Embryos derive nutrients from the fluid in which they are bathed; moreover, of perhaps more importance, embryos depend on the ambient fluid to maintain their physiological integrity. To date, no chemically defined medium has been formulated to support normal development of bovine embryos satisfactorily for longer than about 24 hours. However, several media are commercially available (or can be prepared easily) that adequately maintain embryos for the usual interval between collection and transfer. For longer periods, cryopreservation is recommended.
For short periods of culture, the nutrient properties of the medium are far less critical than pH, osmolality, temperature, sterility, and lack of toxicity. An embryo's range of tolerance for these properties is narrow. Inadequate control of these aspects of culture accounts for many failed transfers. In addition, embryos are damaged if exposed to excess light (e.g. microscope lights) for prolonged periods, but normal lighting or daylight in the work area is not harmful if embryos are not exposed for more than half an hour.
Body temperature of cattle is 39°C, and for certain purposes this temperature is appropriate. However, for routine commercial embryo transfer we do not recommend storage above 37°C because thermometers, even those on incubators, are frequently incorrect by 1° or 2°C. Culturing embryos at 41°C is extremely damaging, whereas storing them for up to 12 hours between 18° and 37°C is not.
The simplest medium is sterile saline (9 gm NaCl/litre of sterilized, deionized or distilled water). Saline will not support embryonic development, but will keep embryos alive for a few hours; it is better to use saline and transfer embryos quickly than to use a more complex medium without a proper buffering system. Moreover, saline can be steam-sterilized (at least 30 minutes at 121°C under a pressure of 104 kilopascals) if a 0.22-μm biological filtration system is not available. A serious problem with saline, however, is that embryos will float or stick to plastic or glass unless a macromolecule such as bovine serum albumin (BSA) or serum is added, both of which require membrane filtration for sterilization. BSA and serum are sterile when purchased.
Recommended culture conditions
|Temperature||Room temperature (15–25°C) or 37°C in incubator|
|Buffer||Phosphate or bicarbonate ion (latter must be maintained under 5 percent CO2 atmosphere)*|
|Sterilization||Filtration of medium through 0.22-μm-pore membranes, aseptic techniques; sterile equipment; addition of 100 IU penicillin G, and 50μg streptomycin sulphate per ml, or 25 μg/ml gentomycin sulfate; addition of antimycotics sometimes indicated|
|Macromolecule||Sterilized, heat-inactivated serum or serum albumin (e.g. Fraction V, bovine serum albumin)|
* There is anecdotal evidence that HEPES buffer is detrimental to bovine embryos.
For most applications, we recommend a modified Dulbecco's phosphatebuffered saline (PBS). It is easy to use because it does not have to be equilibrated and maintained in an atmosphere of 5 percent CO2 in air. It can be prepared from stock reagents (Table 4) or purchased in either ready-to-use or concentrated form, which must be diluted with sterile, deionized or distilled water. A frequent error is failure to dilute concentrated medium; embryos contract markedly when exposed to the high osmolality. Commercially available Dulbecco's PBS does not usually contain antibiotics, sodium pyruvate, glucose or macromolecules.
The least important ingredient is the Na pyruvate; neither Na pyruvate nor glucose is needed for most applications. The CaCl2 and MgSO4 can also be eliminated if embryos are to be kept in vitro only a short time. If these are omitted, osmolality should be adjusted by adding extra NaCl.
Modified Dulbecco's phosphate-buffered saline (to make 10 litres)
|CaCl2.2H2O||1.32 g||Membrane/enzyme function|
|MgSO4.7H2||1.21 g||Membrane/enzyme function|
|The above may be weighted in advance and stored indefinitely in a sterile bottle under refrigeration|
|NaCl||80.0 g||Osmotic balance; neutralize charge cell membrane|
|Na2HPO4||11.5 g||Buffer to maintain pH|
|KH2PO44||2.0 g||Buffer to maintain pH|
|Glucose||10.0 g||Energy source|
|Na pyruvate||0.36 g||Energy source|
|Streptomycin sulfate||0.5 g||Prevent growth of microorganisms|
|Na penicillin G||1 000 000 units||Prevent growth of micro-organisms|
|Mixture Two may be weighed in advance and stored dry in a sterile bottle under refrigeration for six months|
|Combination of mixtures One and Two|
|Dissolve the reagents in mixture Two in 8 litres of deionized or distilled water. Dissolve mixture One in 2 litres of deionized or distilled water. Add these 2 litres to the 8 litres stirring constantly. Other methods of dissolving these ingredients often result in the formation of a precipitate. Sterilize medium by passage through a 0.22-μm bacteriological filter.|
Other media used to culture bovine embryos include Tissue Culture Medium-199 (TCM-199), Ham's F-10 medium, and Brinster's Mouse Ova Culture Medium-3 (BMOC-3). All of these are commercially available. TCM-199 with Hank's salts does not depend on CO2 for buffering, but TCM-199 with Earle's salts as well as Ham's F-10 and BMOC-3 must be maintained under an atmosphere of 5 percent CO2 in air.
