D .A. Stringfellow, M.D. Givens Livestock Production Science 62 2000 237 –251
243
at 50 3 magnification. Trypsin treatment has the not associate with zona-pellucida-intact embryos
same general requirements as washing except that after in vitro exposure when the 10-wash protocol
there are 12 washes. Embryos are pre-washed five described above was used Bowen et al., 1982; Singh
times, exposed to trypsin in a 6th and 7th wash for a et al., 1982a. In comparing this 1990 report to
total of 60 to 90 s and then washed five more times previous reports, the logical conclusion would seem
without trypsin. to be that inadequate washing allows a greater
Depending on circumstances, donor testing might potential for the transmission of disease with em-
still be used in conjunction with or in lieu of embryo bryos.
treatment to certify health of transported embryos The reputed value of the 10-wash protocol for
Anonymous, 1994. However, it is noteworthy that ensuring freedom from foot-and-mouth disease virus
the Research Subcommittee of the IETS has con- was reaffirmed in a report by Camaano et al. 1993.
cluded that sufficient evidence has accrued to show In their study, 94 zona-pellucida-intact bovine em-
7.5
that the risk of transmission of certain diseases is bryos and ova were exposed to high titers 10
of negligible if embryos are properly treated between
foot-and-mouth disease virus for 16 h and washed as collection and transfer. These diseases are blueton-
recommended in the IETS protocol. Then 79 em- gue, Brucella abortus, enzootic bovine leukosis,
bryos ova were assayed immediately for infectious foot-and-mouth
disease, and
infectious bovine
virus while 15 embryos were cultured for 24 h before rhinotracheitis IETS to the OIE; Anonymous,
testing. No virus was isolated from any of the 1998a.
properly washed embryos ova, and exposure to the virus had no apparent effect on embryonic develop-
ment in the cultured group.
5. Embryo–pathogen research in the 1990s
5.2. Testing novel treatments for bovine embryos The emphasis of embryo–pathogen research in the
after artificial exposure to pathogens 1990s has shifted to deal more with the newer
in-vitro-derived embryo
technologies. However,
Since early studies identified prokaryotic patho- there have been some additional reports with rele-
gens that adhered to the zona pellucida after artificial vance to in-vivo-derived bovine embryo production,
exposure and washing or trypsin treatment e.g. and some questions still remain to be answered.
mycoplasmas; Britton et al., 1988; Bielanski et al., 1989; Riddell et al., 1989 there has been only slight
5.1. Additional ‘in vitro exposure and in vitro progress towards development of effective treatments
assay ’ studies with bovine viruses
to deal with these pathogens. Use of antibiotics in embryo culture and wash media has continued to be
In a report by Gillespie et al. 1990, the infective based on conventional recommendations for their use
status of bovine embryos after artificial exposure to in cell culture media Riddell and Stringfellow,
seven viruses was described with results seeming to 1998, but some insight has been gained into how
contradict those in previous studies. They declared antibiotics could be applied to combat prokaryotes.
that pseudorabies virus, bovine herpesvirus-1, ves- Increasing the concentration and time of exposure to
icular stomatitis virus, bluetongue virus, and bovine currently used antibiotics as well as increasing the
viral diarrhea virus all adhered to the bovine zona temperature of treatment media have all been tried.
pellucida after in vitro exposure but that bovine Riddell et al. 1993a reported that treatment with
enterovirus and parainfluenza-3 virus did not adhere. kanamycin 1000 mg ml or tylosin 200 mg ml
Unfortunately, embryos were either not washed or was effective for producing Mycoplasma bovis-free
washed only five times between exposure and assay, bovine embryos when the artificially exposed em-
indicating that their methodology may have been bryos were washed 10 times and then incubated
responsible for results that conflicted with those in 378C for an additional 4 h in media containing the
previous reports. For example, in early studies antibiotic. The concentrations of antibiotics in these
bluetongue virus and bovine viral diarrhea virus did treatments were higher than recommended by their
244 D
.A. Stringfellow, M.D. Givens Livestock Production Science 62 2000 237 –251
suppliers, but treatments had no apparent detrimental were effectively removed when embryos were col-
effects on the embryos. In a similar study, Otoi et al. lected from bovine leukemia-infected donor cows.
1993 demonstrated that bacteria-free bovine em- Subsequently, 585 embryos collected from naturally
bryos could be produced after artificial exposure to infected donors were washed four times and trans-
Escherichia coli if they were incubated 38.58C for ferred, resulting in 278 pregnancies. Four calves
2 h in medium with gentamicin 50 mg ml prior to dying at parturition were not tested, but the other 274
washing 10 times. In an attempt to develop a novel were free of bovine leukemia virus.
