Livestock (1993)

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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

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 zona°pellucida 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

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,

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

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.

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,

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

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.

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

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

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.

182 / Appendix B Results and Conclusions Brucella abortus was not isolated from any of 96 embryos that were exposed to this agent and washed. Similarly, 39 embryos trans- ferred from B. abortus-seropositive donors to uninfected recipients did not result in seroconversion of the recipients, and 309 embryos from infected donors were not associated with this agent when as- sayed. Of 116 uterine flush fluids from B. abortus-infected donors, the organism was isolated from only 9. This evidence indicates that B. abortus is unlikely to be transmitted through embryo transfer. Caprine Arthritis and Encephalitis Virus Some in viva work has been carried out with caprine arthritis and encephalitis virus (CAEV) (Wolfe et al., 1987~. Embryos from CAEV-seropositive donors were transferred to uninfected recipients. Results and Conclusions When 16 embryos were collected from CAEV-seropositive donors and transferred to susceptible recipients, the recipients remained CAEV seronegative. Additional transfers are required to support these pre- liminary results indicating that CAEV is not transferred using em- bryo transfer. Foot-and-Mouth Disease Virus The transmission of foot-and-mouth disease virus (FMDV) has been studied in both cattle (McVicar et al., 1986; Mebus and Singh, 1988; Singh et al., 1986) and pigs (Mebus and Singh, 1988; Singh et al., 1986), and both in vitro and in viva studies have been carried out. Bovine and porcine zone pellucida-intact embryos and bovine zona- free 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. Some of the embryos were transferred, and the balance were assayed in tissue culture or steer tongue. Results and Conclusions 1. No infectious virus was detected on any of the 169 bovine zone pellucida-intact embryos exposed in vitro to FMDV, washed,

Appendix B / 183 and assayed. Thus, proper washing was effective in rendering bo- vine embryos free of FMDV. This was not true for porcine embryos. Five percent of 194 zone pellucida-intact porcine embryos were asso- ciated with FMDV after exposure and washing. Incubation of the embryos resulted in a decrease in the amount of virus detected, which indicates that FMDV was not replicating in the embryonic cells of the porcine embryos and that the Virus was most likely on the zone pel- lucida. 2. Once the zone pellucida was removed from bovine embryos, proper washing was no longer effective in removing this virus from the embryos. ~~ ~~ ~ ~ Thirty percent of bovine zone-free embryos carried FMDV after in vitro exposure and washing. Infectivity titers were higher in hatched embryos after culture than in those assayed imme- diately, although there was no increase in titer in embryos incubated for 2 days compared with those incubated for 1 day. The virus may have entered the embryonic cells, although it is also possible that the virus had merely become trapped in the folds of the embryo. 3. One hundred forty-nine bovine zone pellucida-intact embryos were collected from FMDV-viremic donors, washed, and transferred into 112 quarantined susceptible recipients. Fifteen of the uterine flush fluids from 22 of the donors were found to contain small amounts of FMDNI. All calves and recipients have remained FMDV seronegative. In addition, 372 embryos and 64 eggs collected from these donors were assayed in steer tongues or tissue culture and found to be nega- tive for infectivity. This evidence indicates that the transmission of FMDV can be controlled by embryo transfer. 4. FMDV was not isolated from any of the 177 embryos and 104 eggs collected from FMDV-viremic pigs, washed, and assayed in steer tongue. In order to mimic embryo transfer conditions, embryos were assayed as group samples (20 embryos per sample). Four of the fifteen uterine flush fluid samples from the infected donors were found to be contaminated with low levels of FMDN7. In addition, 436 em- bryos and eggs were collected from viremic pigs, washed, and trans- ferred into 13 quarantined recipients. All of the recipients remained FMDY seronegative. This evidence indicates that embryo transfer is useful in controlling the transmission of foot-and-mouth disease in pigs. Haemophilus somnus Some in vitro experiments have been carried out with this agent (Thomson et al., 1987~. Bovine embryos were exposed to Haemophilus somnus and then washed in the presence or absence of antibiotics.

184 / Appendix B Results and Conclusions Haemophilus somnus was isolated from 10 of 38 embryos after washing in the absence of antibiotics and from none of 9 embryos after wash- ing in the presence of antibiotics. The results show that antibiotics must be included in the flushing and washing media to prevent the multiplication of the organisms that adhere to the zone pellucida of embryos. Hog Cholera Virus Both in vitro (Dulac and Singh, 1987) and in viva experiments have been carried out with this disease agent. Embryos were ex- posed to hog cholera virus (HCV) in vitro, washed, and assayed. In addition, embryos were collected from HCV-viremic donors, washed, and transferred to uninfected quarantined recipients. Results and Conclusions 1. 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 noninfec- tious if they had been exposed to less than 106 infectivity units of HCV. If the virus is excreted into the reproductive tract of infected animals, trypsin treatment would be required to render the embryos noninfectious. 2. Three hundred and eighty-five porcine embryos were collected from HCV-viremic donors, washed, and transferred to uninfected quar- antined recipients. Nineteen embryos and seventy-four unfertilized eggs were also assayed in vitro. The recipients remained HCV seronegative and the embryos/eggs were all found to be negative for HC infectivity. These preliminary results indicate that embryo trans- fer could be used to control the transmission of hog cholera. Infectious Bovine Rhinotracheitis Virus Both in vitro (Bowen et al., 1985; Singh et al., 1982b) and in vivo (Bondioli et al., 1988; Echternkamp and Maurer, 1988; Hasler and Reinders, 1988; Singh et al., 1982b, 1983) studies of infectious bovine rhinotracheitis virus (IBRV) have been carried out. (1) Various stages of bovine embryos were exposed for 1 or 24 hours to IBRV in vitro, washed, and either assayed or cultured and assayed. In addition,

