Click for next page ( 172


The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement



Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.

OCR for page 171
APPENDIX B Embryo Transfer: An Assessment of the Risks of Disease Transmission Elizabeth Singh To date, the most extensive use of embryo transfer has been to increase the number of offspring from genetically superior fe- males. Other uses include planned matings, genetic testing for Mendelian recessive traits, twinning (in cattle), and the salvage of desirable genetic resources from infected animals. When embryo transfer is used in conjunction with cryopreservation, it also enables the stor- age and international movement of genetic material. The advantages that embryos have over semen in this regard is that they provide the complete genotype. Before techniques of embryo transfer can be used to conserve the genetic resources of all domestic species, however, additional research is required. 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 em- bryos and to identify those factors that can influence that potential. A background on the status of embryo transfer technology for the various species is also provided. For a more detailed review, the reader should consult other articles (for example, Betteridge, 1977; Hare, 1985; Mapletoft, 1987~. Elizabeth Singh is acting director of the Animal Diseases Research Institute, Ontario, Canada. 171

OCR for page 171
172 / Appendix B EMBRYO TRANSFER TECHNOLOGY IN DOMESTIC SPECIES Embryo transfer involves the collection of embryos from the ge- netic mother (donor) for transfer to surrogate females (recipients). Although single embryos can be collected and transferred, donors are generally superovulated to allow the collection of several em- bryos from each donor. A number of protocols are available for in- ducing superovulation in most livestock species. The gonadotropic follicle-stimulating hormone (FSH) and pregnant mare serum gona- dotropin (PMSG) have both been used for this purpose; they are given during the luteal phase of the cycle, the duration of which is con- trolled by the administration of prostaglandins, or at the end of a period of progesterone administration. 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. Generally, embryos are collected at the morula or early blastocyst stage. Using surgical methods, earlier stage embryos can be collected, although their cryopreservation is less successful. Regardless of their embryonic stage, embryos are usually collected and transferred while their zonapellucida is intact. Optimal pregnancy rates are obtained only when the preceding es- trus of the donor and recipient occur at about the same time. Prior to cryopreservation or transfer, embryos are evaluated on the basis of their morphology. Embryos rated good or excellent pro- duce the highest pregnancy rate or survival rate following cryo- preservation. The freezing process involves the slow cooling of em- bryos to an appropriate temperature and then direct transfer into liquid nitrogen. Thawing takes place by warming rapidly. At present, technologies for embryo transfer have allowed for in vitro fertilization, the sexing of embryos, and the splitting of em- bryos into parts to produce clones. Although there has been limited use of these techniques to date, they are bound to become increas- ingly important. The remainder of this section is a summary of the state of embryo transfer technology for the various domestic species. Cattle generally respond well to superovulation; they produce an average of 8 to 10 embryos/eggs per treated animal. The use of FSH would appear to be superior to the use of PMSG. Embryos are col- lected nonsurgically 6 to 8 days after estrus. Cryopreservation is extensively used in bovine embryo transfer programs, and transfers are now generally carried out nonsurgically. Generally, one embryo is transferred per recipient, and synchrony between the estrus cycle of the donor and the recipient is within 1 day. With existing technol

OCR for page 171
Appendix B / 173 ogy, five or six of the embryos collected per superovulated cow would be of transferable quality and they would produce three or four preg- nancies. Pregnancy rates are generally around 60 percent with fresh embryos and 40 to 50 percent with frozen embryos. In sheep and goats the average number of embryos/eggs obtained with superovulation is 10. Because fertilization failure frequently occurs in superovulated ewes, semen is often deposited into the tip of each uterine horn surgically. Generally, because of the tortuous nature of the cervix, embryo collection and transfer is carried out surgically. Recently, however, nonsurgical and laparoscopic methods of collection have been used successfully. Depending on the method of collection, embryos are collected either 3 to 4 (8- to 16-cell stage) or 5 to 7 (morula to blastocyst stage) days after estrus. Both freezing and micromanipulation are successful with ovine and caprine em- bryos. When embryos are to be frozen, they should be collected at the late morula and early blastocyst stage. Generally, two embryos are transferred per recipient. The requirement for synchrony in the estrus of the donor and recipient is the same for sheep and goats as it is for cattle. The survival rate of good embryos is 50 to 60 percent, and the number of offspring produced per collection averages five. In pigs synchronization can be difficult because prostaglandin is luteolytic only after day 10. Methods developed to overcome this problem include weaning piglets from sows, aborting sows 16 to 45 days after breeding, or administering prostaglandin to pseudopreg- nant sows. Donors can then be successfully superovulated with one injection of PMSG, but because pigs are multiple ovulators, this is unnecessary. Embryos are usually collected surgically 4 to 6 days after estrus, at which time the embryos range from the four-cell to the expanded-blastocyst stage. Although it is difficult to assess the quality of morula-stage embryos, it would appear that the best preg- nancy rates are obtained with this stage or are most successful when the donor's estrus is synchronous with or occurs either 1 or 2 days prior to or 1 day after that of the recipient. On the average, 30 embryos are produced with superovulation and 20 offspring are pro- duced per collection. Pig embryos are successfully frozen at expanded blastocyst (entire zone pellucida) and early hatched stages, but preg nancy rates are very low. In the horse, gonadotropins are generally not useful for inducing superovulation. Equine pituitary extract has been used, but the em- bryos produced resulted in poor pregnancy rates. Thus, single em- bryos are usually collected from donors following spontaneous ovu- lation. For reproductively sound mares bred to a fertile stallion, embryo recovery rates can be expected to be from 50 to 80 percent. Generally,

