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.A. Stringfellow, M.D. Givens Livestock Production Science 62 2000 237 –251
Embryo Transfer Society. In addition, results of of limiting factors work to prevent the occurrence of
more recent research are summarized with emphasis this complete sequence of events.
on bovine viral diarrhea virus. Finally, we discuss the impact that results of more recent studies and
2.1. Factors limiting exposure of embryos to accumulated experience with commercial embryo
pathogens transfer have had on our current view of strategies
for pathogen-free-embryo production. Under ordinary conditions, numerous factors tend
to restrict exposure of embryos to pathogens. Espe- cially important are, limited mobility of embryos,
2. Epidemiological view of embryo transfer limited distribution of pathogens and precautions
implicit in or applied to management of donor Details of agent, host and environmental factors
animals. which collectively support the use of embryo transfer
Between conception and collection, embryos are as a method for controlling the spread of pathogens
restricted to the uterine tubes and uterus of the donor were reviewed previously Stringfellow, 1985; String-
cow. Consequently, a cow that is uninfected serves fellow and Wright, 1987; Stringfellow et al., 1991
as an isolation unit for the preimplantation embryo and are only summarized here.
prior to collection. If herds or regions of origin of If a pathogen was to be transmitted by transfer of
donor cattle are free from specific pathogens, the in-vivo-derived bovine embryos, an uninterrupted
security of the isolation is enhanced. If a donor cow sequence of events would have to occur Fig. 1.
is infected with an agent of disease, it is still possible Key elements in this hypothetical chain of events
that embryos might remain unexposed. The classic include: 1 exposure of embryos to the pathogen,
example is a cow infected with Brucella abortus. 2 continued association of pathogen with the
While this bacterium is a reproductive pathogen, an embryos, 3 maintenance of infectivity of pathogen
accumulation of evidence from studies in the 1980s throughout embryo manipulation and processing, and
see review by Stringfellow and Wright, 1989 finally, 4 delivery of an infective dose of pathogen
indicated that exposure of preimplantation embryos to a susceptible recipient. It is evident that a variety
in the uterus of an infected, superovulated cow is
Fig. 1. Theoretical chain of events for a pathogen to be transmitted through transfer of an in-vivo-derived bovine embryo.
D .A. Stringfellow, M.D. Givens Livestock Production Science 62 2000 237 –251
239
highly unlikely. The primary reason is that brucella Riddell et al., 1993c; Betteridge, 1995 when com-
do not remain in the postpartum uterus after multiple pared to the murine zona pellucida about 5 mm
estrous cycles and will only return to the uterus long Gwatkin, 1967, and there is no current proof of
after conception when the fetus and placenta are well true vertical transmission via hereditary incorpora-
developed. tion of retroviruses or other pathogens in cattle. To
Donor cattle, representing the highest genetic illustrate the latter, a retrovirus of cattle, bovine
merit, are normally afforded the best available health leukemia virus, is not incorporated into the genome
care. The herds of origin are often free of many of gametes Divers et al., 1995. Without concern for
pathogens due to participation in disease-control infectious disease transmission via the gametes, it
programs and enforcement of judicious herd-replace- was especially important to confirm that the rela-
ment policies. The extent to which preventive man- tively thick bovine zona pellucida could serve as an
agement and medicine is practised can be evaluated effective barrier. In various studies, summarized
by the veterinarian who is a member of an official below, it was confirmed that pathogens would not
embryo collection team Evans, 1998. The team penetrate this barrier, and only a few would adhere
veterinarian also can clinically assess the health of to it. Still, an important concern was that pathogens
the donor cow at or around the time of embryo found in body fluids or as contaminants in media
collection. Finally, some national regulatory au- might remain in close proximity to the embryo until
thorities may request specific tests of donor cattle to the time of transfer.
further enhance safety when embryos are to be Techniques for collection, processing and transfer
moved internationally. of in-vivo-derived embryos vary, but in each situa-
tion, there are dilution factors associated with the 2.2. Factors limiting continued association of
sheer volume of recovery and holding medium that pathogens with embryos
would serve to dilute any pathogen that might be present in the embryo’s environment. Also, pre-
If embryos were to be exposed to pathogens, scribed procedures for washing, with or without
several factors tend to prevent continued association. trypsin, ensure that certain pathogens will be elimi-
These include inherent resistance of the embryo nated by dilution, dislodging, or inactivation String-
provided by the zona pellucida and natural or fellow, 1998. Finally, the use of antibiotics in
prescribed cleansing associated with embryo collec- media for recovery, culture and storage of embryos
tion and transfer. effectively deters the spread of some prokaryotic
The mammalian zona pellucida is a distinctive pathogens and suppresses nonpathogenic microbial
extracellular matrix that ensures species specificity of contaminants Riddell and Stringfellow, 1998.
fertilization, block to polyspermy and protection of the embryo during very early stages of development
2.3. Factors limiting infectivity and delivery of an Dunbar, 1983. However, it is well known that the
infectious dose of pathogens to recipients zona pellucida functions as a microporous mem-
brane, allowing traffic of relatively large molecules Media and techniques for collection, storage and
Sellens and Jenkins, 1975. This permeability and transfer of bovine embryos are intended to ensure
results of several early studies in laboratory animals that embryos maintain their developmental compe-
led to concerns that the zona pellucida might not tence, but they do not necessarily ensure that infec-
provide a significant barrier to pathogens. As exam- tivity of associated pathogens is maintained. An
ples, in one report, the passage of Mengo virus example of a negative impact of embryo processing
through the murine zona pellucida was demonstrated on viability of pathogen was illustrated in an early
Gwatkin, 1967, and in another report, the genetic study in which a standard embryo-cryopreservation
spread of the retrovirus that caused murine mammary procedure resulted in 64 or 99.9 reduction in
tumor was reported Bentivelzen et al., 1970. viability of Brucella abortus in the absence or
However, it is important to note that the bovine zona presence of antibiotics, respectively Stringfellow et
pellucida is substantially thicker up to 15 mm al., 1986.
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.A. Stringfellow, M.D. Givens Livestock Production Science 62 2000 237 –251
The last link in the hypothetical chain of infection recipient cows resulting in viable pregnancy with a
is delivery of an infectious dose of pathogen along high degree of consistency. The potential usefulness
with a viable embryo into the uterus of a susceptible of these procedures in international trade led to
recipient Fig. 1. It should not be presumed that all questions by regulatory authorities and scientists
recipients are susceptible via the intrauterine or any about the role of embryo transfer in transmission of
other route. Some may be resistant to infection based infectious diseases. Consequently, embryo–pathogen
on naturally acquired immunity through prior expo- interactions became an important topic of inves-
sure or induced immunity through vaccination. tigation.
Even if recipients are susceptible to infection with a Selection of infectious agents for study was some-
specific pathogen, the amount of infectious agent that what serendipitous; but usually, the pathogens were
is associated with a single embryo may not constitute either objects of national prevention, control and
an infective dose. The natural and prescribed obsta- eradication efforts, or there was some reason to
cles such as dilution factors and washing that were believe that embryos might be exposed to them.
described above would all tend to reduce the in- Most studies utilized one of four standard ex-
oculum. In reality, there has been no report of a perimental approaches Bielanski and Hare, 1998.
comprehensive study to deal specifically with in- In one of these approaches, zona pellucida intact
trauterine, day 7 inoculations with any bovine patho- embryos were exposed to pathogen in vitro, washed
gen. and then assayed in vitro to determine if infectious
agent was present. In Table 1, collective results are presented for some initial studies of this type. As
3. Early research providing the basis for regards the studies summarized in this table, it is
