ZIRC Sperm Thawing Procedure
Sperm Thawing Solutions
Hank's Stock Solutions:
Stock #1
8.0 g NaCl
0.4 g KCl
in 100 ml ddH2O
Stock #2
0.358 g Na2HPO4 Anhydrous
0.60 g KH2PO4
In 100 ml ddH2O
Stock #4
0.72 g CaCl2
in 50 ml ddH2O
Stock #5
1.23 g MgSO4x7H2O
in 50 ml ddH2O
Stock #6 (make fresh daily)
0.35 g NaHCO3
10.0 ml ddH2O
Hank's Premix:
Combine the following in order:
10.0 ml Solution #1
1.0 ml Solution #2
1.0 ml Solution #4
86.0 ml ddH2O
1.0 ml Solution #5
Store Hank's Premix in the refrigerator along with the Hank's solutions.
Hank's (Final):
9.9 ml Hank's Premix
0.1 ml Stock #6
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Materials needed for sperm thawing
Night before:
• P100 pipetman and tips (set on 70µl)
• 35 mm Petri dishes
• Paper towels
• Aspirator tube assembly
• Spatulas
• Finger bowl for MESAB/Tricaine
• Plastic spoon
• Sharpie markers
• Scissor-type forceps
• 2L glass flask filled with fish water
• measuring pipettes (1 and 5ml)
• Sort healthy females by looking for opaque area around urogenital opening
(about 15 per 1 gallon tank)
Day of thaw & IVF:
• Get females and nets
• Fill Dewar with liquid nitrogen
• Fill fiberglass tray (insulated in Styrofoam cooler) with liquid nitrogen
• Put labeled 5 ml cryogenic vials in tray with liquid nitrogen
• Measure out 4.2 ml MESAB and add 100ml fish water to a fingerbowl
• Make Hanks Final by measuring Hanks premix (9.9ml), then prepare
sodium bicarbonate stock #6 (0.35g to10ml water) and add 0.1ml to Hanks
premix
In vitro Fertilization using Cryopreserved Sperm
We find that this works best with two people or more. One person thaws the
sperm and fertilizes while the other(s) squeeze eggs from females.
1. OBTAIN SPERM SAMPLES: Remove cryogenic vials from liquid nitrogen
freezer and transfer to liquid nitrogen-containing fiberglass tray until ready
to thaw. Using cryogloves unscrew cap and remove capillaries that are to
be thawed, keeping them under liquid nitrogen.
2. ANESTHETIZE FEMALE: Place female(s) in a finger bowl containing
MESAB/Tricaine diluted in fish water. Once gill movement has slowed,
remove fish with spoon and dip in fish water.
3. Place female on a paper towel and blot dry(excess water will swell the
eggs and prevent fertilization).
4. SQUEEZING FEMALES: Squeeze eggs from females into 35mm plastic
Petri dish. With damp fingers, place two fingers on the dorsal side of the
fish to support the back. With the other hand, use one finger to 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
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fails to produce eggs do not continue to squeeze harder because it may
injure the fish.
5. EGGS: 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 and into a
container of fish water for recovery. 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 2 hours after "dawn".
Possible characteristics of eggs:
good eggs - slightly yellowish, granular-looking eggs, not watery
bad eggs - eggs already broken down, whitish and rather like baby
cereal
mixed eggs - some good eggs mixed with bad eggs
6. COMBINE CLUTCHES: If possible, try to obtain 3 clutches of eggs and
combine into one dish. If you cannot get three good clutches within 5 min
of your first clutch, then proceed to step 4 (one good clutch is sufficient).
Keep dish covered while sperm is thawed.
7. HANKS: Quickly add 70µl room temperature Hanks beside the eggs (not
touching).
8. THAW: Remove capillary from liquid nitrogen. Insert capillary into rubber
nosepiece end of aspirator tube assembly hold in air for a few seconds to
thaw. Expel sperm into puddle of Hanks and mix with the tip of the
capillary. Then gently mix Hanks/sperm with the eggs.
9. FERTILIZE: Without delay, activate sperm and eggs by adding 750µl fish
water. Swirl to mix. Incubate 3-5 min at room temperature.
10. Fill dish with fish water and incubate dish at 28ºC. After 2-3 hours count
and transfer fertile embryos to 100mm dishes (50/dish). Count infertile
eggs so that fertilization frequency can be determined.
11. Take good care of the larvae! For first 5 days, change water daily and
remove dead embryos.
References
Draper, B.W., McCallum, C.M., Stout, J.L., Slade, A.J. and Moens, C.B. (2004) A HighThroughput method for identifying ENU-induced point mutations in zebrafish. Method
Cell Biol. 77, 91-112.
Walker, C. and Streisinger, G. (2000) The Zebrafish Book 7, 41
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