(Source: C. Walker and G. Streisinger from Zebrafish Book 5th Edition)
Overview of in vitro fertilization
Large numbers of synchronously developing embryos can be obtained by in vitro fertilization. Maintain breeding males and females on a daily schedule as described in the Zebrafish Breeding Schedule for Maximal Embryo Production. Gametes are expressed from breeding adults by gentle pressure. The sperm are maintained in Hank's saline. The gametes are mixed together in a Petri dish. When water is added to the egg-sperm mixture, fertilization takes place very rapidly in 20 to 60 seconds. After 1 minute, the sperm are no longer active. The clutch of embryos is fertilized essentially synchronously and develops synchronously during the early cleavage stages if maintained at constant temperature.
Depending on health or unknown factors, females will produce:
It is possible that females produce and reabsorb their eggs every day and the majority of females are synchronized to lay their eggs at "dawn".
After being used in an in vitro experiment, males should be rested for 3 weeks and females rested for 4 weeks before being squeezed again. They may be used for natural crosses during that rest time.
Occasionally fish die in an in vitro experiment. Care should be taken not to leave fish in tricaine for too long. Sometimes fish can be revived from too long exposure to tricaine by irrigating their gills with water. Also, extra care should be taken not to damage the gills. Handling fish "front-to-back" when using spoons and spatulas is helpful. One characteristic of sick fish is that their gills hemorrhage easily and the fish die after exposure to tricaine.
General procedures and overview
Why Do We Squeeze?
- To obtain large numbers of synchronously developing embryos. (Average clutch size: 100 embryos)
- To obtain haploid embryos, such as when identifying heterozygous mutants.
- To obtain embryos for Early Pressure.
- To obtain embryos for Heat Shock.
Setting Up For A Squeezing Experiment
Place a liter beaker of fish water at the fertilizing station with a 25 ml pipette and 2-1 ml pipettes in it.
Make sure there is Hank's Premix solution, and tricaine in the refrigerator and premeasured sodium bicarbonate near the fertilizing station. If not, see RECIPES, Chapter 10.
Morning of the Squeeze
Squeezing males
Materials Needed
Preparation
1. Estimate the number of egg clutches to be obtained.
2. Measure 0.05 ml of Hank's for every clutch of eggs anticipated.
3. Put the measured Hank's in the small test tube, cover with cork and put in ice.
4. Put some of the remaining Hank's solution into the 10 ml beaker.
5. Put the capillary tube of the sperm collecting apparatus into the Hank's solution in the 10 ml beaker when not collecting sperm during the procedure.
Procedure
1. Remove two fish from the plastic holding container with net and place into the 250 ml beaker containing the tricaine solution. Repeat this for the other 250 ml beaker.
2. When gill movement has slowed, remove one fish with the plastic spoon.
3. Rinse this fish in the water in the finger bowl and place it upside down in the small sponge in the plastic dish.
4. Gently wipe the region of the anal fin with the corner of a Kimwipe®.
5. Place the dish with the fish under the objective of the microscope, with the light illuminating the fish, especially the region of the anal fin.
6. With the capillary tube of the sperm collecting apparatus, gently push aside the anal fins to expose the anus.
7. Using the forceps gently squeeze the sides of the fish at a point just anterior to the anal fins, collecting the sperm with the capillary tube. When finished return the fish to the finger bowl or recovery container.
8. When you have collected sperm from 2‑3 fish, add them to the Hank's solution in the small test tube in the ice bucket.
9. Repeat until you have collected the required amount of sperm.
10. Keep the small test tube with the sperm and Hank's solution in the ice. This helps prolong the viability of the sperm.
Squeezing females
Materials needed
Preparation
1. Fill the large test tubes with egg water.
2. Put one plunger into each test tube.
3. Fill finger bowls with clean tank water.
Procedure
1. Place two females into the tricaine solution in each of the two 250 ml beakers.
2. When gill movement has slowed, remove one of the fish with the plastic spoon.
3. Rinse the fish in the water in the finger bowl.
4. Gently place the fish on a paper towel to dry briefly.
5. Using the spoon, transfer the fish into a small plastic dish.
6. Slightly dampen your fingers.
7. Place one finger of one hand on the dorsal side of the fish.
8. Using one finger of the other hand express the eggs by gently pressing on the ventral side of the fish, starting just behind the pectoral fins and moving toward the tail. Only gentle pressure is needed. If the fish has eggs, they will come out easily. If gentle pressure fails to produce eggs, do not continue to squeeze harder. Extra squeezing may injure the fish.
9. If eggs are obtained, use the metal spatula to gently move them away from the fish's body. Then slide the fish out of the dish.
10. Put the fish into a recovery container to revive.
11. When eggs are obtained by squeezing, cover them with the lid to the dish.
12. Repeat this for the remaining fish.
In vitro fertilization
Procedure
1. Move to the fertilization station and remove a plunger from the water in the test tube. Remove a capillary tube from its container and insert the plunger into the end of the capillary with the green mark.
2. Push the plunger down almost to the end of the tube, leaving about 0.5 cm of air space.
3. Remove the cork from the test tube containing the sperm and insert the capillary tube with the plunger into the sperm.
4. Draw the plunger part way out of the tube to draw sperm up to the black line on the capillary. Be sure to leave air space between the end of the plunger and the sperm.
5. Expel the sperm onto the eggs by pushing the plunger all the way through the capillary so that the tip of the plunger extends out of the end of the capillary.
6. Gently mix the sperm and eggs with the tip of the plunger.
7. Using the 1 ml pipette, add 1 ml of egg water to the egg/sperm mixture. This activates the sperm so that they can fertilize the eggs. The time of fertilization occurs when the WATER is added, not when the sperm is added.
8. Cover the dish with its lid.
9. Allow a few minutes for fertilization to complete, then add more egg water, approximately 2 ml.
Experiment clean up
When fish are returned to their tanks, they should be marked with a sticker indicating the date used. Rinse spatulas, Wiretrol, and spoons with tap water. Put away all materials in original places. Enter the number and quality (G,B,M,N) of eggs obtained in Experiment Donenotebook.
Follow up of embryos
Embryos should be left alone for one hour after fertilization. Then:
1. Count fertile embryos.
2. Remove dead and infertile embryos.
3. Transfer groups of 25 embryos into 300 ml beakers with 100 ml of fish water each.
Morning Following Fertilization
1. Sort, count, and record the numbers of AA/BB, B/CD and dead embryos.
AA = Embryos are perfect diploids.
A = Perfect haploid embryos.
BB = Diploid embryos that have small imperfections such as a bent tail or one eye.
B = Very abnormal embryos that have a head, body axis and some sort of tail, but obviously will not survive.
C/D = Embryos that are yolks with masses of cells on them.
2. Screen according to particular experiment or mutation.