Bovine embryos can be cryopreserved easily. If procedures are carried out correctly, pregnancy rates are 75–85 percent of those for unfrozen embryos transferred under similar circumstances. The following protocol has worked well in a variety of settings, but attention to detail is required.
Start with good to excellent quality embryos recovered six to eight days after the donor's oestrus. Embryos should be frozen within three to four hours of recovery.
Wash embryos through at least three changes of medium (ten washes if embryos are to be exported or if it is suspected that embryos have been exposed to infectious disease; see Chapter 16) of sterile Dulbecco's phosphate-buffered saline plus 0.4 percent bovine serum albumin (BSA) or 10 percent heat-treated serum (steer serum, newborn calf serum, or foetal calf serum are all satisfactory; however, serum should not be used if embryos are to be exported).
Standard antibiotic concentrations should be used; added pyruvate and glucose are optional.
Embryos should be evaluated morphologically and then placed into PBS plus 0.4 percent BSA (or 10 percent serum) plus 10 percent glycerol (freezing medium) for 10–20 minutes. All of the above steps are done at room temperature. At this point, containers should be labelled and relevant data recorded (see Chapter 16).
Rinse pre-labelled 0.25- or 0.5-cc French straws twice with freezing medium (up to, but not including, the cotton plug) to remove any toxic residues. Next, fill the straw half-way with freezing medium, then an air bubble of 4–6 mm, then another column of freezing medium containing the embryo so that the straw is 90 percent full when the cotton end is wetted (see Figure 26). An optional step is to add 1.5–2 mm of non-toxic paraffin oil to the top of the column. The end is then sealed with heat (for example, by heating a haemostat with a cigarette lighter and then clamping the end of the straw) or polyvinyl chloride powder (PVC) (see Figure 28). The straw is placed into the freezing machine horizontally or, if a vertical system is used, with the heat- or PVC-sealed end down so that the embryo sinks and rests on the paraffin oil.
One function of the paraffin oil is to flatten the meniscus to prevent mechanical damage to embryos that get caught in the angle of the meniscus and the wall of the straw when ice forms. This is critical for the smaller mouse embryos but of minor importance for bovine embryos. Paraffin oil is of no value for this purpose if straws are frozen in a horizontal position. A second benefit of paraffin oil is to prevent embryos from entering the air space next to the heat seal. This results in death of the embryo during freezing. Without paraffin oil, embryos enter this air space easily unless straws are handled very gently.
FIGURE 28
Illustration of sealing a plastic straw by heat (A) and by tamping polyvinyl chloride powder into
the end of the straw before wetting (B); completed seals (C)
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Cool straws to -7°C. The rate of cooling during this step can be slow or rapid.
Seed straws after they have been at -7°C for five minutes, and keep them at -7°C for an additional 10 minutes. Be sure that they remain seeded. Seeding is accomplished by touching the side of the embryo container with forceps dipped into liquid nitrogen (Figure 29A). Automatic seeding occurs with some freezing machines, but not all self-seeding systems are reliable in all circumstances.
Cool straws from -7°C to -30°C at 0.5°C/minute. When straws reach -30°C, plunge them into liquid nitrogen (within two to three minutes) and store in liquid nitrogen (Figure 29B). The equipment to cool embryos can be simple or complex. The only advantage of complex equipment is saving labour. The cooling rate should average 0.5°C/minute (it can fluctuate briefly between 0.3° and 0.7°C).
Thaw 0.5-cc straws by holding them quietly in the air for exactly 20 seconds followed by 20 seconds in a 37°C water bath; 0.25-cc straws should be thawed for 15 seconds in the air plus 15 seconds in 37°C water. After thawing, do all the steps at room temperature.
FIGURE 29
Inducing formation of ice crystals by touching the walls of the straw with forceps cooled in
liquid nitrogen (A), and transferring a straw with a frozen embryo in an insulated container of
liquid nitrogen from the freezing machine to the liquid nitrogen tank (B)
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Next, isolate the embryo. This is done by cutting the heat-sealed end of the straw with clean scissors and expelling the embryo by pushing on the cotton plug. Glycerol may be removed from embryos in several ways. The standard method is to dilute in six steps: PBS plus 0.4 percent BSA plus 8.3 percent glycerol, 6.7 percent, 5 percent, 3.3 percent, 1.7 percent and then 0 percent glycerol, six minutes per step at ambient temperature. Theoretically, a better approach is to use unequal steps, e.g. 7 percent, 5 percent, 3.5 percent, 2 percent and 1 percent; although this is rarely done, perhaps it should be.
An alternative is four steps: (1) 6 percent glycerol plus 10 percent sucrose; (2) 3 percent glycerol plus 10 percent sucrose; (3) 10 percent sucrose (all in PBS plus 0.4 percent BSA); and then (4) PBS plus 0.4 percent BSA with no sucrose or glycerol. Instead of 0.4 percent BSA, 10 percent serum can be used. Each step should take six minutes, or five minutes in warm conditions (above 25°C). Both procedures lead to similar results, but the four-step method is faster.
Glycerol can also be removed in one step by placing embryos in 20–30 percent sucrose plus 0.4 percent BSA with no glycerol for five minutes. We have little direct experience with this method, but others have used it successfully. With some modifications in strategy, the dilution of cryoprotectant can be done directly in the straw, thus circumventing the need to manipulate the embryo between thawing and transfer (Leibo, 1988).
Evaluate embryo and transfer as soon as feasible, preferably within a few minutes of removing the cryoprotectant, especially if the one-step procedure is used. Discard degenerate embryos (should be less than 5 percent if procedures are done properly). If recipients are available, transfer the degenerate embryos anyway (non-surgically). A few will turn into calves.
Variations in procedures have been used successfully by others. For example, glycerol can be replaced by 1,2 propanediol (propylene glycol). Some people use glass containers instead of plastic straws. These thaw more slowly, and embryos should therefore be cooled to -35° or -38°C before plunging when glass containers are used.
Further details about principles of cryopreservation may be found in Seidel (1988b). There are also some new approaches to cryopreservation that are much simpler, for example, vitrification. However, these cannot yet be recommended for routine cryopreservation of bovine embryos.
