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Bovine viral diarrhea virus (BVDV) can associate with in vitro fertilized (IVF) bovine embryos despite washing and trypsin treatment. An antiviral compound, DB606 (2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan), inhibits the replication of BVDV in bovine uterine tubal epithelial cells, Madin Darby bovine kidney cells, and fetal fibroblast cells. As well, DB606 in in vitro culture medium does not affect embryonic development. Antiviral-treated-IVF embryos placed into recipients developed into clinically normal calves. The objective of this project was to determine if these resultant heifer calves were capable of reproducing. Seven heifers from each of the treatment groups (natural breeding, IVF embryo, and IVF embryo cultured in DB606) of the previous study were used. At 20-27 months of age, the heifers were exposed to a fertile bull in a single pasture during a 63 d breeding season. Five of the seven heifers originating from natural breeding were pregnant 35 d after removal of the bull and calved. All of the heifers resulting from transfer of untreated IVF embryos were pregnant at 35 d; however, one aborted the fetus at 5-7 months of gestation. All of the heifers derived from transfer of IVF embryos cultured in DB606 were pregnant and calved. Offspring from dams of all treatment groups were clinically normal at birth. Adjusted 205 d weaning weights were not significantly different among the offspring of the treated and untreated dams. These results indicate that culture of bovine-IVF embryos in DB606 does not impair future reproductive capacity of resulting heifers.

Bovine herpesvirus 1 (BoHV-1) is widely distributed among cattle populations and has been associated with cells, fluids, and tissues collected from donor animals for use in reproductive technologies. The purpose of this study was to determine if lactoferrin would inhibit BoHV-1 in cell culture and to evaluate if embryos could develop normally when cultured in vitro with lactoferrin. In Experiment 1, lactoferrin (10 mg/mL) inhibited up to 25,000 plaque forming units (PFU)/mL of BoHV-1 in Madin Darby bovine kidney (MDBK) cell culture. In Experiment 2, lactoferrin (10 mg/mL) combined with cidofovir (62.5 microg/mL) inhibited up to 100,200 PFU/mL of virus in cell culture. In Experiment 3, following fertilization, presumptive zygotes were cultured in media containing lactoferrin (10, 5, and 2.5 mg/mL). Embryonic development and quality were assessed, and embryonic viability was determined by counting the nucleated cells of developed blastocysts. While lactoferrin did not affect the nucleated cell count of the treated embryos, it did significantly decrease blastocyst development. In conclusion, lactoferrin from bovine milk can inhibit BoHV-1 in cell culture. However, supplementation of in vitro culture medium with lactoferrin inhibits blastocyst development of in vitro-produced embryos.

The purpose of this study was to determine whether or not embryos derived from in vitro fertilization of oocytes from persistently infected (PI) cattle would contain infectious virus.Three in vitro embryo production treatment groups were assessed: 1) oocytes and uterine tubal cells (UTC) free of bovine viral diarrhoea virus (BVDV) (negative control), 2)oocytes free of BVDV fertilized and cultured in media containing UTC obtained from PI heifers, and 3) oocytes from PI heifers fertilized and cultured in media containing UTC free of BVDV. The developmental media, UTC and embryos (individual or groups of five) were assayed for virus.Virus was not isolated from any samples in treatment group 1.As shown in previous studies, a proportion of embryo samples were positive for BVDV in treatment group 2. In treatment group 3, the virus associated with the oocytes contaminated the developmental media and infected susceptible co-culture cells used during fertilization and culture. In addition, 65% (11/17) of the degenerated ova from treatment group 3 had infectious virus associated with them. While none of the ova developed into transferable embryos, the study did confirm that use of oocytes from PI cows could lead to amplification of BVDV and cross contamination during in vitro embryo production.

Although porcine-origin trypsin will effectively remove bovine herpesvirus 1 (BHV-1) associated with in vivo-derived embryos, TrypLE, a recombinant trypsin-like protease, has not been evaluated. In Experiment 1, 17 groups of 10 in vivo-derived embryos were exposed to BHV-1, treated with TrypLE Express or TrypLE Select (10x concentration) for varying intervals, and assayed as 2 groups of 5 embryos. TrypLE Select treatment for 5 and 10 min (two and seven groups of five embryos, respectively) effectively inactivated BHV-1. In Experiment 2, 22 groups of 10 IVF embryos were treated and assayed. Treatment with TrypLE Select for 7 and 10 min (six groups of five embryos each) and with TrypLE Select diluted 1:2 for 10 min (seven groups of five embryos) was also effective. In Experiment 3, 17 groups of 10 IVF embryos were further evaluated with TrypLE Select undiluted and diluted 1:2 for 10 min. Treatment with the diluted product was effective (18 groups of five embryos), whereas the undiluted product was not completely effective (virus isolated from 2 of 16 groups). In Experiment 4, IVF embryos were treated as described in Experiment 3 and then cultured individually or as groups of five on uterine tubal cells (UTCs) for 48 h; 60% of UTC samples associated with groups of embryos and 35% of UTC associated with individual embryo samples were positive for BHV-1. Therefore, although TrypLE Select appeared to have promise for the treatment of in vivo-derived embryos, it cannot be recommended for treatment of in vitro-derived embryos.