OCR for page 171
174 / Appendix B embryo collection is carried out nonsurgically 6 to 9 days after ovula- tion. The equine embryo loses its zone pellucida and is surrounded by a capsule about the seventh day. Transfers can be carried out surgically or nonsurgically. Pregnancy rates of about 50 percent are achieved with either method. Although equine embryos can be suc- cessfully frozen or bisected, limited use has been made of these tech- nologies in this species. THE DISEASE TRANSMISSION POTENTIAL OF EMBRYOS On purely theoretical grounds, the disease transmission potential of embryos is much less than that of either the live animal or semen. Depending on the species, embryos are usually collected and trans- ferred when they are 4 to 7 days old. Thus, prior to collection, there is a very short period of time in which an embryo can become in- fected. Moreover, the embryo is limited in terms of exposure to only those pathogens that are found in the reproductive tract of its mother. In addition, because embryos are collected at the zone pellucida-in- tact stage in most species, any pathogens in the reproductive tract must also be capable of penetrating this structure to gain access to the embryonic cells. 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 protec- tive barrier. Other factors that help to reduce the disease transmission poten- tial of early embryos are inherent in the techniques that are used in the collection and processing of embryos. Embryo transfer technol- ogy involves flushing embryos out of the reproductive tract with several hundred milliliters of fluid. This volume helps to dilute pathogens that might be present in the uterus. In addition, this technology allows for embryo washing and the antimicrobial and enzymatic treat- ment of embryos in order to enhance their freedom from disease. Finally, the majority of embryos involved in international trade are frozen, and cryopreservation has been found to be effective in inacti- vating low levels of the viruses that can adhere to embryos. There are no guidelines that can be used to predict which of the disease agents might be transmitted by embryo transfer. Each patho- genic organism has to be investigated individually. In addition, be- cause there are inherent differences in the zone pellucida of the dif- ferent species of embryos, the potential for disease transmission by embryo transfer of each pathogen must be investigated in each spe- cies. In terms of the major diseases of concern that might be transmit

OCR for page 171
Appendix B / 175 ted by embryo transfer, however, a number of conclusions are pos- sible. Embryos will be free of parasites, and in all likelihood, they will be protected from both bacterial and fungal agents by the pres- ence of an intact zone pellucida. These disease agents are too large to be able to cross this structure, and even if they do adhere to it, the presence of antibiotics and antimycotics in the-washing media will most likely inactivate them. Thus, the major diseases of concern will most likely be viral in nature. Each viral pathogen must, therefore, be investigated to determine its ability to penetrate the zone pellu- cida 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) in the embryo, (2) in or on the zone pellucida of the embryo, or (3) in the fluids in which the embryo is transferred. Each of these transmission mechanisms is described below. 1. The transfer of an infected embryo. If either the oocyte or spermatozoon is infected, the resulting embryo will be infected at the time of fertilization. It is generally accepted, however, that gametic infection is not a significant factor in embryonic infection. Few pathogens have been demonstrated in oocytes, and the majority of pathogens found in semen are in the seminal fluid and not associated with the spermatozoa (Eaglesome et al., 1980~. Even if agents do become adsorbed to the surface of the spermatozoa, it is unlikely that those agents would infect an embryo at fertilization because most of the outer membrane and contents of the acrosome are lost from spermatozoa that penetrate the zone pellucida. Thus, if embryos do become in- [ected, they do so by coming in contact with a pathogen in the repro- ductive tract of their mother. This pathogen-may have been intro- duced into the reproductive tract in the seminal fluid of the semen used to breed the donor or be a contaminant of the uterine excre- tions. 2. The transfer of an embryo with a pathogen in or on the zone pellucida. After in vitro exposure, some pathogens can adhere to the zone pellucida of embryos so strongly that washing fails to remove them (Singh, 1987~. Although the embryo itself is uninfected, the transfer of an embryo carrying a pathogen on the zone pellucida can result in infection of the recipient and possibly infection of the fetus.

