Table1: Listing of all used chemicals with related supplier informations (s.m. = selfmade).

Table 2: Listing of all used equipment and materials with related supplier information (n.s. = not specified)

Method is adapted from (1).

PMSG is available in 5000 I.U. vials. Working solutions are made up by adding 400 mL of isotonic saline to two vials PMSG for a total of 10000 I.U. of powdered hormone which is then solubilized with a magnetic stirrer for 5 to 6 minutes at room temperature. HCG is available in 10000 I.U. vials. It is diluted in the same way as PMSG described above by adding 400 mL of isotonic saline to one vial hCG for a total of 10000 I.U. of powdered hormone. The hormone solutions are aliquoted into 2 mL tubes (maximal 1.6 mL) and stored at – 80°C until the day of use. Frozen preparations are not kept longer than three months.

The prepared solutions of PMSG and hCG yield a working concentration of 2.5 I.U. hormone per 0.1 mL for an appropriate injection volume. They are thawed slowly the day of use at + 4°C. Once thawed the hormone solutions are stored at + 4°C and must be used within 4 days.

Superovulation in three to maximum four weeks old female mice is induced by giving an intraperitoneal injection (i.p.) of PMSG followed approximately 48 hours later by an i.p. injection of hCG as shown in Table 1. The thawed working solution of hormones is inverted slowly several times, not shaken. It is drawn up with a syringe without needle to avoid shearing forces. The needle is attached and air bubbles removed. Enough syringes are filled to dose all animals as needles are not recapped and removed to refill the syringes. Depending on the purpose of superovulation – either oocyte or embryo collection – injections are timed as shown in Table 6. For embryo collection the females are mated to male mice directly after hCG injections. Observing a vaginal plug the next day indicates successful pairing and ensures collection of embryos at the 8-cell cell stage.

Table 1: Time schedule for superovulation of female mice for collection of oocytes or 8-cell stage embryos. The given dose of the hormones is based on the experience discovered with animals of the C57BL/6J strain.

Embryo collection

On day 3 post mating, according to 1, the female is humanely euthanized by cervical dislocation, placed on its back on absorbent paper and the abdomen is generously sprayed with 70% EtOH for disinfection. With the 1^st pair of dissecting forceps a small fold of skin is lifted in the center of the abdomen level with the top of the legs and a small cut is made with scissors to open the abdominal cavity. The skin is pulled in opposite directions towards head and tail until the abdomen is completely exposed. Using the 2^nd pair of forceps a small fold of the peritoneum is lifted and a small cut using 4 inch scissors is made to allow air to enter the abdominal cavity. The peritoneum is torn open in opposite directions towards head and tail to expose the body cavity, the coils of the gut are pushed towards the head or aside until the uterus can be located. By lifting the uterus with a clean pair of forceps the attached ovary and oviduct become visible. Fat tissue and blood vessels are peeled away form uterus and oviduct. Oviducts and part of the anterior horn of the uterus are then removed. When dissecting the oviducts it is important to cut between the ovary and the oviduct according to Figure 1. Any remaining ovarian tissue left attached to the oviduct makes finding the infundibulum very difficult. Enough uterus is left attached for easier manipulation of the oviduct. Furthermore this keeps any embryos from being lost during dissection.

The dissected oviducts are placed into a 60 x 15 mm culture dish and covered with a drop (5 µL) of FHM to prevent from desiccating. They are flushed by introducing a 30 G^1/2 needle (blunted to avoid carving the tissue and curved in an appropriate working angle), attached to a 5 mL syringe filled with medium, into the infundibulum of the oviduct and applying a small volume of approximately 100 μL with medium pressure so that the oviduct blows up and embryos are flushed out. This procedure is carried out using a microscope at 12 x magnification. After examination at 45 x magnification all embryos are picked up with a capillary transfer pipette drawn to fine point and transferred into a Petri dish filled with approximately 500 µL of fresh FHM for washing and selecting 8-cell stage embryos. Handling and transfer of embryos in general is schematically displayed in Figure 2. Depending upon the strain or further experiments, embryos from related females may be pooled into a single dish. At this point embryos are ready to be transferred into pseudopregnant females or cryopreserved.

Freezing embryos in straws

For the cryoprotective solution (CPA 1.5 M PROH in FHM) 4.4 mL of FHM is measured and 0.6 mL of 1, 2-propanediol (PROH) is pipetted into a 15 mL Falcon tube. PROH is highly viscous; hence, to ensure the correct concentration of the CPS, excess PROH has to be removed from the outside of the pipette and no propanediol remains in the pipette when expelling the liquid. The solutions are then mixed by gently rocking the media and filter sterilized through a 0.22 μm filter membrane into a new 15 mL Falcon tube. For the diluent/thawing medium (1.0 M sucrose in FHM) 1.71 g sucrose is diluted in 5 mL of FHM in a 15 mL Falcon tube by gently rocking the media until sucrose completely dissolves. The solution is then filter sterilized through a 0.22 μm filter membrane into a new 15 mL Falcon tube. The prepared solutions of 1.5 M PROH and 1.0 M sucrose are stored at + 4°C. They expire one week from preparation.

To ensure the right temperature for freezing embryos, the controlled-rate freezer is cooled and set to hold at – 7°C prior to the cryopreservation procedure. For each strain of embryos, one wash dish with 500 µL FHM and one incubation dish with 300 µL cryoprotective solution are prepared. The used media are provided at room temperature.