Artificial insemination and embryo transfer are used commonly in cattle production and exchange of germplasm between populations of cattle. If properly monitored, assisted reproductive techniques can be used to prevent the spread of infectious agents. However, these techniques potentially represent unnatural routes for transmission of diseases. Bovine viral diarrhea virus (BVDV) is broadly distributed among the world's populations of cattle. Fluids, gametes and somatic cells from infected animals are likely contaminated with the virus. Thus, use of semen or embryos from infected animals could result in spread of BVDV. This paper provides an overview of the risks of transmitting this virus by AI or production and transfer of embryos and summarizes the precautions needed to prevent such transmissions of disease from occurring.

Embryo technologies have been integrated into production systems for a variety of livestock species. As relates to transmission of infectious diseases, our working hypothesis has been that use of embryo transfer for distribution of germ plasm within and between herds and flocks is likely safer than the movement of postnatal animals. Indeed, research and experience generally have been supportive of this hypothesis. However, the relative risks of transmitting infectious agents via embryo transfer vary among donor species. Further, different methods of producing embryos appear to present different risks. This paper provides a comparative overview of the risks of transmitting infectious diseases via transfer of both in vivo- and in vitro-derived embryos in common domesticated livestock species. Also discussed are universal approaches to biosecurity in embryo production and transfer.

The objectives of this study were to develop techniques to detect BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using either virus isolation (VI) or real time quantitative polymerase chain reaction (RT-QPCR) assays. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed to SD-1, a high affinity strain of BVDV, for 2 h and then processed according to the International Embryo Transfer Society (IETS) guidelines prior to testing. Groups of five or two in vivo-derived embryos, and single in vivo-derived embryos, were VI positive for BVDV 100, 50, and 33% of the time, and were RT-QPCR positive 100, 75, and 42% of the time, respectively. The virus was detected by the VI technique in all of the groups of five or two in vitro-produced embryos and in all of the single in vitro-produced embryos, and it was detected in 100, 80, and 50%, using RT-QPCR. Techniques for RT-QPCR were sufficiently sensitive to detect 10 copies of viral RNA in a sample and to detect BVDV associated with single embryos. Application of this new technology, RT-QPCR, will facilitate additional studies to further assess the risk of transmission of BVDV through embryo transfer.

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 μM 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.

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.

Bovine viral diarrhea virus (BVDV) can be found in cells and fluids from ovaries collected at the abattoir. On the other hand, immunoglobulins are also found in the fluid of ovarian follicles. Anti-BVDV antibodies in follicular fluid might reduce cross-contamination of COCs at the time of collection or hinder the use of virus isolation to test for the presence of virus. One objective of this study was to determine the frequency with which BVDV could be found in pooled follicular fluid collected during the periodic aspiration of COCs from abattoir-origin ovaries. A second objective was to determine the prevalence and neutralizing activity of anti-BVDV antibodies in these blended samples. We collected samples of pooled follicular fluid (n = 55) over a 20-month period as part of our routine oocyte collection activities. We assayed each sample for BVDV using virus isolation as well as reverse transcription nested polymerase chain reaction (RT-nPCR) procedures. We also tested follicular fluid for antibody that would neutralize four representative strains of BVDV (SD-1, a genotype 1a strain; NY-1, a genotype lb strain; CD-87, a genotype 2 strain, and PA-131, a divergent genotype 2 strain). We detected no BVDV by virus isolation, but we did identify the virus by RT-nPCR in one of the 55 samples of follicular fluid. Automated dye terminator nucleotide sequencing of the amplified portion of the viral genome indicated a genotype 1 strain that was distinct from any of our laboratory strains. In addition, each of the samples of follicular fluid contained sufficient antibody to neutralize large quantities of each of the four laboratory strains that were used. Finding BVDV in just 1 of 55 samples was consistent with reports of similar studies in which the occurrence of BVDV in abattoir-origin materials ranged from 0.9 to 12%. We presumed that failure to isolate the virus was due to neutralizing antibody in the sample. Thus, the incidence of BVDV contamination of our IVF system at the level of pooling of follicular fluid was low for the 20-month period. The presence of anti-BVDV antibody in pooled follicular fluid provided a coincidental means of neutralizing BVDV when it was introduced in fluid aspirated from infected ovaries.

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.

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.

Evidence indicates low potential for transmission of pathogens with in vivo-derived embryos of cattle when appropriate precautions are taken. In apparent contrast, results of research with in vivo-derived embryos of small ruminants and swine and with in vitro-derived embryos of cattle suggest a greater tendency for their association with pathogens after artificial exposure. However, regardless of donor species, investigations involving collection of embryos from artificially or naturally infected animals and assessment of health of recipients and offspring after transfer of these embryos have indicated low potential for transmitting disease. In this paper, results of embryo–pathogen research are summarized, emphasizing potential for spread of pathogens under natural circumstances. Also, safe embryo handling practices and their application to multiple species are discussed.

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 embryosor = 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 embryosor = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage toor = 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.

In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 106 to 108 cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 105 CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P=0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.

Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 104 to 106 TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100 % ( 9 9 ) and 78 % ( 7 9 ) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33 % ( 3 9 ) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44 % ( 4 9 ) of groups of M/B and 67 % ( 6 9 ) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22 % ( 2 9 ) of the groups of M/B and 44 % ( 4 9 ) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05).