Supplementation of media with a large protein molecule decreases surface tension, which reduces the tendency of the embryo to float or adhere to plastic or glass, and helps to inactivate heavy metals and other toxins. However, macromolecules derived from serum are also a possible vector for viral infection of embryos. BSA and bovine serum are currently recommended sources of macromolecules. Polyvinyl alcohol and polyvinyl pyrrolidone are being studied as possible non-biological macromolecules to circumvent the danger of infection. These macromolecules do not function as well as BSA in reducing surface tension or chelating toxins.
BSA (Fraction V) should be added to medium just before use at a concentration of 0.05–0.1 percent for flushing and 0.4 percent for culture. The powder should be poured very gently on the surface of the medium and be allowed to dissolve for about 20 minutes without stirring or shaking (otherwise it turns into a glutinous blob or makes the medium unusably frothy). After the BSA has dissolved, the container should be inverted gently five or six times to mix in the BSA completely. The medium should then be sterilized by passage through a 0.22-μ bacteriological filter. Purer types of BSA are also acceptable. BSA can also be purchased as an aqueous solution, e.g. 7 percent BSA in water.
If serum is used, steer or calf blood should be harvested into sterile, sealable containers directly from the vein (to avoid contaminants from skin and hair). After coagulation, clots are cut every 2–3 cm with a sterile knife, and the blood stored overnight at 5°C. Clots are then filtered out and the serum centrifuged at 2000 X g for 12–15 minutes. The sediment is discarded and the supernatant recentrifuged. This step is repeated once again. The final supernatant is sterilized by passage through a 0.22-μ bacteriological filter into sterile containers of convenient size. In order to inactivate the protein complement, which can be toxic to embryos, serum must be treated in a 56°C water bath for exactly 30 minutes after the serum has reached 56°C. Serum can be frozen and stored for up to eight months provided that the containers are tightly sealed. For quality control, one aliquot from each batch of serum should be incubated overnight in a sterile, sealed container at 37°C and examined the next day for bacterial contamination. For some situations, such as international movement of embryos or maintenance of specific-pathogen-free herds, additional sterilization of serum by gamma irradiation is recommended (Manual of the International Embryo Transfer Society, 1987). Heat-inactivated, sterilized serum is added to medium instead of BSA at a concentration of 0.5–1 percent for flushing and 10 percent for storage.
Embryos should be stored in small (<5 ml), sterile, transparent, sealable, inert containers. Small test tubes, Petri dishes or multi-well plates are convenient for routine use (Figure 13), but small test tubes are recommended if embryos must be moved any distance before loading into straws. If the embryos are to be stored in uncovered containers for more than 20 minutes, however, the medium should be covered with a thin layer of non-toxic paraffin oil to prevent evaporation and contamination and to regulate the rate of gas exchange between the medium and the surrounding atmosphere. Paraffin oil can also be used with other types of containers and is recommended if sealable containers are not used. Except while they are being manipulated, embryos should be stored in a dark, dust-free incubator or cabinet (e.g. a refrigerator or an ice chest with a tightly-fitting lid set at room temperature or 37°C).
Left: Plastic multi-well plate with 16 × 17-mm wells (lid to left);
Right: 60 × 15-mm and 35 × 10-mm culture dishes, and a 12 × 75-mm test tube
Many devices have been used to manipulate embryos. Standard Pasteur pipettes are much too large and the tips require fire polishing to prevent damage to embryos. We prefer to make our own pipettes from Pyrex glass tubing with a 4-mm outside diameter. Glass, in 15-cm lengths, is heated in the centre with a Bunsen burner and pulled to make an outside diameter of less than 1 mm. This is then scored with a diamond pencil and broken to make two pipettes. All ends are fire polished. After pipettes have been washed and rinsed thoroughly, they are placed in clean glass test tubes with screw tops and sterilized (and dried) by dry heat.
For use, pipettes are connected to a 0.5- or 1-cc syringe or plastic mouthpiece with rubber tubing (see Figure 32). Pipettes are made in batches of several hundred. They can be reused if debris does not adhere and they are placed in a soapy water bath immediately after use. In practice, we discard most of them after one use. Alternatives to making pipettes are to use tomcat catheters, 0.25-cc French straws (which, however, are too large for good washing procedures), disposable micropipettes, or pipette tips of various sorts. Care must be taken to wash, sterilize, and rinse these devices with sterile medium prior to use (see Chapters 15 and 18).