitatively in their heart rate.  As the embryos develop faster, their heart rate increases.  This activity will use zebrafish heart rate and relate it to the temperature of water the embryo's are reared in.
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• To relate heart rate and development to environmental conditions
• To create a standard curve to predict the temperature that a group of embryo's were raised at.

• Biology or Environmental science students
• 9-12th Grade

Notes to the Teacher: to top

1. Each group should have two to three students
2. The fish can be raised at temperatures ranging from 24^oC to 33^oC.  The optimum temperature for growth is 28.5^oC.
3. The heart in zebrafish develops 24 hours after the eggs are fertilized. The embryos should be collected and observed for heart development.  The embryos will hatch into fry in 2 to 3 days.
4. The unknown temperature could be any temperature between 24^o and 33^oC.
5. Embryo's can be raised in petri dishes as long as the water is changed daily.
6. This activity will take one 45 minute period.
7. Embryo media:

                        1.  Make stock salt solution by mixing 40g "Instant Ocean" sea salts
                             with 1 L distilled water.
                        2.  Make egg water by mixing 1.5 ml stock salt solution with 1 L distilled
                            water.

36-48 hour Zebrafish embryos
Compound or Dissecting Microscope,  with 4X and 10X objectives.
medicine dropper or pasteur pipette
stopwatches or watches with a second hand
counters
metric rulers
concave microscope slides or small petri dishes

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The Zebrafish website is a complete guide to care and lab use of Zebrafish.  Zebrafish (sometimes referred to as "danios")  are one of the easiest breeds of fish to maintain.  Zebrafish can be purchased from almost any pet store.  They are easy to keep if you treat them well. Here are some tips:

Zebrafish need dechlorinated water which can be created by aging the water for 24 hours before use or you can buying bottled water.  If your area puts chloramine in the water you need to buy bottled water because aging will not remove this substance. (To find out if your water contains chloramine you need to call your local water authority.)  To keep producing embryos for future experiments you will need at least 2 - 10 gallon tanks.  A ten gallon tank can hold anywhere from 25 to 50 fish. One tank for breeding adults, and one for embryos/fry.  Zebrafish will lay eggs but they will cannibalize them.  A dish of marbles should be placed on the floor of the tank.  Fertilized eggs that fall between these marbles will not be eaten.   The eggs can be siphoned or pipetted from the marbles and put into petri dishes of  embryo media. (This can be obtained from a pet store or made, see recipe in teacher information.  For further info, see fish net website )
    To lay eggs the fish need to be on a 14 hour light cycle (14 hrs of light/ 10 hours of dark).  They will lay eggs in the morning when the light is first presented to them.  If you keep them covered you can get them to spawn when you want them to but the photoperiod must remain constant.  The eggs have an 85-100% fertilization rate so once you get them your on your way.  If you feed the fish 2-3 times a day with fish food obtained from your local pet store you can keep them happy and producing eggs every second or third day.  The fry need to eat baby fish food that can be obtained from your local pet store (you are going to get to know the guy at the pet store really well) or you can feed them paramecium until they are 9 days old.  At 9 days you can begin to wean them to brine shrimp.  They should remain separated from the adults until they are 3 months old.  At this point they are ready to begin breeding and will not be eaten by the older fish.
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Purpose
    1. To observe the heart rate in developing zebra fish
    2. To relate the environmental conditions to the zebra fish development
    3. To create a standard curve to predict the temperature an unknown group of zebrafish were
         raised at.

Equipment
    Compound or Dissecting Microscope (or magnifying glass with base)
    Stopwatches
    Counter
    Metric Ruler
    Small petri dish
    Pasteur pipette or medicine dropper
    Samples of ZebraFish raised at room temp (17^oC to 22^oC), 28^oC and 35^oC  and one raised at an unknown temperature.

PROCEDURE
   1. Read the entire lab and all questions you will be responsible for answering before starting
    the lab.
   2. Remove a single embryo with a pasteur pipette or medicine dropper from a stock of zebrafish
    that have been  raised at room temp (17^oC to 22^oC).  Place the embryo in a petri dish with
    embryo media from the original stock.
   3. Place the dish under the dissecting microscope or magnifying glass.
   4. Locate the heart of the embryo which should be slightly below the head.

    5. Designate who in the group will be the timer, counter and recorder.
                Timer: ________________________________
                Counter: ______________________________
                Recorder: _____________________________
    6. Practice counting the heart beat for one minute.  You should do this 3 times and alternate the
    activities so everyone in the group has had a change to be the timer, counter or recorder.
    7. Average the three counts at room temperature and put that in your data table.
    8. Return the embryo to the original tank and obtain an embryo raised at 28^oC.
    9. Have each student again take turns timing, counting and recording the heart rate at 28^oC and
    place the average in your data table.  Return the embryo to the appropriate tank.
    10. Repeat this procedure for the embryo at 35^oC.
    11. Obtain an embryo raised at an unknown temperature and repeat the same procedure rotating
    tasks and using the average in your data table.

OBSERVATIONS
 

6. According to the graph, what is the unknown temperature that some of the embryos were raised at?
7. Predict the heart rate at 30^o C.

ANALYSIS

1. Could you repeat this experiment for chicken embryos and expect to see the same results?
2. Factories once used water to cool their machinery and dumped the warm water back into the lakes and streams which it came from.  This is called thermal pollution.  What effect do you think thermal pollution could have on the stream ecosystem?

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•   Discuss conclusion questions
•  Assess use of proper laboratory technique:  use of microscope, labeling, and proper data collection.
•   Graphing and predicting

•  Study other types of fish to make standard curves for their development.
• Find upper and lower limits in development temperature