As various embryo technologies in livestock were developed and evolved to a state of usefulness over the past 40 years, scientists with a specific interest in infectious diseases sought to determine the epidemiologic consequences of movement, especially international movement, of increasing numbers of embryos. Many of the foundational studies in this area were reported in Theriogenology , beginning in the 1970s and especially throughout the 1980s and 1990s. Unquestionably, Theriogenology has been a widely used venue for dissemination of basic information on this subject, which ultimately led to the development of the now universally accepted techniques for certification of embryo health. Today it is well-recognized that movement in commerce of embryos, especially in vivo –derived embryos, is a very low-risk method for exchange of animal germ plasm. This paper chronicles the evolution of strategies for health certification of embryos. An overview is provided of the calculated efforts of practitioners, scientists, and regulators to organize, forge necessary partnerships, stimulate needed research, provide purposeful analysis of the results, and, through these processes, guarantee the universal acceptance of efficient protocols for certifying the health of embryos intended for movement in international commerce.

Techniques for in vitro production of bovine embryos have evolved to the extent that applications for the commercial production of calves have been proposed. However, little is known about the epidemiological implications of the procedures. One concern is the introduction of noncytopathic bovine viral diarrhea virus (BVDV). In this study, follicular oocytes (n=247) collected from 10 cows were matured and fertilized in vitro and presumptive zygotes were cultured for 7 d. Primary cultures of bovine oviductal epithelial cells for use during in vitro fertilization and culture were divided into 2 groups. Treated oviductal cells were infected with BVDV while control cells were not exposed to the virus. Two approximately equal groups of mature oocytes from each cow were inseminated, and the presumptive zygotes were cultured with infected or noninfected oviductal cells. After 7 d in culture, zona pellucida-intact morulae/blastocysts and degenerated ova were washed, sonicated and assayed for the presence of virus. The rates of cleavage and development were also compared by Chi-square analysis. After washing, virus was not isolated from morulae and blastocysts but was isolated from some groups of degenerated ova. Infections of oviductal cells were inapparent and did not significantly (P>0.05) affect rates of cleavage or development.

A model for the prediction of bluetongue virus seroconversion was developed using weather variables and results from serum samples collected from a research herd of Hereford, Angus, Holstein and mixed breed beef cows at 12 different times over 2 years. The six weather variables analyzed were: mean daily air temperature; mean daily soil temperature at a depth of 10 cm; mean daily hours of wet vegetation; total days of rainfall ≥ 0.13 cm; total rainfall for each 7 day period; mean daily solar energy (W m−2). A maximum R2 multiple linear regression technique was applied to meteorological data collected during the four weekly intervals prior to each sample collection date (48 sets of weekly meteorological data). The best predictors for seroconversion were mean daily hours of wet vegetation and total rain days during the second weekly period prior to sample collection. The bluetongue virus seroconversion was related to mean daily hours of wet vegetation, total rain days, and total precipitation as expressed by the equation: Seroconversion=7.1+4.0 (mean daily hours of wet vegetation)−1.3 (total precipitation) (R2=0.62, P

Thirty superovulatory treatments were administered to 19 mixed-breed, nonlactating cows. In 10 superovulatory treatments, the cows were primed with follicle stimulating hormone (FSH) on the second and third day of the estrous cycle, and in another 10 superovulatory treatments, the cows received no priming dosage of FSH. Initiation of the superovulatory treatments in both groups was determined by ultrasonically monitoring for regression of the dominant anovulatory follicle. Still another 10 superovulatory treatments were begun on Day 10 without regard for regression of the dominant anovulatory follicle and without a priming dosage of FSH. The mean days for starting the superovulatory treatment in the FSH-primed cows, in the nonprimed cows and in the controls were 10.5, 11.9 and 10 days, respectively. All cows were treated with eight injections of FSH at 12-hour intervals in a declining dosage (36 mg total). Cows were bred naturally and embryos collected nonsurgically seven days later. There was no significant difference (P>0.05) between the total number of embryos or transferable embryos in the three treatment groups. In this study neither priming on Days 2 or 3 nor initiating the superovulatory treatment, based on the morphologic regression of the dominant anovulatory follicle, was an effective means for improving the superovulatory response in cattle.

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7d post-fertilization were exposed (for 2h) to 2 x 10(5-7) cell culture infective dose (CCID(50))/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was

Oviductal-stage embryos were surgically collected from 27 superovulated adult cows of various breeds, ages, and parity. A total of 88 surgeries was performed via a caudal flank grid approach, with the animals in lateral recumbency and the reproductive tract irrigated with sterile glycerol solution prior to surgical closure. Eight cows were operated on twice and five cows were operated on three or more times. The maximum number of surgeries for a single cow was five. Successful ova collection was accomplished in each surgical attempt, and all cows submitted to this procedure subsequently became pregnant following return to the breeding herd. This technique provided greater exposure of the ovary, uterine tube, and uterine horn, with less adhesion formation than traditional ventral midline techniques.

Nineteen nulliparous, sexually mature heifers free of antibody to infectious bovine rhinotracheitis (IBR) virus were given two 35 mg intramuscular injections of Lutalyse 10 days apart to synchronize estrus and randomly divided into control and vaccinated groups. On the day of the last Lutalyse injection, modified live IBR virus vaccine was administered intramuscularly to each animal in the vaccinated group and each group was placed with a proven sire for 35 days. After the vaccination, samples were taken for isolation of virus and for determination of serum neutralizing antibody titers. No virus shedding occurred after intramuscular vaccination. However, the conception rate was markedly lower in the vaccinated group than in the control group. These results suggest that the intramuscular inoculation of modified live IBR virus vaccines into cattle during estrus is contraindicated.