Appendix B / 185 IBRV-exposed embryos were washed, trypsin treated, and assayed. (2) Embryos were collected from IBRV-viremic donors, washed, trypsin treated, and transferred to susceptible quarantined recipients. (3) Embryos were exposed to IBRV in vitro and transferred to suscep- tible recipients after being washed or washed and trypsin treated. (4) Embryos were collected from uninfected donors bred with IBRV-in- fected semen, washed, and assayed in tissue culture. (5) The effect of treating embryos with trypsin both on the pregnancy rate and the ability of embryos to survive cryopreservation was determined. Results and Conclusions 1. IBRV was isolated from 60 percent of virus-exposed embryos, although it had no effect on the in vitro embryonic development of the embryos. Trypsin (0.25 percent, 60 seconds) and IBRV antiserum were found to be capable of rendering the IBRV-exposed embryos noninfectious. Both the low level of the virus isolated from the em- bryos 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. If very high levels of the virus are excreted into the reproductive tract of infected animals, the data indicate that trypsin treatment would be necessary to render the embryos noninfective. 2. Sixty-four embryos collected from twenty-two donors infected with IBRV were transferred after washing and trypsin treatment to forty-nine uninfected quarantined recipients. Twenty-three pregnan- cies resulted from these transfers, and all of the recipients and result- ing calves remained IBRV seronegative. Thus, embryos can be trans- ferred from IBRV-infected donors (viremic) without transmitting the disease if the embryos are trypsin treated prior to transfer. Since all embryos were treated, it could not be determined whether this treat- ment is essential in order to prevent disease transmission. 3. Thirty-eight embryos were exposed to 107 infectivity units of IBRV, treated with trypsin, and transferred to twenty-two uninfected recipients. All of the recipients and resulting calves remained IBRV seronegative. When eight IBRV-exposed and washed embryos were transferred without trypsin treatment into four recipients, one of the four recipients seroconverted (Singh, unpublished data). Thus, when embryos are exposed to high levels of IBRV in vitro, trypsin treat- ment is required to prevent disease transmission. 4. Forty-seven embryos were collected from six donors bred with IBRV-infected semen. The embryos were washed ten times and then

186 / Appendix B assayed in tissue culture. Twelve embryos (from four donors) were found to be associated with small amounts of IBRV (Singh, unpub- lished data). This experiment indicates that if IBRV-infected semen were used to breed donors for embryo transfer, the embryos collected would require trypsin treatment to render them noninfectious. 5. A number of studies have examined the effect of trypsin treat- ment on embryo viability (Bondioli et al., 1988; Echternkamp and Maurer, 1988; Hasler and Reinders, 1988~. Following trypsin treat- ment, 80 embryos were transferred fresh and 416 were transferred after cryopreservation and thawing. The data demonstrated that trypsin treatment had no effect on the embryo's ability to survive freezing or on the pregnancy rate obtained with treated embryos. Porcine Parvovirus In vitro experiments have been carried out with porcine parvovirus (PPV) (Wrathall and Mengeling, 1979a,b). Embryos were exposed to the virus in vitro, washed, and then either assayed or transferred to uninfected recipients. 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. These data would indicate that if this virus is excreted into the reproductive tract, embryo transfer might result in the transmission of this disease. Pseudorabies Virus Both in vitro (Bolin et al., 1981, 1982) and in viva (Bolin et al., 1982; lames et al., 1983) studies of pseudorabies virus (PrV) have been carried out in swine. (1) Embryos were exposed to PrV in vitro, washed, and assayed for infectivity. (2) Embryos were exposed to high levels of infectivity of PrV in vitro, washed, trypsin treated, and then assayed. (3) Embryos were exposed to PrV in vitro, washed, or washed and trypsin treated, and then transferred to uninfected re- cipients. (4) Embryos were transferred from Pry-infected donors to uninfected recipients. (5) Embryos were collected from PrV-seropositive donors and transferred to uninfected recipients.

Appendix B / 187 Results and Conclusions 1. In one study 155 embryos exposed to 104 to 108 infectivity units of PrV were not associated with infectivity after washing. All em- bryos were cultured for 24 to 48 hours prior to assay, however, which may have resulted in some loss of viral infectivity. In another study 127 embryos were exposed to similar levels of the virus, washed, and then assayed. 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 non- infectious. 2. When 45 embryos were exposed to 106 infectivity units of Pry, washed, and trypsin treated, none of the embryos was associated with infectivity. Thus, trypsin is effective in removing PrV that ad- heres to the zone pellucida of embryos. 3. When 45 embryos were exposed to 104 infectivity units of Pry, washed, and transferred into 4 uninfected recipients, none of the re- cipients seroconverted; when 79 embryos were exposed to 108 units of the virus, washed, and transferred, all 5 of the recipients seroconverted. These results confirm earlier work indicating that washing is not ef- fective in rendering embryos noninfectious when they have been ex- posed to high levels of Pry. In another study (Singh and Thomas, unpublished data) embryos were exposed to 107 units of Pry, washed or washed and trypsin treated, and then transferred to uninfected recipients. The 10 pigs that received the trypsin-treated embryos remained PrV seronegative, but the 6 recipients that received the washed embryos became PrV seropositive. Thus, trypsin was shown to be effective in rendering in vitro-exposed embryos noninfectious. 4. When 45 embryos were collected from donors infected intrana- sally with Pry, washed, and transferred into 3 uninfected recipients, none of the recipients seroconverted. When 70 embryos were col- lected from donors that had been infected intrauterinely and intrana- sally, washed, and transferred, 2 of 5 recipients became PrV seropositive. Again, these results testify to the fact that if embryos come into con- tact with high levels of Pry, the virus adheres to the embryos. 5. A total of 805 embryos from 38 PrV-seropositive donors have been collected and transferred to 34 uninfected recipients to produce 208 piglets. The recipients and all of the piglets remained uninfected. These results indicate that PrV-seropositive donors do not excrete significant amounts of virus into their reproductive tracts and that embryo transfer can be used for the control of Pry.

