Chapter 2 - Breeding
Embryo Production By In Vitro Fertilization
(Source: C. Walker and G. Streisinger)
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 (page 2.4). 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1 minute, the sperm are no longer active.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.
1. Transfer the males and females into separate
holding tanks during the late afternoon of the day before beginning
the in vitro fertilization.
2. One half hour before "dawn" for the females, collect sperm from the males.
- Anesthetize the male fish by immersion in
tricaine (see RECIPES, Chapter 10).
They can be lifted out of the anesthetic with a plastic
spoon.
- Rinse in fish water, and place belly up in a slit
in a damp sponge.
- Gently blot the genital region with a kimwipe
so that no water is present (very important:
water activates sperm).
- Stroke the sides of the fish gently but firmly
with smooth (Millipore) forceps.
- As the milky sperm come out of the genital
pore, collect them in a microcapillary using gentle suction.
- Pool the sperm from several males in ice-cold,
full-strength Hank's saline. Sperm from 5-10 males is
adequate for fertilization of several hundred eggs. Sperm in
cold Hanks's will continue to fertilize eggs efficiently for up to
90 minutes. "Eyeball" the concentration of sperm, collecting
enough to make a cloudy suspension.
3. When the normal light cycle begins in the morning,
start collecting eggs.
- Anesthetize a female in
tricaine.
- Rinse in fish water and blot "damp-dry" on a
paper towel. Excess water will swell the eggs and prevent
fertilization.
- Place the female in a 35 mm plastic petri dish
and with damp (not wet) fingers, press gently but firmly on the
belly. If she is prepared to lay eggs, they will come out quite
easily.
- Gather the eggs with a spatula and return the
female to water. Good eggs are a yellowish, translucent color,
whereas eggs that have remained in the female too long are
white and watery (see below). To ensure getting good eggs,
collect them during the first 90 min after
"dawn".
4. Fertilize the eggs.
- Add 30-50 µl of the sperm suspension in
Hank's to the eggs.
- Mix gently.
- Add about 0.5-0.75 ml of system water and
after 1-2 minutes, add 2 mls more water. The actual time of
fertilization is when the first water is added to the eggs.
Typically, 60-90% of the eggs are fertilized in an in
vitro cross.
Depending on health and unknown factors, females will produce:
good eggs (slightly
yellowish, granular-looking eggs);
bad eggs (eggs already
broken down, whitish and rather like baby cereal);
mixed eggs (some good
eggs mixed with bad eggs);
and
no
eggs
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?
1. To obtain large numbers of
synchronously developing embryos. (Average
clutch size: 100 embryos)
2. To obtain haploid embryos, such as when
identifying heterozygous mutants.
3. To obtain embryos for Early Pressure.
4. To obtain embryos for Heat Shock.
Setting Up For A Squeezing Experiment
1. Set up is done after 3:30 p.m. the afternoon before the experiment.
2. Tanks of prospective fish for squeezing are chosen by:
Appearance of fish: Females should look large and fat in the belly. Males should look yellow and spry.
Elapsed time since fish have been squeezed: Fish should not be squeezed more than once/month. Longer intervals between squeezing are better.
3. Fish are separated during the night in
holding tanks. Males are put into tanks on a light cycle
½ hour earlier than females to allow time for
obtaining sperm in the morning.
4. Materials to gather:
Male squeezing station:
Place on 2 paper towels the following:
2 - 300 ml beakers for tricaine
1 - 10 ml beaker for Hank's solution
1 - finger bowl for rinsing fish
1 - plastic spoon
1 - pair of forceps for squeezing males
2 - plastic dishes with a (red) sponge with a slit to hold fish while squeezing
1 - small "single burst" test tube
Female squeezing station: (One per
person squeezing)
Place on 2 paper towels the following:
1 - finger bowl for rinsing fish
(beakers of tricaine from males can also be used for females)
1 - 50 µl wiretrol
1 - spatula or 2, if dividing clutches
1 - plastic spoon
1 - "Sharpie" marker pen
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".
Morning of the Squeeze
1. Get ice in the ice bucket.
2. To premeasured sodium bicarbonate (0.35 g), add 10 ml dH2O.
3. Measure 9.9 ml of Hank's Premix solution
into a clean test tube with a screw cap.
Add 0.1 ml fresh bicarb solution to the Hanks Premix.
Put the Hank's completed solution on ice.
4. Cut "single burst" test tube for sperm. If
doing UV sperm, make 2 test tubes.
Label the top of one cork for UV sperm.
Put empty corked test tubes on ice.
Squeezing Males
Materials Needed:
Tricaine solution in two 250 ml beakers
Stereo microscope
Sperm collection apparatus
20 ml beaker of Hank's solution
Finger bowl with fish water
Lamp
Kimwipes
Male fish
A fish net
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 Hanks 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:
Tricaine solution in two 250 ml beakers
Sperm in Hank's solution
One package, 35 mm petri dishes
50 µl micropipettes (Drummond Wiretrol)
Plungers for micropipettes
Egg Water
2 18mm x 150mm test tubes
2-1 ml pipettes and a 25 ml pipette in a liter beaker of fish water
Clean tank water
Female fish
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, wiretrols, 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 Done notebook.
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.
The Zebrafish Book
