Two recent studies demonstrated that a high-affinity isolate of BVDV (SD-1), remained associated with a small percentage of in vivo-derived bovine embryos following artificial exposure to the virus and either washing or trypsin treatment. Further, the embryo-associated virus was infective in an in vitro environment. Therefore, the objective of this study was to determine if the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos could cause infection in vivo. Twenty zona-pellucida-intact morulae and blastocysts (MB) were collected on day 7 from superovulated cows. After collection, all MB were washed according to International Embryo Transfer Society (IETS) standards, and all but 4 MB (negative controls) were exposed for 2h to 105–106 cell culture infective doses (50% endpoint) per milliliter (CCID50/mL) of viral strain SD-1. Following exposure, according to IETS standards, one half of the MB were washed and one half were trypsin treated. All MB were then individually sonicated, and sonicate fluids were injected intravenously into calves on day 0. Blood was drawn to monitor for viremia and(or) seroconversion. Seroconversion of calves injected with sonicate fluids from washed and trypsin-treated embryos occurred 38% and 13% of the time, respectively. Therefore, the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos was infective in vivo.

Bovine viral diarrhea virus (BVDV) replicates in embryo co-culture systems and remains associated with developing IVF bovine embryos, despite washing and trypsin treatment. Previous research demonstrated that 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) inhibits replication of BVDV in cultured cells. The objective of this study was to evaluate the capability of IVF embryos to develop into normal, weaned calves after exposure to antiviral concentrations of DB606 during IVC. Oocytes were obtained from cows via transvaginal, ultrasound-guided follicular aspiration. Presumptive zygotes (n = 849) that resulted from fertilization of these oocytes were cultured for 7 d in medium supplemented with 0.4 microM DB606 or medium lacking antiviral agent. All blastocysts (n = 110) were transferred individually into the uterus of a synchronized recipient. The pregnancy status of recipients was determined using transrectal ultrasonography at 21-23 d after embryo transfer. Additional pregnancies as controls (n = 21) were initiated by natural breeding. Developing fetuses and resulting calves were evaluated every 27-34 d. Blastocyst development, pregnancies per transferred embryo, pregnancies maintained per pregnancies established, gestation length, gender ratio, birth weights, viability of neonates, complete blood counts, and serum chemistry profiles at 3 mo of age and adjusted 205 d weaning weights were compared for research treatments. Development to weaning after exposure to DB606 did not differ significantly from controls. In conclusion, bovine embryo cultures can be safely supplemented with antiviral concentrations of DB606; addition of DB606 agent might prevent viral transmission if BVDV were inadvertently introduced into the embryo culture system.

Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus.

Bovine viral diarrhea virus (BVDV) has been shown to replicate in embryo culture systems and remain associated with bovine embryos developing in vitro. In this study, novel antiviral agents were evaluated for capability to inhibit replication of BVDV without affecting embryonic development. Serial concentrations of 2-[5(6)-{2-imidazolinyl}-2-benzimidazolyl]-5-(4-aminophenyl)furan (DB456) or 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) were prepared in IVC medium. Then, bovine uterine tubal epithelial cells (UTC) were placed in IVC media with varying concentrations of DB456 or DB606. Within 1h, a genotype I or II strain of BVDV was added to the cultures. Cultures were maintained for 7 days. Infectious virus was quantitated in IVC media collected on days 3 and 7 and in UTC lysates harvested on day 7. The effective antiviral concentrations of DB606 were much lower than effective antiviral concentrations of DB456. In subsequent experiments, IVF presumptive zygotes were cultured in IVC medium with or without DB456 or DB606 at multiple concentrations for 7 days to evaluate effect of the compound on conceptus development. On day 7, stage of embryonic development was observed, and blastocysts were harvested and stained using Hoechst 33342 to enumerate embryonic cells. While DB456 inhibited blastocyst development, DB606 at 20 times the effective antiviral concentration did not hinder blastocyst development or reduce the mean number of cells per blastocyst. These preliminary results indicated that bovine embryo cultures might be safely supplemented with effective concentrations of an antiviral agent.