188/Appendix B Rinderpest Virus Both in vitro and in vivo experiments (Mebus and Singh, 1988) have been carried out with rinderpest virus (RPN7~. A total of 61 zone pellucida-intact bovine embryos were exposed to high levels of infec- tivity of RPAI in vitro, washed, and assayed in tissue culture. Em- bryos were also collected from RPV-infected donors, washed, and then assayed by animal inoculation, in tissue culture, or by transfer to recipients. Results and Conclusions 1. Preliminary results indicate that 3 percent of zone pellucida- intact bovine embryos are associated with REV after in vitro expo- sure and washing (Dulac and Singh, unpublished results). 2. None of 170 zone pellucida-intact bovine embryos collected from RPV-viremic donors was associated with this virus after wash- ing. The embryos were assayed by animal inoculation and in tissue culture. In addition, 17 embryos from RPV-viremic donors were trans- ferred into 15 recipients. All of them remained REV seronegative (Singh and Mebus, unpublished data). These data indicate that rinder- pest is not transmitted through the embryo. S. craple In viva experiments have been carried out with this agent (Foote et al., 1986~. Embryos were transferred from scrapie-infected sheep to uninfected recipients. Results and Conclusions The transfers from scrapie-infected recipients have resulted in 69 lambs. No scrapie has been identified in these animals, who at this writing now range in age from 41 to 82 months. Scrapie was diag- nosed in the positive controls at approximately 40 months. Although this evidence is promising, additional studies are required before these results can be extrapolated to naturally infected animals. Swine Vesicular Disease Virus Both in vitro (Singh et al., 1987a) and in viva (Singh et al., 1987b) experiments have been carried out with swine vesicular disease virus

Appendix B / 189 (SVDV). In one study embryos were exposed to SVDV, washed, and assayed. In the other study embryos were collected from viremic donors and transferred to uninfected quarantined recipients. Results and Conclusions 1. Infectious SVDV was isolated from all of the embryos after in vitro exposure and washing. Culturing the embryos for 24 or 48 hours or treating the embryos with pronase, trypsin, or antiserum after viral exposure and washing reduced the number of embryos carrying the virus and lessened the amount of virus on each of the embryos. None of the treatments, however, was capable of decon- taminating all of the embryos. Thus, if SVDV is excreted into the reproductive tract of infected animals, transmission of this disease by embryo transfer is possible. 2. Two hundred and five embryos were collected from thirteen viremic donors and transferred to nine uninfected quarantined re- cipients. Seventeen embryos and ninety-five unfertilized eggs were also tested in vitro. The recipients, piglets, and embryos were all negative for SVDV infectivity, which indicates that control of this disease through embryo transfer is possible. Vesicular Stomatitis Virus Experiments have been carried out with both bovine and porcine zone pellucida-intact embryos to determine their in vitro susceptibil- ity to vesicular stomatitis virus (VSNI) (Lauerman et al., 1986; Singh et al., 1987~. The embryos were monitored for both infection and the effect, if any, the virus had on embryonic development. Results and Conclusions VSV was found to adhere to the zone pellucida of both bovine and porcine embryos, although it had no effect on the in vitro embry- onic development of these embryos. 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. If infected donors do excrete high levels of this virus into their reproductive tract, these data would indicate that trypsin treatment would be necessary to render the em- bryos noninfectious.

190 / Appendix B CONCLUSIONS An analysis of the data supports a number of conclusions regard- ing the disease transmission potential of embryos. Of all the disease agents that were tested, none replicated in the embryonic cells of zone pellucida-intact embryos. It was shown that some viruses (in- fectious bovine rhinotracheitis virus and bluetongue virus) could replicate in the embryonic cells of zone pellucida-free embryos, and thus, the integrity of the zone is an important factor in the health status of embryos. 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 vi- rus, bovine leukemia virus, bovine viral diarrhea virus, and foot-and- mouth disease virus, as well as Brucella abortus and Haemophilus somnus). Two, and possibly three, bovine viruses (infectious bovine rhinotracheitis virus, vesicular stomatitis virus, and possibly rinderpest virus) and all of the porcine viruses tested were found to adhere to the zone pellucida of embryos after in vitro viral exposure and wash- ing. If these agents can also adhere to embryos under in viva condi- tions, transmission of the diseases by embryo transfer will depend on whether these agents are ever excreted into the reproductive tract of infected animals. Trypsin treatment was effective in removing or inactivating some of these viruses, and the interpretation can be made that, generally, enveloped viruses (hog cholera virus, infectious bo- vine rhinotracheitis virus, pseudorabies virus, and vesicular stomatitis virus) are susceptible to trypsin treatment, whereas nonenveloped viruses are not. There would appear to be a limit to the effectiveness of using trypsin to render embryos noninfectious. Embryos exposed to high levels of certain viruses (higher than 106) in vitro are not rendered completely noninfectious with trypsin treatment (Dulac and Singh, 1987~. This level of pathogenicity, however, greatly exceeds that which might be found in the reproductive tract of infected ani- mals to which embryos would be exposed in viva. None of the bovine viruses was transmitted when embryos were transferred from infected donors (bluetongue virus, bovine leukemia virus, foot-and-mouth disease virus, or infectious bovine rhinotracheitis virus) to susceptible recipients. All embryos transferred from IBRV- viremic donors were trypsin treated prior to transfer. The necessity of carrying out this procedure, however, has not yet been clearly established. Certainly, embryos collected from donors bred with IBRV- infected semen or embryos exposed in vitro to IBRNT require trypsin treatment to render them noninfectious. However, large numbers of embryos have been transferred out of IBRV-seropositive donors into

