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Appendix B: Embryo Transfer: An Assessment of the Risks of Disease Transmission
Appendix B: Embryo Transfer: An Assessment of the Risks of Disease Transmission
Pages 171-214
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From page 171... ...
Even if existing methods were optimal, the conservation of genetic resources by these means would only be useful if the stored germplasm were free from infectious disease. The main purpose of this appendix is to assess the disease transmission potential of embryos and to identify those factors that can influence that potential.
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From page 172... ...
Depending on the species, embryo collection is carried out either surgically or nonsurgically. Nonsurgical methods are preferable in that they do not damage the reproductive tract, are repeatable, and can be carried out on the farm.
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From page 173... ...
Pig embryos are successfully frozen at expanded blastocyst (entire zone pellucida) and early hatched stages, but preg nancy rates are very low.
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From page 174... ...
After the seventh or eight day, equine embryos are protected by a capsule and not the zone pellucida, and thus, equine pathogens would have to be capable of crossing this protective barrier. Other factors that help to reduce the disease transmission potential of early embryos are inherent in the techniques that are used in the collection and processing of embryos.
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From page 175... ...
Each viral pathogen must, therefore, be investigated to determine its ability to penetrate the zone pellucida and infect the embryo or to adhere to the zone so firmly that it cannot be removed by washing. MECHANISMS OF DISEASE TRANSMISSION BY EMBRYO TRANSFER For infectious disease transmission to occur through an embryo, a disease agent has to be transferred (1)
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From page 176... ...
Thus, it is essential that all substances that come into contact with the embryo be sterile. STUDIES OF THE DISEASE TRANSMISSION POTENTIAL OF EMBRYOS Most of the research on the disease transmission potential of embryos has involved either the in vitro exposure of embryos to pathogens or the collection of embryos from acutely infected donors (in viva experiments)
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From page 177... ...
TRANSMISSIBILITY OF SPECIFIC AGENTS BY EMBRYO TRANSFER The following is a comprehensive review of the work that has been carried out on the disease transmission potential of embryos. Much of the work was undertaken before development of the procedures for washing and processing embryos that are recommended by the International Embryo Transfer Society and endorsed by the Office International des Epizooties (see Annex Bob.
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From page 178... ...
and lowered the amount of virus on the embryos. The evidence suggested that most, if not all, of the virus was on the zone pellucida.
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From page 179... ...
Both zone pellucida-intact and zone pellucida-free bovine embryos were exposed to BTV, washed, and either assayed directly or cultured and then assayed. In addition, embryos were collected from bluetongue viremic donors bred with uninfected semen and from uninfected donors bred with BTV-infected semen.
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From page 180... ...
. Zona pellucida-intact and zone-free bovine embryos have been exposed to the virus in vitro, washed, and then either assayed or transferred to uninfected recipients.
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From page 181... ...
B~DV did not infect zone pellucida-intact bovine or ovine embryos, nor did it have any effect on in vitro embryonic development. Proper washing was effective in removing BVDV from all zone pellucida-intact embryos.
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From page 182... ...
Bovine and porcine zone pellucida-intact embryos and bovine zonafree embryos were exposed to high levels of infectivity of FMDV, washed, and assayed in tissue culture and steer tongue. Both bovine and porcine embryos were collected from FMDV-viremic donors.
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From page 183... ...
2. Once the zone pellucida was removed from bovine embryos, proper washing was no longer effective in removing this virus from the embryos.
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From page 184... ...
HCV was found to adhere to the zone pellucida when 171 porcine embryos were exposed to the virus and washed. This virus had no effect on the in vitro development of the embryos, and trypsin treatment was found to be effective in rendering embryos noninfectious if they had been exposed to less than 106 infectivity units of HCV.
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From page 185... ...
Both the low level of the virus isolated from the embryos and the susceptibility of this virus to trypsin and antiserum suggest that IBRV attaches to the zone pellucida of embryos and cannot penetrate it to gain access to the embryonic cells. Once the zone is removed, IBRV replicates in the embryonic cells.
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From page 186... ...
Results and Conclusions When 38 porcine embryos were exposed to PPV in vitro, washed, and assayed, the virus was found to adhere to the zone pellucida of all of the embryos. When 76 embryos were exposed to the virus in vitro, washed, and transferred into four uninfected recipients, the recipients became PPV seropositive.
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From page 187... ...
All embryos were negative if the exposure level was below 106 infectivity units and positive if above 106. These results suggest that at high levels of infectivity, PrV can adhere to the zone pellucida and that washing is not effective in rendering embryos noninfectious.