OCR for page 171
176 / Appendix B Thus, if a pathogen adheres to the zone pellucida under in vitro conditions, it is essential to determine whether the pathogen is ever excreted into the reproductive tract of infected animals. If it is, it might adhere to embryos in viva and be transmitted using embryo transfer. 3. The transfer of an embryo in media contaminated with a dis- ease agent. Recipients could become infected if embryos were trans- ferred in contaminated media or if procedures to ensure the sanitary health of embryos were not adhered to. Proper washing (see Annex B-1) is effective in removing very high levels of infectivity from em- bryos as long as the pathogens do not adhere to the zone pellucida. However, the sterility of the washing, freezing, and transfer media is essential to ensuring that pathogens are not introduced and trans- ferred along with the embryo. Products of animal origin, such as serum or bovine serum albumin (BSA), that are used in the various media may create problems. There is some evidence that bovine viral diarrhea (BVD) may have been transmitted to recipients, not from the embryos transferred but from the BSA used in the transfer media (Anderson et al., 1988~. 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 em- bryos has involved either the in vitro exposure of embryos to patho- gens or the collection of embryos from acutely infected donors (in viva experiments). The embryos are then either assayed in tissue culture or transferred to susceptible quarantined recipients. The in vitro experiments have provided much useful information and have facilitated the development of washing and enzymatic treatments of embryos to enhance their health status. It should be remembered in assessing this work, however, that embryos are being exposed to preparations of pathogens that usually contain high levels of proteins and enzymes. These latter substances might alter the adherence of the pathogen to the zone pellucida of embryos. In addition, embryos are also being exposed to much higher levels of the pathogens in vitro than they would be under the most extreme in vivo conditions. If embryos never come into contact with a specific disease agent in viva, demonstration of adherence under in vitro conditions is of little significance. For these and many other reasons, it is probably unwise to extrapolate from an in vitro to an in viva situation. The in viva experiments have their own limitations. Generally,

OCR for page 171
Appendix B / 177 the best data for determining the disease transmission potential of embryos are data on the transfer of embryos from seropositive do- nors to quarantined susceptible recipients. Certainly, these are the donors that would most likely undergo embryo collection in the field. However, only a very few seropositive animals are actively infected and therefore capable of potentially transmitting the disease agent with their embryos. A very large number of transfers would be re- quired to assess fully the potential of seropositive donors to transmit a particular disease by embryo transfer. These numbers would be difficult to generate at most research establishments. For this reason transfers are often carried out from actively infected (viremic) donors to maximize the possibility of disease transmission through the em- bryo. Since these donors have high titers of virus in their blood and other tissues, however, their embryos could become contaminated and must therefore be washed thoroughly prior to transfer. The other variable that should be considered in assessing the research is the method by which embryo infectivity is determined. This is usually carried out by transferring in Gino- or in vitro-ex- posed embryos to recipients, by assaying the embryos in tissue cul- ture, or by animal inoculation. Depending on the disease agent, the sensitivity of each of these detection methods will vary. It is gener- ally accepted, however, that tissue culture systems are more sensitive for the detection of virus than are animals when only one embryo is transferred per utero. In fact, tissue culture assays have been sensi- tive enough to detect the small number of virions that adhere to the zone pellucida of a single embryo (Singh and Thomas, 1987b; Singh et al., 1982b, 1984, 1987~. Thus, before a final conclusion can be reached on the transmissibility of each disease agent, the assay sys- tem used to generate the results must be assessed in terms of its sensitivity and specificity. 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 proce- dures 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. Thus, there is consider- able variation in the studies in how embryos were handled after ex- posure and prior to being assayed. Unless otherwise indicated, how- ever, the methodology used in each study was shown to be effective under the conditions used. Similarly, unless otherwise indicated, all