Appendix B / 191 uninfected recipients without disease transmission. Thus, the requirement of many importing countries that embryos derived from IBRV-seropositive donors be trypsin treated warrants further investigation. Recent evi- dence shows that if trypsin treatment is necessary, it does not inter- fere with embryo viability or freezability (Bondioli et al., 1988; Echternkamp and Maurer, 1988; Hasler and Reinders, 1988~. Although some porcine viruses (African swine fever, hog cholera, and swine vesicular disease) adhered to embryos under in vitro con- ditions, embryos collected from donors infected with those viruses were noninfectious when assayed in groups of 15 to 20. In addition, embryos from infected donors (HCV or SVDN7) did not transmit the diseases when they were transferred to uninfected recipients. Since the viruses were found in the uterine flush fluids of infected animals, it is possible that the total amount of virus that adhered to 15 to 20 embryos from infected pigs was below the minimum level detectable by both the recipient animals and the in vitro assay system. This supposition is very unlikely to be valid, however, because the in vitro assay systems have been shown to be sensitive enough to detect the small amount of virus that adheres to the zone pellucida of a single embryo (Singh et al., 1982a, 1984, 1986, 1987~. The other possibility is that the binding or adherence of viruses to embryos is an in vitro artifact. Viral preparations grown and harvested in vitro contain a number of cellular components which might increase the affinity of viruses for embryos. Only one disease agent (pseudorabies virus) is known to have been transmitted when porcine embryos were transferred from in- fected donors to susceptible recipients. The donor pigs were infected by introducing a large amount of virus into the uterus. Disease trans- mission did not occur when donors were infected by the intranasal route. Since it has been demonstrated that PrV adheres to the zone pellucida of pig embryos, these results are not surprising. 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 sus- ceptible pigs than from infected cattle to susceptible cattle; conse- quently, a final conclusion on the safety of using embryo transfer for disease control in the pig is not possible. It is important to remem- ber, however, that fewer transfers will be required in the pig because the chances of detecting any disease transmission via embryo trans- fer are much greater: 15 to 20 porcine embryos are transferred per recipient in contrast to 1 per recipient in cattle.

192 / Appendix B Limited work has been carried out on the disease transmission potential of sheep and goat embryos. Preliminary evidence would indicate that it may be possible to control scrapie using embryo transfer. There is also no final conclusion regarding the feasibility of using this technique to control bluetongue in sheep. In one study disease transmission did not occur. In the other study bluetongue was trans- mitted when embryos were transferred from bluetongue-infected do- nors or exposed to the virus in vitro and then transferred to suscep- tible recipients. Unfortunately, the embryos were washed a maximum of four times in the study in which disease transmission occurred. 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 uter- ine flush fluids of infected donors, proper washing of embryos is essential. Before a final conclusion can be reached regarding the transmissibility of BTV through the ovine embryo, further experi- mentation is required. Finally, goat embryos have been transferred from herds infected with bluetongue and caprine arthritis encephali- tis to uninfected herds with no resulting disease transmission. CONSTRAINTS TO THE INTERNATIONAL EXCHANGE OF EMBRYOS At present, there is no technology for determining the health sta- tus of embryos without destroying them. Thus, until very recently, regulations for the importation of embryos into most countries re- lated to the donor and sire and to the herd of origin rather than to the embryo itself. Recently, based on the research that has been carried out on the disease transmission potential of embryos, the Office In- ternational des Epizooties has modified its recommendations. The recommendations recognize that there are two main ways to ensure that embryos are free of pathogenic organisms. The traditional method is based on the testing of donor animals over extended periods of time. The health of a donor is usually assessed on the basis of the animal's serological status, not on the absence or presence of disease agents in the donor. Problems can arise with this method of assessment, however. Animals can be per- sistently infected with some disease agents and yet remain serologi- cally negative for those agents (immunotolerant). Immunotolerance has been conferred on animals that are congenitally infected with bovine viral diarrhea/mucosal disease, border disease, or bluetongue. The converse of this situation also exists. Many donors have been exposed to disease agents and become serologically positive to them.

Appendix B / 193 Those donors are, however, no longer infected with the disease agent, although their embryos would remain ineligible for export. The other method is based on research on the transmissibility of agents by embryo transfer and requires that embryos be processed in accordance with the procedures of the International Embryo Transfer Society. It obviates long periods of isolation and repeated testing of donor animals. This new method is based on the proper collecting, processing, and transfer of embryos (see Annex Beg. Although the protocols have been demonstrated to be reliable for a number of dis- ease agents, the health status of the embryo is entirely dependent on the care taken during the embryo collection and processing proce- dures. For the more serious diseases, many countries might insist on having their own people involved in all stages of the flushing, collec- tion, washing, and freezing to monitor quality control. In addition, the recipients receiving the embryos might be quarantined in a maxi- mum security quarantine station. Some countries, in an effort to monitor the health status of im- ported embryos, also require the testing of the uterine flush fluid, the embryo washes, or unfertilized eggs/degenerating embryos from the same collection. The testing of these samples is more complex than carrying out serological tests on donors. Serum samples are stable and many laboratories have experience in performing such tests. The isolation of a pathogen from the uterine flush fluid or embryos/eggs will depend not only on the assay system used but also on the stabil- ity of the agent, the treatment of the sample prior to testing, and the volume tested. RECOMMENDATIONS Additional research is required to improve the basic techniques involved in embryo transfer. Better and more reliable methods of superovulation, embryo assessment, and cryopreservation would be of enormous benefit. In addition, improvements are also possible in the techniques used to collect and transfer embryos. Finally, research is required in related areas of embryo transfer, such as embryo split- ting, cloning, sexing, and in vitro fertilization. If these techniques are to be developed to their full potential, this research must be sup- ported. Because this appendix is primarily concerned with the po- tential of embryos to transmit disease, the specific recommendations for research that follow are confined to that area. · Additional research is required to enable a conclusion on the transmissibility of all disease agents of concern through embryo transfer.