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From page 188... ...
Results and Conclusions 1. Preliminary results indicate that 3 percent of zone pellucidaintact bovine embryos are associated with REV after in vitro exposure and washing (Dulac and Singh, unpublished results)
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From page 189... ...
Trypsin treatment was found to be effective in rendering both porcine and bovine embryos noninfective for NISNI. The need for treating embryos derived from VS\T-infected donors with trypsin remains unknown.
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From page 190... ...
In addition, proper washing in the presence of antibiotics was shown to be effective in rendering zone pellucida-intact bovine embryos free of many disease agents (Akabane virus, bluetongue virus, bovine leukemia virus, bovine viral diarrhea virus, and foot-andmouth disease virus, as well as Brucella abortus and Haemophilus somnus)
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From page 191... ...
However, since over 800 embryos have been transferred out of PrV-seropositive donors without any disease transmission, it would appear that under normal circumstances this virus is not excreted in significant amounts into the reproductive tract. Fewer transfers have been carried out from infected pigs to susceptible pigs than from infected cattle to susceptible cattle; consequently, a final conclusion on the safety of using embryo transfer for disease control in the pig is not possible.
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From page 192... ...
This is far below the 10 washes recommended by the International Embryo Transfer Society, which have been proven to be effective. Since low levels of many pathogens have been isolated from the uterine flush fluids of infected donors, proper washing of embryos is essential.
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From page 193... ...
Although the protocols have been demonstrated to be reliable for a number of disease agents, the health status of the embryo is entirely dependent on the care taken during the embryo collection and processing procedures. For the more serious diseases, many countries might insist on having their own people involved in all stages of the flushing, collection, washing, and freezing to monitor quality control.
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From page 194... ...
· The disease agents that can be excreted into the reproductive tract of infected animals must be identified. Some disease agents adhere to the zone pellucida of embryos in vitro, and yet embryos collected from donors infected with those agents do not transmit disease when they are transferred to susceptible recipients.
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From page 195... ...
virus on preimplantation bovine embryos: A preliminary study. Theriogenology 11:81-89.
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From page 196... ...
XII. The in vitro exposure of zone pellucidaintact porcine embryos to hog cholera virus.
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From page 197... ...
1986. In vitro exposure of preimplantation bovine embryos to vesicular stomatitis virus.
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From page 198... ...
IX. The in vitro exposure of zone pellucidaintact porcine embryos to swine vesicular disease virus.
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From page 199... ...
1987. In vitro exposure of reimplantation bovine embryos to Haemophilus somnus.
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From page 200... ...
Research has shown, however, that many of the disease agents that adhere to the zone pellucida can be removed by briefly treating the embryos with trypsin. This treatment, which minimizes the risk of disease transmission, does not affect embryo viability adversely.
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From page 201... ...
Micromanipulation of Embryos As long as embryos are micromanipulated after being washed properly, the actual micromanipulation itself should not alter the health status of the embryo. This might not hold true, however, if foreign zone pellucidae were used in the embryo micromanipulation process.
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From page 202... ...
If embryos are properly processed, the evidence indicates that disease transmission will rarely, if ever, occur. What may turn out to be more important to the health of the embryo (fetus)
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From page 203... ...
Appendix B / 203 ANNEX B-2 Tables Summarizing Research Data The following seven tables summarize the research data presented in Annex B-1.
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From page 204... ...
204 cn o x to Cal 5 ¢ cn of 5 Cal o ·, ._ C)
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From page 205... ...
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From page 206... ...
206 Cal o 50 u o a, cn o C x au be o o a; a' Em ·_4 5k o u a, Cal 1 Cal ¢ EM _` U)
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From page 207... ...
207 be o a; U)
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From page 208... ...
208 _` a, a' cn o o au o ·_' ._, CO o o 50 a, Cal o V)
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From page 209... ...
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From page 210... ...
210 in o o a i_ o · · V)
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From page 211... ...
211 ~ ' v ~ ~ ~ cJ - ~ ~ ~ ~ ~ ~ .m ~ .m ~ .m -o - ~ - ' ~ - ~ id, ~ =4 ~ cL, au .
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From page 212... ...
abortus = Brucella abortus; BLV = bovine leukosis virus; BTV = bluetongue virus; CAEV = caprine arthritis and encephalitis virus; FMDV = foot-and-mouth disease virus; HCV = hog cholera virus; IBRV = infectious bovine rhinotracheitis virus; SVDV = swine vesicular disease virus.
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Key Terms
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