Two types of media are used for the recovery of embryos, phosphate-or bicarbonate-buffered solutions. The pH of most body fluids is 7.2 to 7.4. A pH range of 6.9 to 7.7 is compatible with normal embryo development in mice. Osmolarity of the medium should be in the range of 270–300 mosm. The most commonly used medium for non-surgical embryo recovery is Dulbecco's phosphate buffered saline (PBS). The formula for preparing 10 litres of a modified solution of PBS is presented in Table 3.
One-percent heat-treated bovine serum (10 ml) is added to each individual 1-litre bottle of flushing medium which has been warmed to a temperature of 30–37°C. Serum may act as a protein source for embryo growth and membrane stabilization, and renders the embryos less sticky. Heat treatment at 56°C for 30 minutes removes complement which is embryotoxic. In lieu of serum, 0.4 percent bovine serum albumin (BSA) may be used for recovery and holding media. The advantage of phosphate-buffered media is that they maintain the pH during exposure to the air.
Bicarbonate-buffered media need a gas phase of 5 percent CO2 which means that they require a closed system. Upon exposure to air CO2 rapidly escapes leading to a rise in pH. Examples of bicarbonate buffered media are Ham's F10, Brinster's BMOC-3 and TCM 199 (Hepe's).
Glucose or dextrose and pyruvate are actually only necessary for longer periods of embryo culture. For flushing, PBS only needs to contain penicillin and 1–2 percent serum. 10–20 percent serum is added to the flushing medium to make a holding medium which can also be used for short-term (less than 24 hours) culture. Holding medium should be sterilized by filtration through a 22-μ or 45-μ millipore filter attached to a large glass syringe (Figure 8E). (The rubber plungers of some syringes have been shown to be coated with an embryotoxic lubricant (Takeda and Hasler, 1986).)
Modified Dulbecco's phosphate-buffered saline solution (to prepare 10 litres)
|Dissolve in 4 litres deionized, distilled water|
|Na pyruvate||0.36 g|
|Na penicillin G||1 000 000 units|
|Dissolve in 4 litres deionized, distilled water|
|Add to part A (check for crystals while adding)|
|CaCl2 2HOH||1.325 g|
|MgSO4 7HOH||1.212 g|
|Dissolve in 2 litres deionized, distilled water|
|Add part C to solutions of A+B|
|Check pH and osmolarity|
|Filter using two Sterivex-GV (0.22-micron pore size) changing filter half way through filtering process|
Factors which may affect embryo viability during short-term culture include pH, osmolarity, (elevated) temperature, contamination and toxicity of the medium (Bondioli and Hill, 1986). Embryos should be stored in the same type of medium that was used for flushing. Changing embryos from a phosphate- to a bicarbonate-buffered medium is undesirable because of the possible changes in osmolarity, pH and energy substrates.
Holding dishes must be kept covered to minimize contamination and evaporation. The latter leads to an increase in the osmolarity of the medium. Changing the embryos to a fresh dish of holding medium from time to time further minimizes the effects of contamination and evaporation.
By the very nature of the procedure, it is vital that all aspects of quality control of media and equipment which come in contact with the embryos are strictly adhered to. While it is advisable for the beginner to use commercially prepared media and sterile disposable supplies, this may not be possible in some situations. Under such circumstances, culture conditions, catheters, tubing and pipettes must be checked with an animal model. The development in vitro of in vivo fertilized two-cell mouse embryos to the hatching blastocyst stage is the most commonly used animal model for this purpose.
Female mice are injected intraperitoneally with 5–10 IU pregnant mare serum gonadotrophin. Forty-eight hours later the mice are injected intraperitoneally with 50 IU human chorionic gonadotrophin, followed by mating one-on-one with the male. Approximately 36 hours after human chorionic gonadotrophin, the mated animals are sacrificed by cervical dislocation, and the oviducts are excised, placed in sterile medium, supplemented with 10–20 percent serum, and taken to the laboratory. Under the stereomicroscope, embryos are flushed from the oviduct by inserting a blunt-tipped 30-gauge needle into the infundibulum and injecting approximately 100 microlitres of culture medium. Two-cell embryos collected by this technique are transferred to a fresh aliquot of medium in another culture dish. The dish is incubated at 37°C. Approximately 24 hours after embryo recovery, most embryos should be at the four-cell stage. Seventy-two hours after recovery, embryos will be in the early blastocyst stage, and embryos should reach the hatching blastocyst stage by 96 hours after recovery.
The medium and culturing conditions are suitable for embryo transfer if more than 75 percent of the two-cell mouse embryos develop to hatching1 blastocysts under these conditions. Mouse embryos culture should be performed periodically to monitor the culture conditions, and is mandatory whenever any changes are made in media, equipment, or culture conditions (Dandekar and Quigley, 1984).