The objective of this study was to develop a duplex quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of bovine herpesvirus 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) type I and type II. Follicular fluid was collected from a BoHV-1 acutely infected heifer, a BVDV I persistently infected heifer, and from 10 ovaries recovered from an abattoir. Both the BoHV-1 and BVDV contaminated follicular fluid were diluted 1:5 to 1:10(7) using the pooled, abattoir-origin follicular fluid. Each dilution sample was analyzed using the duplex qPCR, virus isolation, reverse transcription-nested PCR (RT-nPCR), and BoHV-1 qPCR. The duplex qPCR was able to simultaneously detect BoHV-1 and BVDV I in the fluid diluted to 1:100 and 1:1000, respectively. These results corresponded with the reverse transcription-nested PCR and BoHV-1 qPCR. Therefore, the duplex qPCR might be used for quality assurance testing to identify these two viruses in cells, fluids and tissues collected from donor animals and used in reproductive technologies.

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 2 h to 10(5)-10(6) cell culture infective doses (50% endpoint) per milliliter (CCID(50)/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) 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 1 h, 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 2h to approximately 10(6) cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of10 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.

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.

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.

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).

The primary objective of this study was to determine the percentage of individual, preimplantation, in vitro-produced bovine embryos which maintained association with virus despite washing following artificial exposure to a high affinity strain of bovine viral diarrhea virus (BVDV). Another objective of this study was to determine the quantity of virus associated with these embryos. A total of eighty-seven zona pellucida-intact, Day 7, in vitro-produced bovine embryos were exposed for 1 h to 2 � 106 cell culture infected doses per mL to the 50 percent endpoint (CCID50 mL–1) of a type 1 noncytopathic strain of BVDV (SD-1). Following exposure, the embryos were washed according to International Embryo Transfer Society standards for in vitro-produced bovine embryos; they then underwent sonication, RNA extraction, and freezing at –80�C until assayed for virus. A real-time quantitative polymerase chain reaction (QPCR) was run in duplicate on each of the 87 embryos. Forty-two percent (39/87) of the embryos assayed were determined to be positive for virus. The quantity of virus associated with the embryos averaged 0.55 viral copies per 5 µL (SD = 0.89 copies/5 µL, SEM = 0.14 copies/5 µL). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos were associated with ≤2.40 viral copies per 5 µL while 99% of contaminated embryos were associated with ≤3.44 viral copies per 5 µL. These findings show that there is a low level of virus associated with in vitro-produced embryos but virus is associated with a significant number of exposed embryos. In conclusion, this study indicates that the potential for transmission of BVDV via embryo transfer of in vitro-produced embryos is small given the amount of virus that was found to associate with individual embryos.

The objectives of this study were to evaluate the efficacy of trypsin treatment for the removal/inactivation of infectious bovine rhinotracheitis virus (IBRV) adhering to zona pellucida-intact (ZP-I) bovine embryos and to determine if bovine herpesvirus-4 (BHV-4) adheres to ZP-I bovine embryos. When adherence of BHV-4 was demonstrated, an additional objective was to determine whether trypsin treatment removes or inactivates this virus. A total of 139 ZP-I embryos was collected from superovulated donor cows at 7 d after estrus. Embryos were exposed to 10(6) to 10(7) plaque-forming units (pfu) of either IBRV or BHV-4 for 1 to 2 h. Subsequently, approximately equal numbers of embryos exposed to each virus were either washed 12 times and the washes and embryos examined for the presence of infectious virus, or they were treated with trypsin and the embryos examined for the presence of infectious virus. Although the fourth wash was the last positive wash, an average of 18 pfu of virus was detected from each of six groups (a total of 24 embryos) after exposure to IBRV and washing. Infectious bovine rhinotracheitis virus was not isolated from any of nine trypsin-treated groups (a total of 43 embryos). The seventh wash was the last positive wash for any group after exposure to BHV-4, yet an average of 2 pfu of virus was detected from each of six groups (a total of 29 embryos) after washing. No BHV-4 was isolated from any of eight trypsin-treated groups (a total of 43 embryos). The study confirmed previous reports that IBRV adheres to the bovine ZP after in vitro exposure and that trypsin treatment is effective in keeping ZP-I embryos free of this virus. Adherence of BHV-4 to ZP-I bovine embryos was demonstrated for the first time. Trypsin treatment was also effective in removing this herpesvirus.

Bovine viral diarrhea virus (BVDV) replicates in embryo culture systems and remains associated with developing IVF bovine embryos despite washing and trypsin treatment. Previous research (Givens et al. 2006 Theriogenology 65, 344–355) determined that 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) can be added to an in vitro embryo production system to prevent replication of BVDV without inhibiting embryonic development. The objective of this research was to assess sexual development and future reproductive capacity of heifers resulting from embryos that had been exposed to DB606 while developing in vitro. Presumptive zygotes that resulted from in vitro fertilization of oocytes were cultured for 7 days in medium supplemented with 0.4 �M DB606 or medium lacking the antiviral agent. All blastocysts were individually transferred nonsurgically into the uterus of a synchronized recipient. Additional control pregnancies were established by natural breeding. At 20 to 27 months of age, 7 heifers resulting from each treatment were simultaneously exposed to a fertile bull in a single pasture during a 63-day breeding season. All but 2 heifers subjected to natural breeding were pregnant 28 days after removal of the bull. One pregnancy was aborted between 5 and 7 months of gestation from a heifer that had undergone IVF without exposure to DB606. All offspring were normal upon clinical observation at birth. One heifer resulting from IVF with exposure to DB606 would not allow its calf to nurse adequate quantities of colostrum; thus, this calf was removed from the study. Adjusted 205-day weaning weights for offspring were evaluated using ANOVA and Tukey Kramer test for pair-wise comparisons. Offspring of dams treated or untreated with DB606 during IVC or produced by natural breeding exhibited mean adjusted 205-day weaning weights of 194, 181, and 170 kg, respectively (not significantly different). Results of this research using a limited number of animals demonstrates that exposure to DB606 during the first 7 days after fertilization does not appear to cause delayed toxic effects that would impair the future reproductive capacity of heifers.