194 / Appendix B At this time a final conclusion on the transmissibility of many patho- gens by embryo transfer is not possible. The evidence does strongly suggest that if disease transmission ever occurs through the embryo, it is a relatively rare event. Additional studies are required, however, especially in the nonbovine species. Since swine embryos do not survive cryopreservation, extensive research into the disease trans- mission potential of swine embryos may be premature. For the other species, research should be directed in terms of disease agents by the disease status of the country of origin of the desired genetic material. It should be recognized, however, that research facilities will never be able to generate sufficient data to prove unequivocally that infec- tious disease agents cannot be transmitted by embryo transfer. In the interim, current data can be substantiated by transferring embryos into recipients that are held in quarantine. This procedure would allow for the generation of valuable information without risking the spread of disease. · 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 dis- ease when they are transferred to susceptible recipients. The most probable reason for this is that the disease agents are not excreted into the reproductive tracts of infected animals and, therefore, do not come into contact with the embryo. For this reason it is extremely important to determine whether pathogens that adhere to the zone pellucida in vitro are ever excreted into the reproductive tract. · The effectiveness and the nature of the antibiotics in both the flushing and washing media must be determined so that embryos will be free of bacterial agents. Very few bacterial or fungal agents have been investigated in regard to their potential for transmission by embryo transfer. The reason for this is that these disease agents are too large to be able to penetrate the zone pellucida to gain access to the embryo. However, bacterial and fungal agents can adhere to the zone pellucida. Thus, it is very important that in vitro studies be carried out to determine the efficacy of antibiotics in the collection and washing media as a means of preventing the transmission of bacterial diseases through the embryo. It is essential that the proper antibiotic be used and that sufficient time be allowed for it to act. · Treatments must be identified that can be applied to embryos to render them free of all infectious agents without hindering embry- onic viability. Potentially, this area of research could provide the greatest benefit. The nature of the interaction between certain dis- ease agents and the zone pellucida should be studied with a view to

Appendix B / 195 identifying me factors that influence this interaction. Win this knowledge it might be possible to expose all collected embryos, prior to transfer, to conditions that would render them disease free. Although trypsin has been used in this regard to a limited extent, it is not the treatment of choice. Trypsin is a biological product that has a potential for contamination itself, and it would appear to be most effective in inac- tivating enveloped viruses. Thus, it is essential to identify other agents that can be used to render embryos disease free. · Research is required into the suitability of using synthetic me- dia in the collection, cryopreservation, and transfer of embryos. To ensure that embryos are not contaminated during collection or pro- cessing, it is essential that serum or any product of animal origin used in the media be free of infectious agents. This requirement can be difficult to meet because serum and BSA fraction ~ can be con- taminated with B)IDV and other viruses. Rigorous testing is required to identify the presence of these agents, which are often missed on initial screening. Thus, if a synthetic medium is developed that en- ables the successful collection, freezing, and transfer of embryos, it would be an important gain irk ensuring the disease-free status of embryos. REFERENCES Acree, J. A. 1988. Report to the U.S. Animal Health Associationts Committee on bluetongue and bovine retrovirus. P. 124 in Proceedings of the 92d Annual Meet- ing of the U.S. Animal Health Association. Richmond, Va.: U.S. Animal Health Association. Anderson, J. B., H. Pedersen, and L. Ronsholt. 1988. A series of calves born after embryo transfer and persistently infected with BVD-virus. P. 29 in Proceedings of the 13th Conference of the OIE Regional Commission for Europe. Paris, France: Office International des Epizooties. Archbald, L. F., R. W. Fulton, C. L. Seager, F. Al-Bagdadi, and R. A. Godke. 1979. Effect of bovine viral diarrhea (BVD) virus on preimplantation bovine embryos: A preliminary study. Theriogenology 11:81-89. Barrios, D. R., D.C. Kraemer, E. Bessoudo, and L. Adams. 1988. Failure to isolate Brucella abortus from embryos or ova from culture-positive superovulated cows. Theriogenology 29:353-361. Betteridge, K. J. 1977. Embryo Transfer in Farm Animals: A Review of Techniques and Applications. Agriculture Canada Monograph 16. Ottawa, Ontario: Agri- culture Canada. Bolin, S. R., L. J. Runnels, C. A. Sawyer, K. J. Atcheson, and D. P. Gustafson. 1981. Resistance of porcine preimplantation embryos to pseudorabies virus. Am. J. Vet. Res. 42:1711-1712. Bolin, S. R., L. J. Runnels, C. A. Sawyer, and D. P. Gustafson. 1982. Experimental transmission of pseudorabies virus in swine by embryo transfer. Am. J. Vet. Res. 43:278-280. Bondioli, K. R., K. R. Gray, and J. B. Gibson. 1988. The effect of trypsin washing on