Straws in which the embryos are frozen are prepared as shown in Figure 3. The plug is pushed into the straw by using a metal rod (with a stopper) so that 56 mm are remaining on the left or shorter end including the plug; 77 mm remain on the right end. For later identification labels are attached on the shorter (left) end of the straw leaving at least 3 mm for attaching the Monoject syringe. By marking the straws with a cryo pen at various intervals (it is convenient to use a ruler or pre-marked tray), they can be loaded with the correct amounts of diluent and cryoprotectant. The 1^st mark is set 20 mm from the plug, a 2^nd mark 7 mm from mark 1 and a 3^rd mark 5 mm from mark 2. A Monoject syringe is applied to the labeled end of the straw for aspirating the liquids provided in 15 mL Falcon tube. The straw is filled with diluent up to the first line (mark 3) followed by aspiration of air until the diluent reaches second line (mark 2). The cryoprotective solution is then filled until sucrose reaches the third line (mark 1). Finally air is aspirated with a steady state until the diluent reaches the powder part of the plug, with this sealing one end of the straw.

The 8-cell stage embryos – either freshly collected or derived from in vitro fertilization – are graded, collected and held at room temperature in the provided washing dish containing 500 µL FHM. To ensure the right concentration of the cryoprotective solution, embryos are moved with a minimal amount of FHM from the wash dish into the prepared dish with 300 µL of cryoprotectant. They are incubated for 15 minutes at room temperature and a timer is set, as time is crucial for this step. After incubation the appropriate amount of embryos (max. 15 for transfer) is loaded into the straw by mouth pipetting (Figure 2). Embryos and air bubbles are expelled evenly along the length of the PROH fraction in the straw with the air bubbles confirming the complete loading. The loading end of the straw is then plugged with Critoseal and the seal is confirmed by pushing a finger against the plugged end of the straw. A straw loaded with embryos and ready for freezing is schematically shown in Figure 3.

The straws are placed into the pre-cooled freezer at – 7°C for 5 minutes. When “seeding” the straw by touching it just below the cotton plug in the diluent section with a cotton swab or forceps submerged in LN₂, small ice crystals at the seeding area should become visible. After keeping the straws for additional 5 minutes at – 7°C, the seeding success is controlled by eye (PROH section is kept in the cooling liquid at any time); ice crystals should have formed completely in both the sucrose and the PROH section of the straw (Figure 4Error: Reference source not found). The freezer is then cooled to – 30°C with a cooling rate of – 0.5°C per minute. After reaching this temperature, straws are held for additional 10 minutes at – 30°C, then quickly submersed in an LN₂ temporary vessel and transferred for longtime storage into the LN₂ tank.

To control the success of the cryopreservation procedure one to two straws are thawed earliest the day after freezing. The embryo viability is evaluated as described below. Long-term storage of cryopreserved embryos requires appropriate record keeping.
 

Thawing embryos from straws

A water bath is set-up at room temperature (20 – 25°C) prior to thawing the straws. The following culture dishes are prepared for each straw: one thawing dish 35 x 10 mm and one 60 x 15 mm washing dish containing 200 µL FHM. Additional washing steps or dishes may be required according to subsequent procedures e.g. embryo transfer.

The straws are quickly transferred from the LN₂ storage tank to a temporary LN₂ container. As incubation times during thawing are crucial for the recovery of embryos with a high viability, only one straw is thawed per time. The straw is removed from LN₂ with forceps and exposed to air for 40 seconds (crucial time). Then it is, labeled end up, submerged and stirred in the water bath for approximately 5 seconds until condensation in the straw disappears completely and the media in the straw becomes clear. The straw is dried by wiping it with a napkin carefully. The critosealed end is cut and the end re-rounded with fingers. A second cut is made between the cotton portions of the cotton plug (sealed area), leaving about half the cotton plug in place to act as a plunger. A pusher (metal rod) is used to expel contents of the straw, the embryos in the cryoprotectant and the diluent solution, towards the critosealed end and stopped when the cotton plug reaches the end of the straw. The thawing dish is covered and embryos are incubated for 5 minutes at room temperature before they are transferred with a minimal amount of medium to the washing dish by mouth pipetting. Again the dish is covered and embryos are incubated for a minimum of 5 minutes until they assume normal appearance. A second wash step may be inserted; five washing steps are optimal to remove any residue cryoprotectant. The embryos are then visually checked for viability (healthy morphology); numbers of found and healthy embryos are recorded. Embryos with a healthy appearance are now ready for transfer into pseudopregnant females or for culturing to blastocyst stage in KSOM.

Culturing frozen – thawed embryos

Culture dishes for embryos are prepared with 200 µL drops of KSOM, one ore more drops per dish, covered with mineral oil and equilibrated approximately 30 minutes before use in the incubator at 37°C and 5% CO₂, 5% O₂, 90% N₂. If frozen embryos are to be cultured, they are thawed at this point. With a working medium volume as small as possible, all embryos are transferred from the working/washing media into KSOM with approximately 20 to 30 embryos per drop; dishes are placed back into the incubator at 37°C and an atmosphere of 5% CO₂, 5% O₂ and 90% N_2.

The cultured embryos are checked for their development according to their stage when frozen.
2-cell stage embryos are evaluated on day 4 (thawing is considered to be day 0); 8-cell stage embryos on day 2 after thawing. The freezing and thawing methods are performed successfully when more than 80% of the frozen/thawed embryos develop into blastocysts.

1. Anon. (2006): Workshop on Shipping and Reconstitution of Cryopreserved Embryos. The Jackson Laboratory, Course book p. 29, 36