TrypLETM (Invitrogen, Carlsbad, CA, USA) is a recombinant, fungal, trypsin-like protease that is used as a substitute for porcine-origin trypsin in cell culture procedures. It is stable at room temperature and does not present the same risk of contamination as animal-origin trypsin. Previously, TrypLE SelectTM (10X) was shown to remove bovine herpesvirus-1 (BHV-1) from Day 7 in vivo-derived embryos (Marley et al. 2006 Reprod. Fert. Dev. 18, 213–214). The objective of this study was to determine if the same treatment would effectively remove BHV-1 from Day 7 zona pellucida-intact, in vitro-derived porcine embryos. Day 7 in vitro-derived morulae and blastocysts and non-fertile or degenerate embryos (NFD) were washed according to the International Embryo Transfer Society protocol. One group of 10 NFD was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106–108 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 10 NFD was washed and served as the positive control. The remaining developed embryos were divided into groups of 10 and washed and treated as described in Table 1. Following treatment, the embryos were sonicated in groups of 5 and assayed by virus isolation. The negative control embryos, as well as the embryos treated with porcine-origin trypsin, TrypLE Select (10X) for 7 min, and TrypLE Select (10X) diluted 1 : 2 for 10 min, were negative for virus. The positive control embryos in addition to the other treatments were positive on virus isolation (Table 1). Although, TrypLE Select (10X) does have some antiviral effect when used for 10 min, it was not completely effective, as shown by the positive virus isolation results of one group of 10 embryos. The groups treated with TrypLE Select (10X) diluted 1 : 2 for 10 min were negative for virus; however, if a larger sample size had been tested, positive groups might have occurred. Though using a recombinant trypsin product would be beneficial over using an animal-origin product, it is not known if TrypLE Select (10X) would render a single IVF embryo free of infectious virus. Further research would also need to be performed to assess the viability of embryos following treatment with TrypLE Select (10X). In addition, other recombinant trypsin products need to be evaluated to determine their efficacy against BHV-1 associated with IVF embryos. Table 1.Effect of recombinant trypsin-like proteases on BHV-1 virus in porcine embryos

Quantitation of bovine viral diarrhea virus (BVDV) associated with individual transferable embryos is prerequisite to a thorough assessment of the risk for transmission of BVDV via embryo transfer. One objective of this study was to determine the proportion of in vivo-derived bovine embryos that remained virus-positive after artificial exposure to a high-affinity strain of BVDV and thorough washing. A second objective was to determine the quantity of virus associated with these individual embryos. A total of 87 zona pellucida-intact, Day 7, in vivo-derived bovine embryos were exposed to a type 1 noncytopathic strain of BVDV (SD-1) and washed according to International Embryo Transfer Society recommendations. Subsequently, individual embryos were sonicated, and the RNA was extracted from the sonicate fluids and stored at -80�C until assayed using a real-time quantitative polymerase chain reaction (QPCR). Twenty-six percent (23/87) of the embryos contained virus. The average quantity of virus associated with individual embryos after viral exposure and washing was 1.12 viral copies per 5 �L (SD = 1.57 copies 5 per �L-1; SEM = 0.33 copies 5 per �L-1). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos will be associated with ≤4.64 viral copies per 5 �L, whereas 99% of contaminated embryos will be associated with ≤6.62 copies per 5 �L. Obviously, only extremely small quantities of virus were associated with less than one-third of the embryos tested. Based on previous research, it is presumed that this virus is associated with the outer layers of the zona pellucida. A logical next step in the risk assessment would be to determine if these quantities of zona-associated virus are sufficient to infect na�ve recipients and/or embryonic cells after embryos are transferred. Further, similar efforts should be made to estimate the quantity of virus associated with in vitro-derived, zona pellucida-intact, bovine embryos after exposure to the same high-affinity strain of virus and washing.