Early research indicated that bovine viral diarrhea virus (BVDV) would not adhere to zona pellucida-intact (ZP-I), in vivo-derived bovine embryos. However, in a recent study, viral association of BVDV and in vivo-derived embryos was demonstrated. These findings raised questions regarding the infectivity of the embryo-associated virus. The objectives of this study were to evaluate the infectivity of BVDV associated with in vivo-derived bovine embryos through utilization of primary cultures of uterine tubal cells (UTC) as an in vitro model of the uterine environment and to determine if washing procedures, including trypsin treatment, were adequate to remove virus from in vivo-derived embryos. One hundred and nine ZP-I morulae and blastocysts (MB) and 77 non-fertile and degenerated (NFD) ova were collected on day 7 from 34, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to International Embryo Transfer Society (IETS) standards and exposed for 2 h to approximately 106 cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of <10 MB or NFD ova were washed in accordance with IETS standards. In addition, an equivalent number of MB and NFD ova were subjected to IETS standards for trypsin treatment. Subsequently, NFD ova were immediately sonicated and sonicate fluids were assayed for presence of virus, while individual and groups of MB were placed in microdrops containing primary cultures of UTCs and incubated. After 3 days, embryos, media, and UTCs were harvested from each microdrop and assayed for BVDV. Virus was detected in the sonicate fluids of 56 and 43% of the groups of NFD ova that were washed and trypsin-treated, respectively. After 3 days of microdrop culture, virus was not detected in media or sonicate fluids from any individual or groups of MB, regardless of treatment. However, virus was detected in a proportion of UTC that were co-cultured with washed groups of MB (30%), washed individual MB (9%) and trypsin treated individual MB (9%), but no virus was detected in the UTC associated with groups of trypsin-treated embryos. In conclusion, virus associated with developing embryos was infective for permissive cells. Further, the quantity of virus associated with a proportion of individual embryos (both washed and trypsin treated) was sufficient to infect the UTC. In light of these results, an attempt should be made to determine if the quantity of a high-affinity isolate of BVDV associated with an individual embryo would infect recipients via the intrauterine route.

A variety of procedures associated with in vivo and in vitro embryo production, as well as cloning and transgenics, are in current use by both researchers and practitioners. Biohazards associated with these procedures could influence clinical proficiency and the outcome of basic research or result in unusual distribution of pathogens in populations of animals. By their nature, embryo technologies are vulnerable to contamination from numerous sources. Although pathogens can originate in the physical environments in which embryo technologies are applied, they are more likely to be introduced via animals or materials of animal origin. However, it is important to note that both the occurrence and consequences of contamination are heavily influenced by environmental circumstances. This paper represents a philosophical description of biohazards associated with three generations of embryo technologies using the cow as a model species. Emphasis is placed on sources of contamination, current or suggested preventive actions and the issue of environmental changes as they relate to the emergence of biohazards and the implementation of biosecurity measures. Some specific pathogens are discussed for illustration. In addition, details of the risks associated with introducing bovine viral diarrhoea virus in each of three generations of embryo technologies are described.

Washing procedures (without trypsin treatment) recommended by the International Embryo Transfer Society (IETS) for use on in vivo-derived embryos effectively removed a cytopathic strain (NADL) of bovine viral diarrhea virus (BVDV) after artificial exposure. However, these washing procedures have not been evaluated using other isolates of BVDV, including representative non-cytopathic strains. Thus, the objective of this study was to evaluate the efficacy of the IETS procedures following artificial exposure of in vivo-derived bovine embryos to two different strains and biotypes of BVDV. One hundred and twenty-nine zona pellucida-intact (ZP-I) morulae and blastocysts (MB) and 56 non-fertile and degenerated (NFD) ova were collected 7 days following exposure to bulls from 32, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to IETS standards. Subsequently, half of the MB and NFD ova were exposed for 1 h to approximately 106-cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1, and the other half were exposed to the same concentration of CD-87. After exposure, groups of ≥3 and ≤10 MB or NFD ova were washed using methods that met or exceeded IETS standards. Then, the washed groups were sonicated, and sonicate fluids were assayed for presence of virus using virus isolation and a reverse transcription nested polymerase chain reaction. No virus was detected in any group of MB or NFD ova that had been exposed to the CD-87 isolate. However, virus was detected in association with 50% of the groups of MB and 33% of the groups of NFD ova that had been exposed to the SD-1 isolate. Therefore, standard embryo-washing procedures recommended by the IETS are more effective for removal of some isolates of BVDV than for others. It remains to be determined if the quantity of a high-affinity isolate of BVDV associated with individual washed embryos would infect recipients via the intrauterine route. Further, it should be determined if an alternative embryo processing procedure, washing and trypsin treatment, would be more effective for removal of high-affinity isolates.