OCR for page 171
178 / Appendix B embryos exposed to pathogens in the studies were zone pellucida- intact embryos. For ease of reference, the disease agents that have been investigated are listed in alphabetical order and not in order of importance. Tables summarizing the research data are presented in Annex B-2. The conclusions reached regarding the transmissibility of each disease agent by embryo transfer are based on the data available. One of the most difficult tasks remains to determine the validity of extending the conclusions derived from these data to field condi- tions. African Swine Fever Virus Both in vitro (Singh et al., 1984) and in viva experiments have been carried out with this virus. Porcine embryos were exposed to African swine fever virus (ASFV), washed, and assayed in tissue cul- ture, and embryos were collected from viremic pigs, washed, and assayed in vitro. Results and Conclusions 1. In vitro: Ninety-five percent of the embryos retained infec- tious ASFV after viral exposure and washing. Treating the embryos with papain, EDTA, or ficin had no effect on the retained virus, whereas treating them with trypsin or pronase reduced the number of em- bryos carrying detectable virus (30 percent instead of 95 percent) and lowered the amount of virus on the embryos. The evidence sug- gested that most, if not all, of the virus was on the zone pellucida. The data indicated that if ASFV is excreted into the reproductive tract of infected animals, the virus could be transmitted by embryo transfer. 2. In viva: A total of 245 porcine embryos were collected from viremic donors, washed, and assayed in vitro in groups of 18 to 20 in order to duplicate embryo transfer conditions. None of the embryo samples was found to be associated with ASFV (Dulac and Singh, unpublished data), and very little virus was isolated from the uterine flush fluids. Thus, these preliminary results suggest that African swine fever can be controlled using embryo transfer. Akabane Virus In vitro experiments have been carried out with the Akabane vi- rus (AV) (Singh et al., 1982a). Bovine embryos were exposed to AV, washed, and then either assayed or cultured and assayed.

OCR for page 171
Appendix B / 179 Results and Conclusions Akabane virus did not infect zone pellucida-intact bovine em- bryos, and it had no effect on in vitro embryonic development. Proper washing was effective in rendering Akabane-exposed embryos free of this virus. Bluetongue Virus Experiments in cattle (Acree, 1988; Bowen et al., 1982, 1983; Singh et al., 1982a; Thomas et al., 1983, 1985), sheep (Gilbert et al., 1987; Hare et al., 1988), and goats (Chemineau et al., 1986) have been car- ried out with bluetongue virus (BTV). The bovine studies involved both in vitro and in viva experi- ments. 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. These em- bryos were washed and then transferred to susceptible quarantined recipients. The ovine experiments involved the transfer of embryos from bluetongue viremic sheep bred by either infected or uninfected rams to quarantined susceptible recipients. In addition, embryos were ex- posed to BTV, washed, and transferred to susceptible recipients. The caprine work involved the transfer of embryos from BTV- seropositive herds to uninfected recipients. Results and Conclusions 1. BTV did not infect zone pellucida-intact bovine embryos after in vitro exposure, nor did it have any effect on in vitro embryonic development. Thus, proper washing was effective in rendering BTV- exposed bovine embryos free of this virus. The integrity of the zone pellucida is important because BTV can infect zone-free bovine em- bryos. Bluetongue was not transmitted by embryo transfer. A total of 334 embryos, collected from infected donors or uninfected donors bred with infected semen, were washed and transferred into 330 quar- antined susceptible recipients. Although BTV virus was isolated from some of the uterine flush fluids, none of the calves or recipients de- veloped BTV antibodies. Thus, embryo transfer was shown to be effective in preventing the spread of bluetongue in cattle. 2. The results obtained when BTV-exposed ovine embryos were