Porcine-origin trypsin will effectively remove bovine herpesvirus-1 (BHV-1) from in vivo-derived embryos. It is not known if TrypLE (Invitrogen, Carlsbad, CA, USA) could be used to remove BHV-1, but this recombinant porcine sequence trypsin-like protease would be an attractive alternative because it is highly stable at room temperature and does not pose the same threat for contamination as animal-origin trypsin. Thus, the objective of this study was to determine if TrypLE Express (1X) for 1.5 min of exposure or TrypLE Select (10X) for 10 min of exposure would be effective at removing BHV-1 from Day 7 zona pellucida-intact, in vivo-derived embryos after they had been exposed to the virus. Day 7 bovine in vivo-derived morulae and blastocysts and non-fertile degenerate (NFD) embryos were collected and shipped overnight to our facility. Upon arrival, the zona pellucida intact embryos were washed according to the International Embryo Transfer Society protocol. Developed embryos were washed separately from NFD embryos. One group of 5 or 10 NFD or developed embryos was not exposed to virus and served as the negative control. The remaining embryos and 10 NFD were exposed to 106 PFU/mL BHV-1 (Colorado strain) for 1 h. Following exposure, one group of 5 or 10 NFD or developed embryos was washed and served as the positive control. One group of 10 developed embryos was washed and treated with porcine origin trypsin. The remaining developed embryos were divided into groups of 5 or 10 and washed and treated with TrypLE Express for 1.5 min or TrypLE Select (10X) for 10 min. Following treatment, the embryos were sonicated in groups of 5 or 10 and assayed by virus isolation. The negative control embryos, porcine origin trypsin treated embryos, and TrypLE Select treated embryos were negative for virus. The positive control embryos and the TrypLE Express treated embryos were positive on virus isolation (Table 1). When it was determined that TrypLE Express was not effective at 1.5 min, TrypLE Select (10X) was used for 10 min. These preliminary results indicate that use of TrypLE Select (10X) for 10 min is effective for removal of BHV-1 associated with Day 7, zona pellucida-intact, in vivo-derived embryos. In addition, TrypLE Select has the advantage of being an animal-origin-free product. However, use of TrypLE Express (1X) for 1.5 min was not effective. Because it is not practical to expose embryos to trypsin for 10 min, further research is needed to determine the ideal treatment concentration and time that will effectively remove BHV-1 without harming in vivo-derived bovine embryos. Table 1. Virus isolation results

Lactoferrin is an iron-binding glycoprotein found in milk, saliva, tears, and other exocrine secretions. It is known to have in vitro antiviral effects against human, feline, and canine herpesviruses. In addition, lactoferrin is known to be safe in cell culture. Bovine herpesvirus-1 (BHV-1) is a likely contaminant of in vitro embryo production. Further, trypsin treatment is not completely effective in removing the virus from these embryos. We hypothesized that a nontoxic concentration of lactoferrin might prevent replication of BHV-1 within in vitro embryo production systems. Thus, the specific objectives of this research were to determine if lactoferrin from bovine milk would inhibit BHV-1 in cell culture and to determine if in vitro-produced embryos could develop normally when cultured in lactoferrin. Two-fold dilutions of lactoferrin (from 10 to 0.625 mg/mL) were added to Madin Darby bovine kidney cells, followed in 15 min by the addition 104 PFU/mL of BHV-1 (Colorado strain). Samples of cell lysate were taken at Day 2 and virus was quantified by plaque assay. The percent of virus inhibited by the antiviral agent at each concentration was determined by comparison to equivalent samples from temporal control cultures in which no compound was added before or after inoculation (Percentage of virus inhibited = [Quantity of virus in the control sample - Quantity of virus in the compound sample]/Quantity of virus in the control sample � 100). Next, the effect of lactoferrin was determined on in vitro-produced embryos. Cumulus oocyte complexes were received from an abattoir, matured in transit, placed in fertilization drops for 6 h, and then placed in culture drops containing lactoferrin (10, 5, and 2.5 mg/mL). At Day 3.5, embryos > 4 cell stage were placed into fresh culture drops containing lactoferrin. On Day 7.5, blastocyst development was noted and the developed embryos were stained to count viable cells. Blastocyst development rate and nucleated cell count of the treated embryos were compared to those of the controls using Chi square test, and ANOVA and Tukey-Kramer HSD, respectively. Lactoferrin (10 mg/mL) inhibited 2 to 5 logs of virus. At concentrations of 5 and 2.5 mg/mL, 1 to 3 logs of virus were inhibited, and concentrations of 1.25 and 0.625 mg/mL inhibited 0 to 2 logs of virus. Lactoferrin did not affect the nucleated cell count of the treated embryos. In addition, unlike 10 and 5 mg/mL, 2.5 mg/mL of lactoferrin did not affect blastocyst development. These preliminary results indicate that lactoferrin from bovine milk can significantly inhibit BHV-1 in cell culture. Furthermore, supplementation of in vitro culture with 2.5 mg/mL of lactoferrin does not affect blastocyst development or cell count of in vitro-produced embryos.