The International Embryo Transfer Society (IETS) was founded in 1974. Early members used the society as a forum for the exchange of scientific and technical information relevant to a newly emerging embryo transfer industry. The impact that embryo transfer could have on the international trade of livestock genetics was clear by 1982, so the IETS commissioned the Import/Export Committee. The initial challenge for this Committee was to deal with concerns about disease transmission via embryo transfer. Many of the early concerns have been dispelled, but at the time they threatened the continued development of a fledgling industry. Over the past two decades, many new critical challenges have been met and managed by this Committee, which was recently renamed the Health and Safety Advisory Committee (HASAC). Assessing risks of animal disease transmission via reproductive technologies and establishing protocols for managing these risks are still major issues for HASAC. However, additional concerns have developed as views of the society changed and as novel applications of biotechnology in farm animals were identified. This paper is intended to chronicle some of the major changes and challenges that were managed by members of the HASAC and its Subcommittees from the early years of embryo transfer to the current millennium with technological advances in molecular biology.

ABSTRACT Bovine viral diarrhea virus (BVDV) is an economically significant pathogen of cattle and a problematic contaminant in the laboratory. BVDV is often used as an in vitro model for hepatitis C virus during drug discovery efforts. Aromatic dicationic molecules have exhibited inhibitory activity against several RNA viruses. Thus, the purpose of this research was to develop and apply a method for screening the aromatic cationic compounds for in vitro cytotoxicity and activity against a noncytopathic strain of BVDV. The screening method evaluated the concentration of BVDV in medium and cell lysates after 72 h of cell culture in the presence of either a 25 or 5 μM concentration of the test compound. Five of 93 screened compounds were selected for further determination of inhibitory (90 and 50%) and cytotoxic (50 and 10%) concentration endpoints. The screening method identified compounds that exhibited inhibition of BVDV at nanomolar concentrations while exhibiting no cytotoxicity at 25 μM concentrations. The leading compounds require further investigation to determine their mechanism of action, in vivo activity, and specific activity against hepatitis C virus.

Bovine viral diarrhea virus (BVDV) is a significant pathogen that can be shed in the semen of infected bulls. Thus, screening for BVDV in semen of bulls is recommended prior to their entry into an artificial insemination center. No previous research has compared the analytical sensitivity of reverse transcription-nested polymerase chain reaction (RT-nPCR) and virus isolation assays for detection of BVDV in semen from an infected bull. Therefore, the goals of this research were to compare the analytical sensitivity of RT-nPCR and virus isolation assays for BVDV in semen and to apply these assays to determine the prevalence in the Southeastern United States of bulls that lack viremia yet shed BVDV in semen. Semen collected from a bull that was persistently infected with BVDV was serially diluted (1/10) in semen from uninfected bulls and frozen in liquid nitrogen as raw, partially extended or fully extended semen. Subsequently, samples of semen were assayed by virus isolation and RT-nPCR. Viral detection was more sensitive in extended semen samples than in raw semen samples and more sensitive by RT-nPCR than virus isolation. After this evaluation of analytical sensitivity, serum and semen were collected from 558 post-pubertal bulls in our region. These samples were tested for BVDV by virus isolation. Partially extended semen was also assayed for BVDV by RT-nPCR. All samples were negative by all assays for BVDV. The application of analytically sensitive assays reveals a very low prevalence (≤0.54%) of BVDV in semen from bulls in the Southeastern United States.

