In Vitro Fertilization Protocol - see following paragraph for Discussion
This protocol is a general protocol for In Vitro Fertilization. If you are doing IVF for Microinjection, please refer to the Microinjection protocol, which have all the steps for Microinjection, including IVF.
For dissection and storage of testes:
Benchcoat (Fisherbrand, 14-127-47)
Latex, powder-free gloves
1 - 2 1.5 ml Eppendorf tube
For collecting eggs:
1008 Falcon petri dishes
marker for marking dishes
Microtube pestle( USA Scientific, 1415-5390)
(Globe Scientific, # 89209-802)
For sorting embryos after de-jelly:
Glass pipettes (Pasteur pipettes, Corning, 2239912)
Hand pipetteman;(Pipette pump, green, VWR, 53502-233)
10 – 20 µl pipetteman
(Seque/Pro capillary tips, Bio-rad, #2239912) for filling injection needle
Fine forceps (Dumont #5, #57-520) for breaking injection needle
Plastic beaker (for liquid waste)
Benzocaine (animal grade: Spectrum, BE130 or chemical grade: Sigma, #E1501-500g), 0.05%, pH 7.4. Need to make 5% stock in 100% EtoH first to dissolve.
1/9X Modified Ringers solution
3% Ficoll (GE Healthcare Biosciences, PM400, 95021- 196) in 1/9X MR, autoclaved
3% Cysteine (Sigma, W326305) by volume in 1/9X MR, pH 7.8-8.0 with NaOH, prepared same day
1/9X MR +/-gentamicin (100 µg/ml)
10X MBS high salts, autoclaved
0.1X MBS pH 7.8 - 8.0 Important: check pH same day
1X MBS + 0.2% BSA, filter sterilized, DO NOT use if cloudy
____ Collect one male, three females
____ Prime females w/ 10 units hCG
____ Set up embryo incubator temp. (18-28°C)
____ FIRST THING IN MORNING: Boost females w/200-250 units hCG (Time 0)
____ Make cysteine (3% in 1/9 MR, Ph=7.8-8.0)
____ After euthanizing male, remove testes and put in 1X MBS + 0.2% BSA
____ Squeeze females for eggs in plates previously coated with 1X MBS + 0.2% BSA.
____ Release sperm from testes using ball crusher (pestle).
____ Pour sperm on eggs, mix well with pestle WAIT 3 MINUTES.
____ Start fertilization by flooding with just enough 0.1X MBS to cover eggs. Important: pH 7.0 -8.0. Be sure to check ph same day.
____ WAIT 10 minutes
____ Cysteine embryos for 5 min or until done.
____ Rinse 3 times with 0.1X MBS, then 2 times with 3% ficoll in 1/9 MR. Leave embryos in 3% ficoll in 1/9 MR.
____ Change solution to 1/9x MR + GENT, put in 25°C or desired temp.
Buffers (From Early Development of Xenopus Laevis, Laboratory Manual, Sive, Grainger, Harland, Cold Spring Harbor Laboratory Press)
MBS (Modified Barth's Saline) Two solutions: 0.1 M CaCl2 and 10X MBS salts.
0.1 M CaCl2= 11.1 g/liter, Autoclave and store aliquots at -20ºC or 4ºC.
10X MBS salts
880 mM NaCl
10 mM KCl
10 mM MgSO4
50 mM HEPES (pH 7.8)
25 mM NaHCO3
Adjust final pH to 7.8 with NaOH, autoclave.
Prepare the final solution mixing 100 ml of 10X salt solution with 7 ml of 0.1 M CaCl2, and adjust the volume to 1 liter with distilled water. This is MBS 1X.
1X MBS + 0.2% BSA
Make 1X MBS and add 0.2% (w/v) of BSA, filter sterilized. Check pH: 7.8-8.0.
0.1X MBS Dilute 1X MBS 10 times with distilled water, check pH: 7.8-8.0 the same day.
In Vitro Fertilization
After the testes are in 1XMBS + 0.2% BSA and the eggs are in the dishes as described above, use a micro-pestle to crush the testes until the solution is milky. Distribute an equal amount of the sperm solution among the dishes with a plastic pipette. Mix the eggs and sperm solution well with the micropestle to distribute the sperm, and start a timer for 3 minutes. During this time, the sperm will attach to the eggs.
After 3 minutes, place the dishes flat on the counter top, flood with enough 0.1X MBS to cover the eggs, and swirl gently once or twice. Set a timer for 10 minutes. The drop in salt content in the solution will activate the sperm to enter the egg.
Fertilization can be checked starting about 3 minutes after flooding with 0.1 X MBS. There are several signs the embryos are fertilized. An approximate indicator is the “turning “ of the embryos. Embryos tend to turn so the pigmented animal hemisphere is facing up, while unfertilized eggs are impartial. A second indicator is the sperm entry point (SEP), which appears as a dot or indentation in the pigmented area. Thirdly, the dark hemisphere of the embryo often contracts during the early part of fertilization, appearing smaller than the dark area of an unfertilized egg. The contraction may relax after approximately 15 minutes, so it may not be evident to aid in selecting fertilized embryos later in the process. The flood solution can be left in the dish for up to 15 min if fertilization does not seem apparent or adequate after 10 minutes. Additional eggs may be fertilized during this time. Proceed to dejellying embryos with cysteine as described in the next section.