Because of its broad distribution among populations of cattle and its association with materials of animal origin used in embryo production, bovine viral diarrhea virus (BVDV) is a potential problem in applications of embryo technologies. While some isolates of BVDV are known to associate with both in vivo-derived and in vitro-produced bovine embryos, it has yet to be determined if the quantity of virus associated with exposed zona pellucida-intact embryos is sufficient to infect susceptible recipient cows via the intrauterine route. Techniques to detect and quantify BVDV associated with single transferable embryos are important to determine the risk of transmitting BVDV via embryo transfer. The objectives of this study were to define reproducible techniques to detect and quantify BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using virus isolation (VI) or real time quantitative polymerase chain reaction (Q-PCR) assays. In vivo-derived and in vitro-produced embryos were exposed for 2 h to approximately 106-cell culture infective doses (50% endpoint) per mililiter of a high affinity strain of BVDV, SD-1, and then washed according to IETS guidelines. Embryos were assayed in groups of five or two embryos, or single. There were 5 replicates of the group of five embryos, 4 of the group of two embryos, and 3 of the single embryos for the in vivo-derived embryos undergoing VI; 5, 4, and 2 replicates, respectively, undergoing Q-PCR, and 2, 5, and 2 replicates, respectively, for the in vitro-produced embryo groups undergoing VI and Q-PCR. Those to be assayed by VI were sonicated and the sonicate fluids were layered onto Madin Darby Bovine Kidney (MDBK) cells and passaged to allow for viral replication; an immunoperoxidase monolayer assay was then used for viral detection. A Roche� RNA/DNA extraction kit (Roche Diagnostic Systems, Inc., Somerville, NJ, USA) was used to extract RNA from virally exposed embryos, and extracted samples were assayed in duplicate Q-PCR reactions consisting of 100 �L. The primers used were L1 and U3 which are specific for conserved areas of the 5 prime nontranslated regions of the viral genome of BVDV. The PCR product was detected using hybridization probes s1 and s2 as in Struder et al. 2002 Biologicals 40, 289-296. In vivo-derived groups of five or two embryos, or single embryos, were positive for BVDV 100, 50, and 30% of the time, respectively, when VI was used and 100, 75 and 100%, respectively, when Q-PCR was used. The virus was detected in all of the in vitro-produced embryo groups of five, or two embryos, or single embryos, 100% of the time using VI, and in 100, 80, and 100% respectively, using Q-PCR. The virus isolation technique is highly sensitive but the need to destroy embryos by sonication to identify any embryo-associated virus precludes its use for embryos intended for transfer. Techniques for Q-PCR were sufficiently sensitive to detect and quantify 10 copies of RNA in a sample and to detect BVDV associated with single embryos.

Numerous genetically similar cattle for use in research or teaching can be produced with chromatin transfer technology (Sullivan et al. 2004 Biol. Reprod. 70, 146–153). Miniature cattle can provide advantageous biotechnological models for teaching and study of human and animal diseases. Miniature cattle are approximately one fourth the size of standard cattle and, therefore, represent a potentially less expensive, safer, and more easily managed animal model. Our limited attempts to reproduce miniature cattle via in vivo and in vitro production of embryos resulted in poor response to superovulation and logistical challenges in recovering embryos or oocytes due to the small size of the animals. Thus, the purpose of this study was to evaluate the potential of embryos derived by chromatin transfer from fibroblasts of a miniature cow and cytoplasts from cows of standard size to produce viable offspring after transfer into recipient cows of standard size. The donor of somatic cells was a heifer that weighed 7.7 kg at birth. Chromatin transfer resulted in 19% (82/428) blastocyst formation. A total of 66 cloned blastocysts (65 excellent/good quality, 1 fair quality) were transferred into 26 synchronized recipients. While ultrasound revealed 13 pregnancies prior to 67 days of gestation, only 4 pregnancies of 5 fetuses were maintained beyond 100 days. Parturition was induced with dexamethasone and prostaglandin on Day 286 of gestation. One singleton (12.3 kg) and a set of twins (10.2 and 11.1 kg) were healthy at birth and normal at 1 week of age. Two fully developed singletons, weighing 21.4 and 13.6 kg, died in utero. The latter fetus exhibited a fixed dorsolateral deviation of the neck that complicated delivery despite a caudal obstetrical presentation. No abnormalities were noted in the size or structure of any placenta. Our results indicate that healthy miniature calves can be gestated by recipient cows of standard size after transfer of embryos derived by chromatin transfer. Unfortunately, the fetal wastage, fetal anomalies, and stillbirths observed with standard sized cattle also may occur.

An early study demonstrated that washing embryos was effective for removal of virus after artificial exposure of in vivo-derived embryos to a cytopathic isolate of BVDV (Singh et al., 1982 Theriogenology 17,437–444). However, a recent study using representative noncytopathic isolates of BVDV demonstrated that washing was less beneficial for removing some isolates of BVDV than for others (Waldrop et al., 2000 Theriogenology 57, 575). Thus, the objective of this study was to determine if the quantity of a high affinity isolate of BVDV that remains associated with single-washed or trypsin-treated embryos is sufficient to cause infection in vivo. Twenty zona pellucida-intact Day 7 morulae and blastocysts (MB) were collected from superovulated cows. After collection, all MB were washed according to International Embryo Transfer Society (IETS) standards, and all but 4 (negative controls) were exposed for 2h to approximately 106 cell culture infective doses (50% endpoint) per mL (CCID50/mL) of viral strain SD-1. Following exposure, one-half of the MB were washed and one-half were trypsin-treated according to IETS standards. All MB were then individually sonicated, and sonicate fluids were injected intravenously into seronegative calves. Blood was collected from each calf on Days 0, 3, 6, 9, 12, 15, 20, 25, and 30, and sera were assayed for BVDV and anti-BVDV antibodies. All cattle used in the study were determined to be virus- and antibody-negative 30d prior to and the day of intravenous inoculation of sonicate fluids into calves. Viremia was not detected in any calf following injection, possibly due to intermittent sampling and/or small amount of embryo-associated virus present. However, seroconversion of 38 and 13% of the calves occurred following injection with sonicate fluids from washed and trypsin-treated embryos, respectively. Findings demonstrated that the quantity of a high affinity isolate of BVDV associated with single-washed or trypsin-treated embryos is sufficient to be infective in vivo as evidenced by seroconversion. These results emphasize the need for studies to determine if the virus associated with exposed individual embryos constitutes an infective dose when placed into the uterus.