OCR for page 171
180 / Appendix B transferred to uninfected recipients varied. In one study 49 embryos were transferred into 27 recipients, and all of the lambs and recipi- ents remained BTV seronegative. In the other study, when 20 em- bryos from BTV-infected sheep were transferred into 15 recipients, 2 of the recipients seroconverted, and when 13 embryos exposed to BTV virus in vitro were transferred, 9 of the 13 recipients seroconverted. The difference in the results might be due to the different strains of virus used in the two studies. In the study in which transmission occurred, however, the embryos were washed only 4 times instead of the recommended 10. Since the investigators did not establish that the four washings were effective, implication of the ovine embryo in the transmission of bluetongue is uncertain. Additional research is required to determine whether BTV can be transmitted to sheep us- ing embryo transfer when the embryos are handled according to the recommendations of the International Embryo Transfer Society. 3. When 63 caprine embryos derived from herds in which 47 per- cent were BTV seropositive were transferred to 19 recipients, the re- cipients and kids remained BTV seronegative. These initial experi- ments indicate that embryo transfer may be successful in preventing the transmission of BTV. Bovine Leukemia Virus Both in vitro (Hare et al., 1985) and in viva (Di Giacomo et al., 1986; Eaglesome et al., 1982; Hare et al., 1985; Kaja et al., 1984; Olson et al., 1982; Parodi et al., 1983; Thibier and Nibart, 1987) experiments have been carried out with bovine leukemia virus (BLV). Zona pellu- cida-intact and zone-free bovine embryos have been exposed to the virus in vitro, washed, and then either assayed or transferred to uninfected recipients. Embryos also have been transferred from BLV-seropositive donors to uninfected recipients. Results and Conclusions 1. When 27 zone-intact and 15 zone-free bovine embryos were exposed to BLV, washed, and assayed, no infectivity was associated with any of the embryos. Similarly, when 48 embryos were exposed to the virus, washed, and transferred into 3 recipients, the recipients remained BLV seronegative. These experiments indicate that proper washing is effective in rendering embryos free of BLV. 2. At least 1,200 embryos (596 from known BLV-seropositive do- nors and the remainder from 1,500 donors in which the majority were BLV seronositive) have been transferred from BLV seropositive do

OCR for page 171
Appendix B / 181 nors to uninfected recipients, and all of the recipients and calves remained BLV seronegative. This evidence demonstrates that BL\7 is highly unlikely to be transmitted by embryo transfer. Bovine Viral Diarrhea Virus Both in vitro (Evermann et al., 1981; Potter et al., 1984; Singh et al., 1982b) and in viva (Archbald et al., 1979) experiments have been carried out with bovine viral diarrhea virus (BVDV). Bovine em- bryos were exposed to BVDV in vitro, washed, and assayed using two assay systems in order to maximize the chances of detecting virus in or on the embryo. Both zone-intact and zone-free ovine embryos were exposed to BVDN7 in vitro, washed, and then assayed. Bovine embryos were collected from donors in which BVDV was inoculated into one of their uterine horns. These embryos were as- sessed and then examined using electron microscopy. Results and Conclusions 1. B~DV did not infect zone pellucida-intact bovine or ovine em- bryos, nor did it have any effect on in vitro embryonic development. Proper washing was effective in removing BVDV from all zone pellu- cida-intact embryos. This virus also did not replicate in zone-free ovine embryos. 2. The results from eight embryos obtained from donors inocu- lated per utero with BVDV are difficult to interpret. The embryos were degenerating and there was evidence of structures that mor- phologically resembled B~DV beneath the zone pellucida. The de- generation was most likely caused by inflammation in the uterine horn. The significance of the particles will require further investiga- tion, however. Because the embryos were degenerating, the zone pellucida might not have been an effective barrier to the virus, which might account for the BVDV-like particles beneath the zone. Brz~celia abortus Both in vitro (Mallek et al., 1984; Stringfellow et al., 1984) and in viva (Barrios et al., 1988; Bolin et al., 1981; Stringfellow et al., 1982, 1983, 1988; Voelkel et al., 1983) experiments have been carried out with this agent. Bovine embryos have been exposed to the agent, washed, and assayed, and embryos have been transferred from seropositive donors to susceptible recipients.