196 / Appendix B post thaw viability of bovine embryos. Pp. 85-89 in Proceedings of the Seventh Annual Convention Meeting of the American Embryo Transfer Association. Reno, Nev.: American Embryo Transfer Association. Bouillant, A. M. P., G. M. Ruckerbauer, M. D. Eaglesome, B. S. Samagh, E. L. Singh, W. C. D. Hare, and G. C. B. Randall. 1981. Attempts to isolate bovine leukemia and bovine syncytial viruses from blood, uterine flush fluid, unfertilized ova and embryos from infected donor cattle. Ann. Rech. Vet. 12(4):385-395. Bowen, R. A., P. Spears, J. Storz, and G. E. Seidel, Jr. 1978a. Mechanisms of infertility in genital tract infections due to Chlamydia psittaci transmitted through contami- nated semen. J. Infect. Dis. 138:95-98. Bowen, R. A., J. Storz, and J. Leary. 1978b. Interaction of viral pathogens with preim- plantation embryos. Theriogenology 9:88. Bowen, R. A., T. H. Howard, and B. W. Pickett. 1982. Interaction of bluetongue virus with preimplantation embryos from mice and cattle. Am. J. Vet. Res. 43:1907- 1911. Bowen, R. A., T. H. Howard, R. P. Elsden, and G. E. Seidel, Jr. 1983. Embryo transfer from cattle infected with bluetongue virus. Am. J. Vet. Res. 44:1625-1628. Bowen, R. A., R. P. Elsden, and G. E. Seidel, Jr. 1985. Infection of early bovine em- bryos with bovine herpesvirus-l. Am. J. Vet. Res. 46:1095-1097. Chemineau, P., R. Procureur, Y. Cognie, P. C. Lefevre, A. Locatelli, and D. Chupin. 1986. Production, freezing and transfer of bluetongue virus-free goat embryos. Theriogenology 26:279-290. del Campo, M. R., R. Tamayo, and C. H. del Campo. 1987. Embryo transfer from brucellosis-positive donors: A field trial. Theriogenology 27:221. Di Giacomo, R. F., E. Studer, J. E. Evermann, and J. Evered. 1986. Embryo transfer and transmission of bovine leukosis virus in a dairy herd. J. Am. Vet. Med. Assoc. 188:827-828. Dulac, G. C., and E. L. Singh. 1987. Embryo transfer as a means of controlling the transmission of viral infections. XII. The in vitro exposure of zone pellucida- intact porcine embryos to hog cholera virus. Theriogenology 29:1335-1341. Eaglesome, M. D., W. C. D. Hare, and E. L. Singh. 1980. Embryo transfer: A discus- sion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents. Can. Vet. J. 21:106- 112. Eaglesome, M. D., D. Mitchell, K. J. Betteridge, G. C. B. Randall, E. L. Singh, B. S. Samagh, and W. C. D. Hare. 1982. Transfer of embryos from bovine leukemia virus-infected (BLV-positive) cattle to BLV-negative recipients. Preliminary re- sults. Vet. Rec. 111(6):122-123. Echternkamp, S. E., and R. R. Maurer. 1988. Capability of bovine embryos to develop in vitro after trypsin treatment and cryopreservation. Theriogenology 29:241. Evermann, J. F., M. A. Faris, S. M. Niemi, and R. W. Wright. 1981. Pestivirus persistence and pathogenesis: Comparative diagnostic aspects of border disease virus of sheep and bovine viral diarrhea virus. Pp. 407~26 in Proceedings of the 24th Annual Meeting of the American Association of Veterinary Laboratory Diag- nosticians. Columbia, Mo.: American Association of Veterinary Laboratory Diag- nosticians. Foote, W. C., J. W. Call, T. D. Bunch, and J. R. Pitcher. 1986. Embryo transfer in the control of transmission of scrapie in sheep and goats. Pp. 413~16 in Proceedings of the 90th Annual Meeting of the U.S. Animal Health Association. Richmond, Va.: U.S. Animal Health Association.

Appendix B / 197 Gilbert, R. O., R. I. Coubrough, and K. E. Weiss. 1987. The transmission of bluetongue virus by embryo transfer in sheep. Theriogenology 27(3):527-540. Guerin, B., J. P. Builly, P. Humblot, M. Nibart, and M. Thibier. 1988. Effets de la contamination experimentale in vitro des embryons de souris et de brebis par Can~pylobacterfefus. Bull. Acad. Vet. de France 61:63-78. Hare, W. C. D. 1985. Diseases Transmissible by Semen and Embryo Transfer Tech- niques. Technical Series No. 4. Paris, France: Office International des Epizooties. Hare, W. C. D., D. Mitchell, E. L. Singh, A. M. P. Bouillant, M. D. Eaglesome, G. M. Ruckerbauer, A. Bielanski, and G. C. B. Randall. 1985. Embryo transfer in rela- tion to bovine leukemia virus (BLV) control and eradication. Can. Vet. J. 26:231- 234. Hare, W. C. D., A. J. Luedke, F. C. Thomas, R. A. Bowen, E. L. Singh, M. D. Eaglesome, G. C. B. Randall, and A. Bielanski. 1988. Nontransmission of bluetongue virus by embryos from bluetongue virus-infected sheep. Am. J. Vet. Res. 49:468~72. Hasler, J. F., and A. M. Reinders. 1988. Pregnancy rate following transfer of bovine embryos treated with trypsin prior to freezing. Pp. 91-93 in Proceedings of the 7th Annual Convention Meeting of the American Embryo Transfer Association. Hastings, Neb.: American Embryo Transfer Association. James, J. E. D., D. M. James, P. A. Martin, D. E. Reed, and D. L. Davis. 1983. Embryo transfer for conserving valuable genetic material from swine herds with pseudorabies. J. Am. Vet. Med. Assoc. 183:525-528. Kaja, R. W., C. Olson, R. F. Rowe, R. H. Stauffacher, L. L. Strozinski, A. R. Hardie, and I. Bause. 1984. Establishment of a bovine leukosis virus-free dairy herd. J. Am. Vet. Med. Assoc. 184:184-185. Lauerman, L. H., D. A. Stringfellow, P. H. Sparling, and L. M. Kaub. 1986. In vitro exposure of preimplantation bovine embryos to vesicular stomatitis virus. J. Clin. Microbiol. 24:380-383. Mallek, Z., B. Guerin, M. Nibart, M. Parez, and M. Thibier. 1984. Consequences de la contamination in vitro des embryons de souris et de vaches par Brucella abortus. Bull. Acad. Vet. Fr. 57:479-490. Mapletoft, R. J. 1987. The technology of embryo transfer. Pp. 2~0 in Proceedings of a Symposium on International Embryo Movement, W. C. D. Hare and S. M. Seidel, eds. Ottawa: Lowe-Martin. McVicar, J. W., E. L. Singh, C. A. Mebus, and W. C. D. Hare. 1986. Embryo transfer as a means of controlling the transmission of viral infections. VIII. Failure to detect foot-and-mouth disease viral infectivity associated with embryos collected from infected donor cattle. Theriogenology 26:595-601. Mebus, C. A., and E. L. Singh. 1988. Failure to transmit foot-and-mouth disease virus via bovine embryo transfer. Pp. 183-185 in Proceedings of the 92d Annual Meet- ing of the U.S. Animal Health Association. Richmond, Va.: U.S. Animal Health Association. Olson, C., R. F. Rowe, and R. W. Kaja. 1982. Embryo transplantation and bovine leukosis virus. Preliminary report. Pp. 361-370 in Fourth International Sympo- sium on Bovine Leukosis, O. C. Straub, ed. Boston: Martinus Nijhoff. Parodi, A., G. Manet, A. Vaillaume, F. Crespau, B. Toma, and D. Levy. 1983. Trans- plantation embryonnaire et transmission de ['agent de la leucose bovine enzootique. Bull. Acad. Vet. Fr. 56:183-189. Potter, M. L., R. E. Corstvet, C. R. Looney, R. W. Fulton, L. F. Archbald, and R. A. Godke. 1984. Evaluation of BVDV uptake by preimplantation embryos. Am. J. Vet. Res. 45:1778-1780.