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.

Most isolates of BVDV cause unapparent infections in cultured cells. Fetuses, postnatal animals or fetal bovine serum are possible sources of the virus for cultivated cells used as karyoplasts in cloning. Routine screening by veterinary diagnostic laboratories of 39 fetal fibroblast cell lines used in cloning research had revealed that 15 (39%) were positive for BVDV by various assays including RT-nPCR. As some were valuable transgenic cell lines, a rigorous protocol for evaluation of each line was undertaken to confirm infection with BVDV. A cryopreserved vial of each line was thawed, medium discarded and cells incubated (38.5°C in 5% CO2 and air) through 2 passages (6–10 days) in α-MEM supplemented with 10% equine serum. At the end of the second passage, cells were separated from medium, washed and assayed for presence of BVDV using virus isolation in 2 sequential passages in Madin Darby Bovine Kidney Cells and RT-nPCR. Available lots of fetal bovine serum and medium that had been used to culture the cells also were tested for BVDV. When the virus was detected, the RT-nPCR products were sequenced and compared. Also, an attempt was made to evaluate the earliest available cryopreserved passage of any positive cell lines. Results indicated that just 5 of 39 of the original cells tested (13%) were positive. Since cryopreserved earlier passages of 4 of the cell lines were available, they were assayed with the result that 2 of the 4 were not infected at the earliest passage. Further, BVDV was isolated from one lot of fetal bovine serum that was used to culture one of the cell lines. Sequence analysis verified that only 2 of these 4 cell lines were infected with the same isolate of BVDV, and one isolate was identical to the virus found in the fetal bovine serum used in medium to culture it. The discrepancy between our viral detection and that of the diagnostic laboratories is explained in part by the presumed test protocols. All BVDV-positive cells, as reported by the diagnostic laboratories, were positive by RT-nPCR. We presume that they did not separate medium from cells before assays. Thus, any noninfectious viral RNA that was in the medium (e.g. as would be expected in many lots of irradiated serum) would have been reported positive. The only possible sources for BVDV in these cell lines were the fetuses from which they originated or fetal bovine serum used in medium. Sequence analysis confirmed that serum was the source of viral infection in one line. The likely source of virus for 2 other lines was serum, since they were not infected at earlier passages. The 2 remaining cell lines were positive at the earliest available passages, so the fetuses from which cells were harvested could not be discounted as the source of BVDV. This report highlights the risks of introducing BVDV in embryo technologies and the difficulties that can be encountered in attempting accurate diagnosis of the presence of infectious virus.

Unnoticed infections with bovine viral diarrhea virus (BVDV) can occur in cultured cells used for somatic cell nuclear transfer. Aromatic cationic molecules have exhibited inhibitory activity against in vitro replication of BVDV. The purpose of this research was to evaluate the ability of aromatic cationic compounds to prevent or treat noncytopathic BVDV infections of fetal fibroblast cells. Aromatic compounds tested were 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606); 2-(2-benzimidazolyl)-5-[4-(2-imidazolino) phenyl]furan dihydrochloride (DB772); and 2-(1-methyl-2-benzimidazolyl)-5-[4’-(2-imidazolino)-2’-methylphenyl]furan dihydrochloride (DB824). To evaluate prevention of BVDV infections, 10 cell lines in the absence or presence of 7 dilutions of each of the 3 compounds were inoculated with BVDV. The concentrations of BVDV in medium and cell lysates were determined by serial dilution and virus isolation. Samples were obtained 72 hours post-inoculation. Bovine viral diarrhea virus in cell culture medium and cell lysate samples was evaluated by comparison to equivalent samples from control cultures in which no compound was added (percent of control=cell culture infective doses (50%; CCID50) of BVDV in compound sample/CCID50 of BVDV in control sample lacking compound). The viral inhibitory concentrations (99%) of compounds were calculated with JMP software by least-squares regression techniques. Cumulatively, the 99% endpoints for inhibition of viral replication in fetal fibroblast cell lines for the 3 compounds were 0.1μM, 0.007μM and 0.028μM, respectively. To evaluate therapeutic treatment of established BVDV infections, the concentration of BVDV in medium and cell lysates of 2 fetal fibroblast cell lines were evaluated. The cell lines were previously determined to be infected with a genotype 1a strain of BVDV. Samples were obtained during 4 sequential passages in the absence or presence of 0.04μM and 4μM concentrations of DB772 or DB824. Presence of BVDV was determined by reverse transcription nested polymerase chain reaction and virus isolation. While BVDV persisted in cultures supplemented with no aromatic compound or 0.04μM, both DB772 and DB824 effectively cured BVDV infections after 1 passage in 4μM, and cells remained viable. Results indicate that BVDV infections can be effectively prevented or treated in fetal fibroblast cultures. Further research is needed to determine if exposed cells are competent for production of normal embryos via nuclear transfer.

Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683–700). On Day 8, degenerated embryos were removed from each well and 400L of modified culture medium (SOFaaci plus 0.5% glucose and 10% fetal bovine serum) were added. At Day 11, hatched blastocysts were selected by scoring them as Quality 1 (Q1: >1.0mm, clear trophoblast, compact inner cell mass), Quality 2 (Q2: 0.5mm, dark spots in the trophoblast, less compact inner cell mass), or Quality 3 (Q3