The risk of transmitting genetic disease via embryo transfer is the same as that involved in natural mating or artificial insemination; wise selection of dams and sires is mandatory, no matter how cattle are propagated. There is no increased incidence of abnormal offspring due to these procedures (King et al., 1985). With this technology, however, there may be greater temptation to amplify reproduction of cows with a very high market, but questionable genetic, value, both because some kinds of infertility with a genetic basis can be circumvented and because the sale of the offspring can be very profitable.
Embryo transfer procedures can be used to control large-scale transmission of genetic diseases such as syndactyly by screening young bulls for undesirable recessive Mendelian characteristics before semen is distributed for artificial insemination (see Chapter 2; Johnson et al., 1980). Also embryos from parents with abnormal karyotypes can be biopsied and karyotyped, and only the normal ones transferred.
If proper procedures are followed, the risk of transmitting infectious disease via embryo transfer is lower than with natural mating or artificial insemination (Stringfellow, 1985; Hare, 1986; Hare and Seidel, 1987). Carelessness in even the smallest of details, however, greatly diminishes the advantage.
Arguments for the inherent safety of embryo transfer procedures are based on the physical characteristics of the embryo and the ability to test the environment of the embryo for the presence of pathogens and to treat the embryo to remove pathogens. Prior to collection, an embryo is exposed only to the oviductal and uterine environment of the donor. Thus, even when the donor is infected, there is little opportunity for infection by pathogens other than those present in uterine fluid and, rarely, by blood-borne pathogens that are introduced into the uterine lumen because of injury to the endometrium. Viruses are rarely found at infective levels in uterine fluid. Furthermore, pathogens present in the uterine fluid frequently do not adhere to, or are only loosely associated with, embryos. Pathogens can be removed entirely by washing embryos experimentally infected in vitro. Of great importance is the vast dilution of any pathogens by the flushing procedure. In addition to dilution and physical removal of infectious agents, antimicrobial agents can be added to the washing medium.
Primary resistance to pathogens is provided by the physical barrier of the zona pellucida. Nevertheless, some pathogens adhere to the zona pellucida (Hare, 1986); in some cases treatment with enzymes removes these agents. The presence of pathogens on or in the embryo cannot be diagnosed by examining the embryo microscopically with current techniques.
The possibility of infection of the embryo between recovery and transfer to a recipient can be controlled by ensuring a sterile environment. Again, conscientious attention to detail must be emphasized.
An issue apart from the infected or uninfected status of the embryo is whether transfer to a recipient of an embryo with adherent pathogens results in transmission of the disease. In most cases, it does not because the very few pathogenic organisms are eliminated by the body without causing a productive infection.
Continuously updated analyses of research on the interaction between embryos and pathogens and the transmissibility of infectious disease via embryo transfer are available (Manual of the International Embryo Transfer Society, 1987). Minimum standards for the sanitary handling of embryos are also presented in the Manual of the International Embryo Transfer Society. This manual is updated regularly to include recommendations based on the latest research and serves as a reference document of the International Zoosanitary Code. Currently recommended handling procedures are outlined below.
Embryo handling area (see Chapter 15).
Equipment and solutions must be sterile and free from contaminants (see Chapters 6 and 15). Care must be taken to avoid contamination of equipment while it is in use. For example, setting a pipette down on the bench for a moment to adjust the microscope could result in the introduction of infectious pathogens into the container of embryos if the pipette is reused (see Figure 32).
Washing embryos. As soon as embryos are isolated from the collection fluid, they should be pipetted in as small a volume of fluid as possible into a container of fresh medium. No more than 10 embryos should be placed in a single container, and embryos from different donors should never be placed in the same container. The container should be agitated gently. The embryos should then be transferred with a new sterile pipette to a second container of fresh medium. It is important to transfer as little fluid as possible from the first wash to the second wash, and the volume of medium in each wash should be sufficient to dilute the volume of fluid transferred in the pipette with the embryos one hundred times (2 ml is standard). The second container should be agitated gently before the embryos are transferred to a third container. These steps should be repeated until the embryos have been washed ten times in fresh medium. Containers should be covered to avoid contamination from dust and aerosols (see Figure 2). Containers and pipettes should not be reused without resterilization.
FIGURE 32
A mouth pipette laid on the work-bench (A) and a pipette attached to a tuberculin syringe laid
on the microscope stage (B) so that the tips of the pipettes do not touch any surface. Pipettes
are made of fine-drawn, fire-polished glass tubing.
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Trypsin treatment. So far, two viruses (infectious bovine rhinotracheitis virus and vesicular stomatitis virus) have been found to adhere to the zona pellucida of bovine embryos so firmly that normal washing procedures do not remove them. It is recommended that embryos exposed to these viruses be washed five times in phosphate-buffered saline to which antibiotics and 0.4 percent bovine serum albumin have been added, but without Ca++ and Mg++ (because they inhibit trypsin), then through two washes of trypsin of 30–45 seconds each, then again through five washes of saline containing antibiotics and 2 percent heat-inactivated serum (not bovine serum albumin) to inactivate the trypsin. The trypsin enzyme used to prepare the washes should have an activity such that 1 g will hydrolyse 250 g of casein in 10 minutes at 25°C and pH 7.6. The sterile trypsin solution should contain a concentration of 0.25 percent trypsin in Hank's balanced salt solution without Ca++ and Mg++. A ready-to-use solution is available commercially.
Evaluation of embryos. After embryos have been washed, they should be examined over their entire surface area at no less than 50X magnification. For purposes of disease control, only embryos that have an intact zona pellucida and that are free of adherent material should be transferred or cryopreserved.
Collection of diagnostic samples. Although current diagnostic procedures kill embryos, an indication of the health status of embryos can be obtained by testing samples of the fluids used to recover and wash the embryos and by testing unfertilized ova and non-transferable embryos recovered from the same flush. If embryos are recovered by examining the sedimented collection fluid, the last 100 ml in the bottom of the cylinder along with any debris should be retained in a sterile, sealable bottle for testing. If embryos are recovered by filtration of the collection fluid, the fluid should be retained in a sterile cylinder, allowed to settle for 30 minutes, and the bottom 100 ml transferred to a sterile bottle for testing. The fluid from the last four washes should be pooled and stored in a sterile, sealable bottle for testing. Degenerate embryos and unfertilized ova should be pooled, washed ten times, and stored for testing. Samples may be stored at 4°C if the tests are done within 24 hours or at -70°C if the interval until testing is longer.
It must be emphasized that the above procedures must be carried out with scrupulous regard for sterility and cleanliness or they will be of little avail. Every detail is important.
Regulations for exporting and importing embryos are usually different for each set of countries, and are often determined in part by political considerations. Nevertheless, most are based on the principles discussed in this chapter.