198 / Appendix B Shelton, J. N. 1983. Prospects for the use of embryos in the control of disease and the transport of genotypes. Aust. Vet. J. 64:6-10. Singh, E. L. 1987. The disease control potential of embryos. Theriogenology 27:9-20. Singh, E. L., and F. C. Thomas. 1987a. Embryo transfer as a means of controlling the transmission of viral infections. IX. The in vitro exposure of zone pellucida- intact porcine embryos to swine vesicular disease virus. Theriogenology 27:443- 449. Singh, E. L., and F. C. Thomas. 1987b. Embryo transfer as a means of controlling the transmission of viral infections. XI. The in vitro exposure of bovine and porcine embryos to vesicular stomatitis virus. Theriogenology 28:691-697. Singh, E. L., M. D. Eaglesome, F. C. Thomas, G. Papp-Vid, and W. C. D. Hare. 1982a. Embryo transfer as a means of controlling the transmission of viral infections. I. The in vitro exposure of preimplantation bovine embryos to Akabane, bluetongue and bovine viral diarrhea viruses. Theriogenology 17:437~44. Singh, L. L., F. C. Thomas, M. D. Eaglesome, G. Papp-Vid, and W. C. D. Hare. 1982b. Embryo transfer as a means of controlling the transmission of viral infections. II. The in vitro exposure of preimplantation bovine embryos to infectious bovine rhinotracheitis virus. Theriogenology 18:133-140. Singh, E. L., W. C. D. Hare, F. C. Thomas, and A. Bielanski. 1983. Embryo transfer as a means of controlling the transmission of viral infections. IV. Non-transmission of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus from donors shedding virus. Theriogenology 20:169-176. Singh, E. L., G. C. Dulac, and W. C. D. Hare. 1984. Embryo transfer as a means of controlling the transmission of viral infections. V. The in vitro exposure of zone pellucida-intact porcine embryos to African swine fever virus. Theriogenology 22:693-700. Singh, E. L., J. W. McVicar, W. C. D. Hare, and C. A. Mebus. 1986. Embryo transfer as a means of controlling the transmission of viral infections. VII. The in vitro exposure of bovine and porcine embryos to foot-and-mouth disease virus. Theriogenology 26:587-593. Singh, E. L., F. C. Thomas, W. C. D. Hare, and M. D. Eaglesome. 1987. Embryo transfer as a means of controlling the transmission of viral infections. X. The in vivo exposure of zone pellucida-intact porcine embryos to swine vesicular dis- ease virus. Theriogenology 27:451~57. Stringfellow, D. A., V. L. Howell, and P. R. Schurrenberger. 1982. Investigations into the potential for embryo transfer from Brucella abortus infected cows without trans- mission of infection. Theriogenology 18:733-743. Stringfellow, D. A., C. M. Scanlan, S. J. Hannon, V. S. Panagala, B. W. Gray, and P. A. Galik. 1983. Culture of uterine flushings, cervical mucus, and udder secretions collected post-abortion from heifers artificially exposed to Brucella abortus. Theriogenology 20:77-83. Stringfellow, D. A., C. M. Scanlan, R. R. Brown, G. B. Meadows, B. W. Gray, and R. R. Young-White. 1984. Culture of bovine embryos after in vitro exposure to Brucella abortus. Theriogenology 21:1005-1012. Stringfellow, D. A., V. S. Panangala, and P. A. Galik. 1988. Recovery and culture of ova from Brucella abortus infected cows. Theriogenology 29:1105-1112. Thibier, M., and M. Nibart. 1987. Disease control and embryo importations. Theriogenology 27:37-47. Thomas, F. C., E. L. Singh, and W. C. D. Hare. 1983. Embryo transfer as a means of controlling viral infections. III. Non-transmission of bluetongue virus from viremic cattle. Theriogenology 19:425~31.

