Kelly Levy                     Andrea Wise    Henry Viscardi                Harlandale    School                            High School    Albertson, NY                San Antonio, TX    11507                             78214

• Laboratory Experiment
• Research

• Biology and Independent Research students
• 9-12th Grade

Notes to the Teacher: to top

1. Groups for this lab should be set up with 2 to 4 students
2. Prepare the ethanol solutions in front of the class. This will illustrate how small a volume of alcohol is needed to cause developmental changes.
3. Prior culturing of zebrafish for 1 week is needed to obtain fertilized eggs.  See the zebrafish web site for information on culturing and maintaining zebrafish for research or other labs.  Also contains ideas to use zebrafish in place of other organisms in common genetics labs.
4. One  45 minute lab period is needed for students to set up and 10-15 minutes the next day for the observations.
5. Retin A (Retin A is found in Vitamin A and can also be referred to as Retinoic Acid) and ethanol have been shown to cause birth defects in pregnant women.  Ethanol is also flammable.
6. Ethanol and Lithium Chloride can be purchased from any chemical supply house.  Retinoic Acid can be purchased from VRW 1-800-932-5000.
7. Egg water:

2 to 3 hour or older zebrafish embryos
Compound and/ or Dissecting Microscope
2.8% Ethanol
0.3M Lithium Chloride
Egg water (see teacher instructions)
Parafilm
Small Petri dishes
Pasteur Pipettes or medicine droppers

Zebrafish need dechlorinated water which can be created by aging the water for 24 hours before use or you can buy 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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INTRODUCTION:
Many chemicals can interfere with normal embryological development.  These chemicals are called teratogens because they cause a variety of developmental anomalies.  Some chemicals we cannot avoid, such as environmental pollutants.  Others can be avoided, such as drugs and alcohol.  Fetal Alcohol Syndrome causes a wide range of developmental problems including growth retardation, facial abnormalities, and neurological disorders.  Alcohol has the most dramatic effect if taken during organogenisis which occurs during the first trimester in humans.  Zebrafish are also affected by alcohol.  Zebrafish are an excellent organism for using to study teratogens in development because they have a clear egg casing.
Your job is to find out what effects alcohol and lithium chloride have on developing embryos.

PURPOSE:

• The student will be able to see the effects of alcohol and lithium chloride on developing zebrafish  embryos.
• The student will predict the effects of alcohol and other drugs on developing human embryos.

3.  Place 5 embryos in a petri dish of embryo media.  Mark this-- CONTROL.
4.  Place 10 embryos in a petri dish of 2.8% ethanol solution.  Mark this--ALCOHOL.
5.  Seal each dish with parafilm or tape.  Place in incubator at 28^oC.

PART 2: EFFECTS OF LITHIUM CHLORIDE
1.  Place 5 zebrafish embryos into a petri dish of embryo media.   Mark this--CONTROL.
2.  Place 10 embryos in a petri dish of 0.3 M LiCl for 9 minutes.
3.  Remove the embryos after 9 minutes and rinse them in fresh embryo media.  Place the embryos in a dish of embryo media marked Lithium Chloride.
4.  Seal the dishes with tape or parafilm and place them in the incubator set at 28^oC overnight. (If an incubator is not available a warm moist place will be sufficient.)

DAY 2
Look at the control embryos and then look at the treated embryos.

OBSERVATIONS:
Describe what you see.   Draw pictures of both control and treated embryos.

CONCLUSIONS:
1. What effect does ethanol  have on the development  of  zebrafish embryos?
2. What effect does LiCl have on the development of zebrafish embryos?
3. When you added the LiCl what observations can you make?
4. Predict what might happen to a human embryo if the mother drank during the pregnancy?
 
 

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

FURTHER QUESTIONS:
1. Look up Fetal Alcohol  Syndrome in a text book or on the internet.  What are some of the effects listed?  How does this compare to your zebrafish embryos?
2. Look up a drug you think may affect the development of a human  embryo.  How would you set up an experiment to test the effects of this drug on zebrafish?  Some drugs you may want to consider are aspirin, Retin A, vitamins, caffeine and nicotine.
 

Teacher: You may wish to test the effects of Retin A in zebrafish development.   Retin A is classified as a retinoid along with all other vitamin A derivatives.  They come from eggs, liver, meat, fruits and vegetables.  Vitamin A is important for many things such as vision, spermatogenesis, conception and normal development of the fetus (Gregor, 1993).  Retin A is commonly used to treat troublesome acne and cancers such as promyelocitic leukemia, breast and ovarian cancer and carcinomas of the respiratory tract.  In rats Retin A causes cleft lip and palate, brachygnathia and eye disorders.  In humans and chickens high doses have lead to abnormal development in limbs, brain and spinal cord (Soprano, 1995).  Zebrafish also show a high toxicity for Retin A.  In concentrations as low as 10^-7  M  zebrafish have very severe side effects.  They can develop without a head or eyes, microcephalic, micro-opthalmic, abnormal somites or abnormal heart (Zhang, 1996).
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• Soprano, Diane.  "Retinoids as Teratogens," Annual Review of Nutrition (1995). 15:111-132.
• Zhang, Zunyi. "Specific Teratogenic Effects of Different Retinoic Acid Isomers and Analogs in the Developing Anterior Central Nervous System of Zebrafish," Developmental Dynamics (1996). 206:73-86.
• Eichele, Gregor. "Retinoids in Embryologic Development," Annals of the New York Academy of Science (1993). 678:22-36.
• Laale, Hans. "Ethanol Induced Notochord and Spinal Cord Duplication in the Embryo of the Zebrafish, Brachydanio rerio," Journal of Experimental Zoology (1971) 177:51-64.
• Saunders, Armant. "Exposure of Embryonic Cells to Alcohol: Contrasting Effects During Preimplantation and Postimplantation Development," Semin Perinatol. (April 1996) 20(2):127-139.
• If link to website is not working try to access it manually through http://zfish.uoregon.edu/

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