OCR for page 171
204 cn o x to Cal 5- cn of 5 - Cal o , - ._ C) a; a) o a; a Cal I o 5 - r9 Fit be 1 ~ En LL1 ~ ~ Cal _% an lo: be . ~0 Lo an, 0~m o of ~ _ U a, o ~ En ~ ~ O z O I.,, O . ~ Cal o U ) _ ~ ~b ~C OCR for page 171
205 ~ be Cat ~ ~ : ~ ~ Hi' 5 ~ 6 E~ ~ ~ ' - ~ .S ~ ~ ~ ~ ~ ~- LO ~ o ~o ~ oCal o Go at Cd ~U :^ al ~ al au ~ a, ._ ~ ~a,, Ct = ~ ~ ~ o 0 0 ~o 0 0 0 0 Zip Zip Z FEZ ZZ O ~ Do ~ ~ di oo ~ ~Go Cat Cal _ _ ~ ~- ~ o En _ =, _ ~ JO ~O ~ O 8 - ~ r ~ ~ ~ ~ ~O ~0 ~ ~ O sS) ~et ~4 ~t~ u~ 0 0 0 0 0 0 0 0 ~ ~ ~ ~ ~ 0 0 u, ~0 ,5 ~ ~ ~ ~ ~ ~ S ~O 11 O.a~_ >~= ~ 5 .= ~ ~ ~ O v' ~ 11 _ ~ O u _ ~ ~ ~ t' ~ .m .~> 5 ~ g =m Q ~ ~ l' .` 60 Q U) O ~ ~0 ~ ~ ~ o ~ ~ ~ ~ O .~% ~: ~ ~ O =: . .m Ct ~ V) ,~ ~ O I! `~5 ~, `- ~ so I' ~ I1 ~ ~ o .~ ~4 11 ~) O t15 c~ ~ u u) ~9 (-, ~ ~ il ~ ~ ~ ~ u d ~ ~ ~ 3 ~ ~ . ~ ~ . .> ~ o ~ U a~ .'- ;:!: ~ = au ~ m. ~ ~ 1 1 ~ ~ V) ~ U) o .~ ~ ~ ~ ~ ~ ~ ~ ~ o o ~ V ~ Lo~ ~ - ~ ~ ~ > ~i 0 ~ .~.~ 11 E~ ~ ~ ~ 3 o O ~ o ,,_ ,,, 1 ~ ~ .= % 5 - a' o a~ u, o co u ._ . ._ et ._ ~5 ~ a, 0 co ~ 0 ~ x u' ~ 0 0 x u: u~ 0 0 ~Q L~ ~ ~: ~

OCR for page 171
206 Cal o 50 u o a, cn o C x au be o o a; a' Em _4 5- k o u a, Cal 1 Cal EM _` U) a' a' ~ ~ to Vm -= - , out o o U) o 1-, o ~ by; .~ ~ oc to Cal ~9 oo Go oo Do Go ~ ~ Cal ~ ~ U) ~ U) CO o ~ ~ ~ o ~ o o I; ~ .= .= cn cn E~ ~s; E~ ' ~a~ t b0 ~ ~ ~ bO . - ._ . - ~ ~ ._' . - ._ cn cn ~ ~ 11 Cl) C~ CJ) 0 0 0 0 ~ 0 ~0 ~ c~ CM 00 ~ di oO ~O c~ ~ ~ ~ di O _ ~ _ ~O__ a~ ~ a ~ ~ a~ ~ ~ ~ ~ C) X ~o ~c~ E~ ~C) ~ ~ O O O ~ ~ O O O ~ ~ V A O O => ~ ~ .= ~ =: ~ ~ a ~ C~ I U. o - cn ~ ~ 11 ~ > m (t t~ U:~` ~ 5-, ~ - ~ ~ - = ~ u, - u, 5 ~ ~ ~ ~ ~ U) U, o _ U cn ~ ~ ~ CJ = 11 ~o O ~11 x `-,, ~ a O ~ ~ ~ ~ ~ ~' X ~ ~ ~ ~ ~ ~ U) .=- ~ ~ U) _ V] ~ ~a ~ ~ ~ . ~ ~ ~ 11 .~ ~ . ~ ~ :~ CJ . ~ ~ O ~ O .e ~ ~ _ CD O ~ ~ .~ . ~ ~ ~ O U) ~ a, ~ ~ ~ a, u O z.= 5.= U) U) - _ CD . - 11 0 ~ i_ ._ U ~ ~ O ~ ~. U) I ~ cn .~ ~ 3 ~o - 11 11 - .` ~; U, ._ 70 b U-) ._ ~ ~, o o ~ o ~ .~ Q' o ~n 11 . 11 5- o - Lr U, ~ ._ - ~ X s.- ~o ~ ~ o . o il

OCR for page 171
207 be o a; U) o 1 o 5 ._ C) a, o au o >~ _1 5- Cal Cal ~ 5 - EM ~ , ~ On o or ~ 5~, Ct is, au o or ~ , o o ~ o or .~ ~ ~4 o $ ~oo . ~i= Do 00 ~Lf) X (t A,-) ~ Hi $ X . ~X {\s ,:'- a v ~ ~ ~ 5 ':, ~ :: a' ~ ~ au a~ .> .> .> ~ .> ~ .> ~.> .> ct (~3 (~S - t~ ~ ~ ~ t~ ~>` ~ u u - ~ ~ ~ ~ ~ = ~ ~ ~ z z ~ z zo ~ ~ ~z zo I oo d ~c~ ~o ~ ___ _ ~ ~> o ~ ~ ~ ~ ~ E- O '- ~ ~ ~ ~di co o o o o o o o o ~c~ ~ ~ ~ ~ ~ D ~O I1 .` - cn ~ . - O .` .m _ i:~ 0 11 ~ O .= ~ ~ - O O ,, ._ " O "D ,_1 11 0 .` ~ ~ U] L:~, s, .` 11 C:: cn ~: ~ - ~ _ .= =: o ~ ~, ._ . ~ ._ ~ O O '~ 1 1 ~ ~0 ~ U] ._ o ~ U) _ ~ - - ~ ~ - 11 ~ o u~ (J,) a~ 11 ` o (e - 11 St <~ cn =: ,~'~ ~ ~ ~ - v) . - o - ~ o ~ ~ 5- 11 c~ .. =, 11 o ~ ~ 7 o o -9 v) co - u] ~ - ~ .~ cJ ~ o o ~s o ~- .= c) -