Appendix B / 199 Thomas, F. C., E. L. Singh, and W. C. D. Hare. 1985. Embryo transfer as a means of controlling viral infections. VI. Bluetongue virus-free calves from infectious semen. Theriogenology 24:345-350. Thomson, M. S., D. A. Stringfellow, and L. H. Lauerman. 1987. In vitro exposure of reimplantation bovine embryos to Haemophilus somnus. Theriogenology 27:287. Voelkel, S. A., K. W. Stuckey, C. R. Looney, F. M. Enright, P. E. Humes, and R. A. Godke. 1983. An attempt to isolate Brucella abortus from uterine flushings of superovulated donor cattle. Theriogenology 19:355-366. Wolfe, D. F., K. E. Nusbaum, L. H. Lauerman, P. W. Mysinger, M. G. Riddell, L. S. Putman, and T. A. Powe. 1987. Embryo transfer from goats seropositive for caprine arthritis-encephalitis virus. Theriogenology 28:307-316. Wrathall, A. E., and W. L. Mengeling. 1979a. Effect of porcine parvovirus on develop- ment of fertilized pig eggs in vitro. Br. Vet. J. 135:249-254. Wrathall, A. E., and W. L. Mengeling. 1979b. Effect of transferring parvovirus-in- fected fertilized pig eggs into seronegative gilts. Br. Vet. J. 135:255-261.

200 / Appendix B ANNEX B-1 Sanitary Techniques for Handling Embryos This annex describes the manner in which embryos should be treated between collection and transfer in order to preclude the pos- sibility of transferring disease agents along with the embryo. A1- though the technique of embryo transfer can be used to limit the disease transmission potential of embryos, if the technique is im- properly carried out, it could be used to introduce disease. The pro- cedures described are recommended by the International Embryo Transfer Society and endorsed by the Office International des Epizooties. They are based on research that has been carried out on the transmissibil- ity of disease pathogens by the embryo. This research has shown that the presence of an intact zone pellucida is an important structure in maintaining the disease-free status of the embryo itself and that proper washing can render embryos free of many pathogens. A1- though the presence of the zone pellucida is an important barrier that prevents many pathogens from gaining access to the embryo, under certain circumstances this structure can have a negative im- pact on the health status of the embryo. That is because some disease agents have been found to adhere to the zone pellucida so firmly that proper washing will not remove them. Thus, although the embryo itself would still be disease free, transfer of an embryo with a patho- gen on the zone pellucida could lead to infection of the recipient and, ultimately, infection of the embryo or fetus. 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. EMBRYO COLLECTION Collections must be carried out using strict aseptic procedures. The vulva of the donor should be scrubbed with an antiseptic and then rinsed well. All collection equipment (catheters, recovery tub- ing, petri dishes, and so on) should be sterile. In addition, all media, solutions, and sera that come into contact with the embryo must be free of contaminants and living microorganisms. EMBRYO WASHING The recommended washing procedure involves transferring em- bryos, in groups of 10 or fewer, through 10 changes of sterile medium

Appendix B / 201 containing antibiotics. A fresh, sterile micropipette must be used to transfer the embryos to each of the washes; each wash must consti- tute a hundredfold dilution of the previous wash. The embryos must be gently agitated in each of the washes; as soon as that has been carried out, they can be moved to the next wash. Only embryos from the same donor are washed together. Trypsin Treatment of Embryos If it is deemed necessary that embryos be treated with trypsin, the following combined washing and trypsin treatment should be followed. Embryos are transferred through five washes of sterile phosphate-buffered saline without calcium (Ca++) and magnesium (Mg++) but with antibiotics and 0.4 percent bovine serum albumin. It is important to remove serum and divalent cations before the embryos are exposed to trypsin to insure the biological activity of this en- zyme. The embryos are then exposed to two washes of trypsin, pH 7.6 to 7.8, for a total time in the trypsin of 60 to 90 seconds. Sterile trypsin (trypsin 1:250 that has an activity such that 1 g will hydrolyze 250 g of casein, at 25°C and pH of 7.6, in 10 minutes) in Hanks' balanced salt solution, without Ca++ and Mg++, is used at a concen- tration of 0.25 percent. After the trypsin treatment the embryos are transferred through five washes of phosphate-buffered saline con- taining Ca++, Mg++, antibiotics, and 2 percent serum. Serum is a potent inhibitor of trypsin and its inclusion in the last five washes ensures that the action of trypsin will be stopped. Inspection of Embryos for Intactness of Zona Pellucida All embryos must have a zone pellucida that is intact and free of adherent material. To ensure this, each embryo must be examined over its entire surface area at not less than 50 magnification. Em- bryos are gently rolled in the dish so that all surfaces on the zone pellucida can be examined. This evaluation should take place after washing and before embryo micromanipulation or cryopreservation. 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. Under these circumstances it would be essential that the foreign zone

202 / Appendix B pellucidae had been obtained from properly washed embryos/eggs and that they were not derived from a donor infected (seropositive) with any pathogen that is known to adhere to the zone pellucida. CRYOPRESERVATION AND THAWING OF EMBRYOS All solutions, cryoprotectants, and containers must be sterile, and a sterile technique must be used throughout these procedures. Embryo Identification Proper identification is essential if embryos are to be related to their health certificates. Thus, containers (straws or ampoules) of frozen embryos must be labeled with an initial code that identifies the embryo transfer company code, the breed code, and the donor cow and sire registration numbers. In addition, the straw or am- poule must also be marked with the freezing date (year/month/day), a straw or ampoule number (for a given collection), and the number of embryos it contains. Goblets and canes holding straws or ampoules of frozen embryos must be labeled with the cane number, the embryo transfer company code, the date of freezing, the breed code, and the registered name and number of the donor female and male. EMBRYO TRANSFER cures. The transfer of embryos is carried out using strict aseptic proce CONCLUSION In terms of disease control, the safest form of genetic material is embryos. 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) than the disease status of the genetic dam and sire is the health of the recipient. It is essential that embryos be transferred to recipients that are free from infectious diseases that can be trans- mitted "in utero." Otherwise, the enormous benefits of embryo transfer in terms of disease control will be lost.

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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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.

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)