OBJECTIVE:To evaluate persistence of bovine viral diarrhea virus (BVDV) in semen after inoculation of postpubertal bulls.ANIMALS:Three 2-year-old bulls and five 6-month-old calves.PROCEDURE:3 seronegative 2-year-old bulls were inoculated intranasally with BVDV. Serum and semen samples were obtained at regular intervals until 7 months after inoculation. Serum samples were tested for BVDV by use of virus isolation (VI) and reverse transcription-nested polymerase chain reaction (RT-nPCR) tests. Semen samples were tested for virus by use of VI and RT-nPCR tests. Testicular biopsy specimens were obtained 7 months after inoculation and tested for BVDV by use of immunohistochemical analysis and VI and RT-nPCR tests. Semen samples collected from 1 bull immediately before and 5 and 7 months after inoculation were administered IV to seronegative calves, which were monitored for subsequent viremia and seroconversion.RESULTS:Use of VI and RT-nPCR tests detected transient virus in serum of all bulls. The VI test detected BVDV in semen of 2 bulls for < 21 days after inoculation, whereas RT-nPCR assay detected BVDV until 7 months after inoculation. Virus was detected in testicular biopsy specimens of these 2 bulls by use of immunohistochemical analysis and RT-nPCR assay but could only be isolated from the biopsy specimen of 1 bull. Of the calves administered semen IV to detect infectious virus, only the recipient of semen collected 5 months after inoculation of the adult bull was viremic and seroconverted.CONCLUSIONS AND CLINICAL RELEVANCE:Bovine viral diarrhea virus can persist in semen of acutely infected bulls for several months after exposure.

It has been reported that bovine herpesvirus-1 (BHV-1) remains associated with in vitro-produced (IVP) bovine embryos after exposure to the virus and either washing or trypsin treatment. However, it is not known if the quantity of virus associated with an exposed IVP embryo is likely to infect a recipient cow after transfer. The specific objective of this study was to determine if IVP embryos that were exposed to BHV-1 would infect uterine tubal cells (UTC) in a co-culture system. In vitro-produced Day 7 embryos were exposed to BHV-1 and then washed or trypsin treated according to the IETS guidelines. These embryos were then co-cultured individually or in groups with UTC in microdrops of tissue culture medium 199 (TCM 199) supplemented with 10% equine serum. Following co-culture for 48 h, virus isolation was attempted on the embryos and the UTC from each drop. Virus was detected in washed individual embryos, groups of washed embryos, groups of trypsin-treated embryos and the UTC co-cultured with each of these treatments. However, BHV-1 was not detected in the individual, trypsin-treated embryos or the UTC co-cultured with them. It is concluded that trypsin treatment might effectively prevent infection of recipients if individual, Day 7, exposed embryos were transferred into the uterus.

Routine quality controls in production of bovine embryos by in vitro fertilization (IVF) should include screening all materials of animal origin for the presence of bovine viral diarrhea virus (BVDV). Using a reverse transcription nested polymerase chain reaction (RT-nPCR) assay, we detected BVDV in primary cultures of uterine tubal cells (UTC) that had been used during IVF procedures. The goal of our ensuing investigation was to determine its source and assess risks associated with the identified contaminant. Sequencing of the amplified 5' nontranslated region (NTR) of the viral genome confirmed a Genotype I BVDV contaminant. This viral contaminant was also identified by RT-nPCR in multiple samples of the same lot of fetal bovine serum (FBS) that was used in transport media by the laboratory that harvested the UTC. Both routine and enhanced roller bottle methods for virus isolation failed to detect BVDV in the FBS. Furthermore, virus neutralization assays did identify antibodies to Genotype I strains of BVDV in the FBS. After 7 days of co-incubation, neither cultured, washed UTC nor exposed, washed embryos were RT-nPCR positive for BVDV. Eight embryos produced in the contaminated system were nonsurgically transferred into eight seronegative cows. None of the embryo recipients seroconverted to BVDV. Thus, contamination of cell culture medium with BVDV did not result in transmission of the virus when IVF embryos were transferred. Failure to transmit disease was likely aided by serendipitous control from anti-BVDV antibodies in the FBS. However, a diagnostic dilemma was created when the RT-nPCR assays used to screen for BVDV were positive, yet attempts to isolate the virus were negative. This case study illustrates that if molecular assays are to be used to confirm the pathogen-free status of IVF embryo production systems, media components of animal origin (e.g. FBS) should be screened with molecular assays for BVDV as well as traditional virus isolation techniques.

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.

Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.

Investigation and experience have demonstrated that movement of in-vivo-derived bovine embryos can be accomplished while effectively limiting spread of infectious disease between populations of cattle. Experimental and theoretical justifications of current strategies for production of specific-pathogen-free, in-vivo-derived embryos are reviewed. Hazards of spreading bovine viral diarrhea virus via in-vivo-derived embryos are dealt with specifically. It is concluded that established sanitary procedures for producing pathogen-free, in-vivo-derived embryos are efficacious if the ethical and technical excellence of those performing the procedures can be assured.

