IVF protocol
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Cryopreservation Protocols Contents | ||||||
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IVF is a very tricky procedure. Our sense is that you are walking a knife-edged path of success, with an abyss of failure yawning on both sides. It doesn't take much to tumble from the straight and narrow. We suggest that you follow these instructions as closely as possible. Some of the issues in the success of IVF are:
- Zona hardening. The zona pellucida will, with time, become inpenetrable to sperm. The longer the eggs sit in medium, the harder the zonas will become. Work fast when dissecting the eggs!
- Temperature is critcal. Keep everything at 37°C as much as possible. We place small gas incubators immediately beside our microscopes, so that fertilization dishes have minimal exposure to room temperature and atmosphere.
- pH is critical. The pH of you medium will begin to rise as soon as it is exposed to air. Try and keep fertilization dishes under gas
- Volatile organic compounds in the air of the laboratory can greatly affect success of IVF and the viability of embryos after transfer. Do what you can to maintain good air quality.
- In our hands, IVF has also failed due to bad media, bad gas, bad oil and bad luck.
Day 1
PMSG (Pregnant Mare Serum Gonadotropin) Injection
19-23 day old females should be injected i.p. between 5 and 7:00 PM depending on time the time oocytes are to be collected in the morning. (Eggs are collected as close to 13 hours post-HCG as possible for good results).The dose is strain dependent and it ranges from 2.5 IU to 5.0 IU.
Day 3
Culture Dish Preparation
It is convenient to prepare culture dishes the day before an IVF. The following types of dishes are used:
- Fresh sperm dish: 1 dish (35X10mm) for each strain w/ 1ml of HTF medium
- Fertilization dish (35X10mm): 1 dish per 3 females; one 500ul drop of HTF medium
- Wash dish (60X15mm): 1 dish per fertilization dish; five 250ul drops of HTF medium
- Culture dish (60X15mm): 1 dish per fertilization dish; five 250ul drops of KSOM medium
Mineral oil (pre-gassed) is added to cover the drops, and the dishes are transferred to an incubator at 37° C in an atmosphere of 5% CO2 5% O2 90% N2.
(Note: We find it convenient to use Billups-Rothenberg modular incubator chambers, which can be sealed and gassed and then transferred to a 37° incubator.)
(Note 2: Don't forget to label the bottoms of the culture dishes; it is helpful to color-code the culture vs. the wash dishes)
HCG (Human Chorionic Gonadotropin) injection:
Inject mice with 2.5 IU ip 48 hours after PMSG.
Day 4
Collect fresh sperm:
Sacrifice a male by cervical dislocation 15 to 45 minutes before the females reach 13-14 hours after the HCG. Dissect out the epididymides along with the vas deferentia. Place them in the sperm dish and mince the epididymis, making 5-7 slashes with the needle of an insulin syringe. Then, using forceps, "walk down" the vas to push out any remaining sperm. Place sperm dish in the incubator under gas and allow sperm to "swim out" for 10 minutes. Count the sperm; you will want to have somewhere between 1 - 25 x 106 sperm per ml.
(Note: With practice, you can gauge the sperm concentration "by eye".)
Thaw sperm (if using frozen sperm)
Thaw frozen samples rapidly by removing from liquid nitrogen storage and place into a water bath at 37°C until all ice crystals are melted ( approximately 2 min). Use the sperm immediately.
Aliquot sperm to fertilization dishes
Using a wide bore pipette tip, add 10 ul sperm (or more if the concentration is low) to each fertilization dish.
Collect eggs
Sacrifice three females, starting with those injected earliest with HCG. Dissect out oviducts and place them into a drop of HTF. Tear the ampullae to release egg clutches. Transfer clutches from three females to a single fertilization dish using a wide bore pipette tip. Repeat until eggs are collected and distributed from all the donors.
Incubate fertilization dishes
At 37° C under gas for 4 hours.
Wash eggs
After four to six hours of incubation, wash the eggs to remove the excess of sperm. Using a capillary transfer pipette, transfer the eggs from the IVF drop to one of the drops of medium in the "wash dish", taking care to leave behind as much debris as possible. All viable cells should be transferred to a second drop. Finally, the embryos should be distributed among the remaining drops of medium.
Incubate wash dishes
At 37° C under gas overnight.
Day 5
Transfer embryos to Pseudopregnant Females or to KSOM for culture to later stages
At this point the embryos can be counted and transferred to pseudopregnant foster mothers, or they can be cultured to later stages. For this latter step, transfer 2-cell embryos from each wash dishes to one of the drops of KSOM medium the corresponding culture dish. They are then washed through the second of the drops and then distributed to the rest of the drops (to remove the HTF) for culture to blastocyst stage.
Notes:
- You should be able to obtain a fertilization rate (the percentage of oocytes that develop to the 2-cell stage) of >90% when using sperm and oocytes from an F1 strain such as B6D2F1 or CB6F1.
- Fertilization rates from frozen sperm will vary by strain. Thawed sperm from F1 strains will do as well as fresh strains, but inbred strains will not do well. In particular, C57Bl/6J has a fertilization rate of only 5%, and 129-derived strains do worse. DBA/2J does well, and FVB does moderately well. See Sztein,-J-M; Farley,-J-S; Mobraaten,-L-E. In vitro fertilization with cryopreserved inbred mouse sperm. Biol-Reprod. 2000 Dec; 63(6): 1774-80.
- Sperm are fragile. Minimize shear forces by pipetting them gently. Centrifugation also reduces viability.
- If you wish, DMEM may be substituted for HTF, but we have found that HTF seems to work better for a wider variety of strains