treatment, Riddell et al. 1993b tried to use organic In a small study, Schlafer et al. 1990 incubated
halamines to inactivate Mycoplasma bovis adhering day 6 embryos in bluetongue virus-infected cell
to bovine embryos. The mycoplasma were effective- cultures for 24 h, washed them three times and then
ly inactivated, but there was insufficient margin for transferred two embryos into each of three seronega-
error between mycoplasmacidal and embryocidal tive recipients. None of the three heifers became
concentrations to recommend regular use of these pregnant, but virus was isolated from blood and a
chemicals. vaginal swab from one of the three heifers taken on
In a novel approach for inactivation of viruses the 7th day after the embryos were transferred.
associated with embryos, Bielanski et al. 1992 Because of the experimental procedures used, it is
successfully used photosensitive agents hemato- impossible to determine if adherence of the virus to
porphrin and hematoporphrin derivative to inacti- embryos or inadequate washing was responsible for
vate bovine herpesvirus-1 and bovine viral diarrhea transmission of the virus.
virus. Their treatment had no apparent negative In a large scale study reported by Acree et al.
effect on the viability of embryos, but there is a need 1991, 60 heifers were artificially exposed to
to determine if normal rates of pregnancy with birth bluetongue virus. Embryos were collected from 59 of
of normal calves could be achieved after transfer of these heifers during either the acute or convalescent
embryos treated in this way. stage of the disease. Embryos also were collected
Certainly, added security could be provided by from serologically positive donors that were pre-
new broad-spectrum treatments for embryos, but sumed to have been naturally infected during the
there appears to be little current interest in research previous year. Thus, they were considered to be
in this area. Presumably, this is because current recovered donors. A total of 169, 141 and 52
protocols for certifying the health of embryos have embryos were collected from acute, convalescent and
been efficient and effective. recovered donors, respectively, and washed 10 times
according to the IETS standard procedure. In vitro 5.3. Collection and assay of embryos from infected
assays cell culture and embryonating chicken eggs donor cows or transfer of embryos after in vitro
of 57, 20 and 25 of the embryos, respectively, were exposure to pathogen
negative for bluetongue virus. Furthermore, 248 embryos 110 from acute, 121 from convalescent and
In additional investigations in the 1990s, embryos 17 from recovered donors were transferred to
were collected from donors that were artificially or seronegative recipients. A total of 95 calves were
naturally infected with bovine leukemia virus, born 36 from acute, 52 from convalescent and seven
bluetongue virus, foot-and-mouth disease virus or from recovered donors. There was no evidence of
bovine spongiform encephalopathy. Also, in one transmission of bluetongue to any recipients or
study, embryos were subjected to long term exposure offspring with embryos from acute or convalescent
in vitro to bluetongue virus. Then, as in earlier stage donors. However, two recipients of embryos
studies, the embryos were evaluated for their po- from recovered donors and subsequently their off-
tential to transmit the pathogens by direct assay in spring seroconverted between the 5th and 9th month
vitro or by transfer to recipients with subsequent after transfer of embryos. Subsequent investigation
evaluation of recipients and offspring for pathogen. yielded evidence that these recipients were naturally
Krolinski et al. 1994 evaluated a four-step, exposed to bluetongue virus in mid to late pregnancy
embryo wash procedure to ensure that lymphoid cells due to a breach in isolation of recipients. Failure to
D .A. Stringfellow, M.D. Givens Livestock Production Science 62 2000 237 –251
245
adequately protect the latter recipients from insect birth and two fetuses aborted at 5 months of gesta-
vectors is unfortunate, but failure to transmit virus tion. Sera from normal calves and one set of
with embryos from all of the acutely and convales- premature twins were negative for anti-foot-and-
cently infected donors is still a strong endorsement mouth-disease virus antibody. Thus, collective re-
for the IETS washing protocol. sults showed that the IETS protocol for washing was
There were two reports of studies evaluating effective for producing embryos without detectable
potential for use of embryo transfer to salvage amounts of associated virus regardless of whether in
genetic material from foot-and-mouth virus-infected vitro or in vivo assays were used.
cattle. Villar et al. 1990 reported on a field trial in Finally, the enormous task of determining if
Argentina in which 253 embryos were recovered properly washed embryos from donor cows with
from 48 foot-and-mouth virus seropositive convales- clinical bovine spongiform encephalopathy BSE
cent cows and washed according to IETS pro- might carry the disease to recipients or their embryo
cedures. The flushing fluids and 171 of the embryos transfer offspring was begun in 1990. Because of the
were tested for presence of virus by isolation in cell long incubation period of the disease, this study is
culture or intradermal lingual inoculation of negative not scheduled to conclude until the year 2001. A
cattle. All results were negative. In addition, the current update of results is found elsewhere in this
remaining 82 embryos were cryopreserved and 42 issue see BSE update by Wrathall. Preliminary
were transferred to seronegative recipients in a foot- progress reported by Wrathall et al., 1997 indicated
and-mouth-disease-free area of the country. Fourteen that bioassays of degenerate embryos ova and
live calves were born. The calves and recipients uterine recovery medium from BSE affected cows
remained seronegative. Thus, the washing procedure were all negative.
and embryo transfer procedures were confirmed to be useful for preservation of germ plasm from infected
populations of cattle.
6. Bovine viral diarrhea virus BVDV