Infectious agents in systems for producing bovine embryos might reduce the number and quality of embryos generated, result in transmission of disease to recipients and offspring, or confound findings of research. Embryo-associated pathogens might also jeopardize human health when the goal of embryo production is creating transgenic animals intended to be a source of pharmaceuticals or organs. This paper addresses risks and resulting hazards of pathogen and microbial contaminant introduction into in vivo or in vitro embryo production systems. Additionally, methods for prevention and quality control are discussed.

Recent studies have shown that exposed, in vitro-derived embryos remain contaminated with bovine viral diarrhea virus (BVDV) after washing. However, introduction of a Genotype II versus Genotype I strain of BVDV into an IVF system was reported to provide greater potential for transmission of disease. The primary objective of this study was to compare the potentials for different strains of noncytopathic BVDV to replicate in an IVF system, associate with IVF embryos and infect co-cultured cells via association with washed embryos. The secondary objective was to compare the effect of different strains of BVDV on embryonic development. Two Genotype I (SD-1 and NY-1) and 2 Genotype II (CD-87 and PA-131) strains of BVDV were evaluated. After IVM and IVF of oocytes, presumptive zygotes were washed and transferred into in vitro cultures containing uterine tubal cells (UTC) and medium that was free of BVDV-neutralizing activity. Immediately before addition of zygotes, the cultures were inoculated with 10(5) cell culture infective doses (50%, CCID50) of a strain of BVDV or maintained as a negative control. Cultures of zygotes were then incubated for 7 d. Embryonic development was observed on Days 3 and 7, and attempts were made to isolate BVDV from UTC and medium on Day 7. Also on Day 7, groups of intact, washed blastocysts were either transferred into virus-free secondary cultures containing UTC or sonicated with sonicate fluid assayed by both virus isolation and single-closed-tube reverse transcription nested polymerase chain reaction (RT-nPCR). After 3 d in secondary culture, hatched embryos were enumerated, and medium from the cultures, washed UTC and embryos were tested for BVDV by virus isolation. In addition, washed UTC and embryos were tested for BVDV using RT-nPCR. All strains of BVDV persisted and replicated in the embryo culture environment, but cleavage beyond the 4-cell stage, blastocyst development and hatching varied among cultures contaminated with different strains of virus. Further, the quantity of BVDV associated with washed embryos from both initial and secondary cultures varied among strains, but the variation was unrelated to difference in genotype (SD-1 and PA-131 greater than NY-1 and CD-87). Although all strains of BVDV replicated in UTC in the initial in vitro cultures and remained associated with washed blastocysts, susceptible UTC in the secondary in vitro cultures were seldom infected by any strain of virus.

In vitro embryo production has been used extensively in research and is now offered as a commercial service, yet the hazards of introducing specific infectious agents into in vitro embryo production systems have not been completely defined. The introduction of noncytopathic bovine viral diarrhea virus (BVDV) is a special concern. One objective of this study was to determine if noncytopathic BVDV-infected uterine tubal cells in IVF and IVC systems affected the rate of cleavage and development. An additional objective was to determine if either degenerated ova or embryos produced in the presence of the infected cells had virus associated with them after washing. Follicular oocytes (n = 645) collected from slaughterhouse ovaries were matured and fertilized in vitro, and presumptive zygotes were cultured for 7 d. Primary cultures of uterine tubal cells for use during IVF and IVC were divided into 2 groups. One-half of the cultures was infected with noncytopathic BVDV while the other half was not exposed to the virus. Approximately equal groups of mature oocytes were inseminated, and the presumptive zygotes were cultured with infected or noninfected uterine tubal cells. After 7 d in IVC, zona pellucida-intact (ZP-I) morulae and blastocysts and degenerated ova were washed and assayed for the presence of infectious virus. Infections of uterine tubal cells were not apparent and did not reduce rates of cleavage and development (P > 0.05; Chi-square test for heterogeneity). After washing, BVDV was isolated at a significantly higher rate from groups of virus-exposed degenerated ova (79%) than from individual virus-exposed morulae and blastocysts (37%; P = 0.0002; Mantel-Haenszel summary, Chi-square).