OCR for page 171
208 _` a, a' cn o o au o _' ._, CO o o 50 a, Cal o V) of o En 5 - E~ do Cal Ed At, ~ ~ o .= ~ U: $ - - Cal O ~ o ~ ~ au or, ._ ~ ._ r_ At, ~ ~ o b o s~ ~ ~ id hi 5- cr~ ~ ~ E~ U) o o ~ t~ ~oo ~ oo oo ~oo ~ C~ ~ ~ ~ ~ ~ ,-D ~ ~, ~ . _ ~ ~T ", ~ ~ ~ ~ ~ ~ au ~ ~ ~ ~ ._ ._ ._ ._._._ ._ ._ ._._ Ct b0 ~ bC a, ~ ~a;~ au a' a'au zz zz ~ZZZ ~ ~ ~0 0C~ CO ~N O C ~ O L~ Vl ~, V V ~: ~ ~: O ~ o ~ O ~ O ~ooCC 10 1O C,)~/LC) C~d~ A ._ ._ O =.> .> ~._ S O ~ U ~ ~ ~ ~U au O Co a, ~.= ~ ~ ~._ ~ ~ ~ ~ ~ ~~ ~

OCR for page 171
209 oo =` X ~ ~Go ~X ~ ~ _ _ (t ~ ~_ ~ _ ~ a'~ ., V ~ ~ o o ~ ~- o ~ o at au 5 - o o at, at, A, at, o ~ ~ ~ Z ZZ Zig Z ZZ I) ~ so CO 0 up En ~ o o oo ~ ~ o ~CO ~ Dot ~- - ID ~ ~ ~. ~ .> At C Cat ,~ ~ An ~ ~ ~ ~ D C .= .= ~ = ~ g ~ ~ ~ - ~ cn O ~ ~Sow U ~ .rn u) ~ - ~ .~ ~ ~ o s~ ~ tO - a, .= ~ ~ o u ,~) 11 ~ ~ .~o u ~ . ~ .- li ~ ::> =, ' a, 11 o BJ ~ o~ U3~ r3; ~ l ca= ~yo u ~ ~ o-> .> O .cn 11 ,= x' ~ o ~ ~ ~ g ~ cn 11 ~ uD' =: o > <:: v~ 11 m:~> E~ ~ ~n o ~ ~ Z a; ~ ~a) .- ;- u) cC 5 - u~ .> . ~ o ~ ~4 . - o o a, ~ ~C~ ~n ._ a, U) U, ._ 5- Ct - ;> U) ~ ~U] ,,,, 0 ~ Ct = ~ 5 - a, C~ ~ 0 ~ cn 5- ~ .m ~ - =, aN ~5 a' ~ a, ._ ._ ~ ~ ~ 0 ~ ~ Q~ a; O ~ ~ >` ~ Co au ~ a; E~ ~ ~ ~: ~ ~ CJ a; di 5 ._ ._ o ._ ._ a' UO o cn o o 50- CD o CJ _ a~ E~ . U) ~ ~ O au ~ ~ Uo) .> ~ ^-m o ~ ~S) = U) ~S 5- o ._ Q N o - Ct ._ ~0 - oo U)

OCR for page 171
210 in o o a i_ o V) o o cry o 5 - ~5 a, ~ a, U) o o U) US Cal EM i_ CC au u V] O O ~ Q U) ~ UO ~ ~ =Q ~ ~ a' a O U -0 0 ~ ^O U o o o in Go Go ~<5 .` .` X ~ X ~ ~ ~ ~ ~ ~ ~ ~ _ ~ ~ ~ Go ~ ~ ~ Go Do ~ U) _ ", O ct , ~ _ ~ ~ ~ ~ ~ _ ", O o O .= O ~.= .= .= .= ~ ~ a, ~ ~ ~ a, . - ._ ._ .~ ._ ._ ._ ._ ._ Ct Ct b4 ~ b4 ~ ~ ~b0 a ~ ~ ~ ~ a; ~ a, Z ZZZZZ ZZZ di ~ ~oo oo CN ~ C~ ~ o ~ ~ ~ ~CO ~ O O a, .> U ~ ._ ~U ~ ~ cn ._ ~ .~ ~= ~ ~ ~ g .> .

OCR for page 171
211 ~ ' v ~ ~ ~ cJ - ~ ~ ~ ~ ~ ~ .m ~ .m ~ .m -o - ~ - ' ~ - - ~ id, ~ =4 ~ cL, au . -. - . - . - cn an aual . -. - As~ ~of z z ~a, . - . - ~a; z z Dad ~ ~of d4 me ~ o ~ .= ~a- .= .~; ~ ~ ~ ~ 11 ~ ~ 1 m O U) 0 5- 1 1 . ._ ~ 0 ~ .~ 11 A: is 11 O Hi: 11 V) Cal . 5- .= us tJ - 11 cn . . z u) .= 11 . ~ .> do a~ o ~n - o - .` ~ - - 11 . - - ~ . ~ - o ~ ,= .= o o ,= 11 11 .` b() ~ o ~ ~ o u) co ~o 5 ~ - - =, a' u) u] - 5- .Q u) a' co 11 cn 5- ~ - s" - co :^ 5- a~ u) ~D

OCR for page 171
212 / Appendix B TABLE B2-6 Assay of Uterine Flush Fluids from Seropositive or Infected Dorrors Status of Donor Flushed Number of Positive Flushes Reference(s) Bovine embryos BLV seropositive BTV infected 4/25 12/30 BTV-infected semen (bred with) FMDV infected 15/22 0/4 IBRV infected 9/33 B. abortus infected 9/116 Bouillant et al., 1981 Bowen et al., 1983; Thomas et al., 1983 Thomas et al., 1985 McVicar et al., 1986; Mebus and Singh, 1988 Singh et al., 1982b Stringfellow et al., 1982, 1983, 1988; Voelkel et al., 1983 Porcine embryos FMDV infected 4/15 Mebus and Singh, 1988 HCV infected 6/33 Singh and Dulac, unpublished data SVDV infected 8/17 Singh et al., 1987 Caprine embryos CAEV seropositive 0/12 Wolfe et al., 1987 NOTE: B. abortus = Brucella abortus; BLV = bovine leukosis virus; BTV = bluetongue virus; CAEV = caprine arthritis and encephalitis virus; FMDV = foot-and-mouth dis- ease virus; HCV = hog cholera virus; IBRV = infectious bovine rhinotracheitis virus; SVDV = swine vesicular disease virus.

OCR for page 171
213 _` U) a, 5- a; _ ~ g An, o .= o U) ~ _ _ ~ ~ ~ o . - C,0 _ on cn O En o o U) o x o Q o 5 - a; V) 5- E~ 1 LO o En ,r, ._ Cat ~ U o ~ ~ ~ ~ X A ~ ~ Ed au en, ._ X ~ Ed o a' ' ~ ~ ~ ~_ _ ~ ~ ~ ~ ~ ~X _ ~ ~ ~ . ~ s ~ ~_ ~ ~_ ,,., tt a~ 4~ a, ~ :5 ~.= .= ~ ~ 0 0 ~ ~ ~ ~~ X cr: cn Z Z O s Z s Z V) ~' co C~ ~di di U) o CO U) - - _ _ _ _ _ 0 ~ ~ ~~ >~a~a~a~ a~a~ a~ a ~ a ~ v U U v' vU 'v C~ ~ ~ ~ ~ r ~O O O O O O O O v ~ ~ ~ ~ O O ~ > ~ ~ a' a' 0 ~ ~ ~ ~=' ~ > O U) ._ ~ . Q 0 ~ 11 ' Loo a, U3~ U) > ~ U: _ ._ ~ ~ ._ ~ a,, 0 ~ U ._ CD ~ .m 0 =\ 11 U, ~ 0 ,o ~ U E~ ._ . ~ 11 (], . ~ ~ =_ U)' 11 . - _ a; ~ . 0 ~ ~ ~o =._. 11 0 E~ a: ` CO _ _ .m ~o . ~ _ ~ a~ .= ~ . =0 Q 11 .. L~ 11 Z ~ a; ~n 7 - o o ._ ~5 .= U) :^ U, o 5- U)

OCR for page 171