Shaping the norms that regulate international commerce of embryos.

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.

Amplification of bovine viral diarrhoea virus introduced into an in vitro embryo production system via oocytes from persistently infected cattle.

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.

Epidemiology of prolonged testicular infections with bovine viral diarrhea virus.

Previously, bovine viral diarrhea virus (BVDV) had been found in prolonged testicular infections following acute infection of immunocompetent bulls. The primary purpose of this research was to evaluate the production and maintenance of prolonged testicular infections after exposure to BVDV of seronegative bulls in varying circumstances. The secondary objective was to initiate assessment of the potential for transmission of BVDV via semen of bulls exhibiting a prolonged testicular infection.

Bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure.

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).

Normal reproductive capacity of heifers that originated from in vitro fertilized embryos cultured with an antiviral compound.

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.

Lactoferrin from bovine milk inhibits bovine herpesvirus 1 in cell culture but suppresses development of in vitro-produced bovine embryos.

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.

Development of a duplex quantitative polymerase chain reaction assay for detection of bovine herpesvirus 1 and bovine viral diarrhea virus in bovine follicular fluid.

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.

Efficacy of a recombinant trypsin product against bovine herpesvirus 1 associated with in vivo- and in vitro-derived bovine embryos.

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.

Comparison of tests for detection of bovine viral diarrhea virus in diagnostic samples.

Currently, a variety of tests are used to detect bovine viral diarrhea virus (BVDV) in persistently infected (PI) cattle. These tests include immunohistochemical staining (IHC), antigen capture enzyme-linked immunosorbent assay (ACE), virus isolation (VI), and reverse transcription-polymerase chain reaction (RT-PCR). However, a lack of methods standardization could compromise the ability to consistently identify animals infected with BVDV.

Bovine viral diarrhea virus (BVDV): epidemiologic concerns relative to semen and 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.

Relative risks and approaches to biosecurity in the use of embryo technologies in livestock.

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.

Detection of bovine viral diarrhea virus (BVDV) in single or small groups of preimplantation bovine embryos.

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.

Use of a modified-live vaccine to prevent persistent testicular infection with bovine viral diarrhea virus.

A commercial vaccine containing modified-live bovine viral diarrhea virus (BVDV; types 1 and 2) was administered to one group of 22 peripubertal bulls 28 days before intranasal inoculation with a type 1 strain of BVDV. A second group of 23 peripubertal bulls did not receive the modified-live BVDV vaccine before intranasal inoculation. Ten of 23 unvaccinated bulls--but none of the vaccinated bulls--developed a persistent testicular infection as determined by immunohistochemistry and polymerase chain reaction.

Noncytopathic bovine viral diarrhea virus can persist in testicular tissue after vaccination of peri-pubertal bulls but prevents subsequent infection.

The objectives of this research were to evaluate the risk of prolonged testicular infection as a consequence of vaccination of peri-pubertal bulls with a modified-live, noncytopathic strain of BVDV and to assess vaccine efficacy in preventing prolonged testicular infections after a subsequent acute infection. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with an approximate minimum immunizing dose or a 10x standard dose of modified-live, noncytopathic BVDV or were maintained as unvaccinated controls. Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV.

Effect of phosphonoformic acid in the development of bovine embryos in vitro.

This research evaluated the ability of phosphonoformic acid to inhibit bovine herpesvirus 1 (BHV-1) in cumulus cells commonly used in co-culture with bovine in vitro-produced embryos. At 200 and 400 microg/ml, phosphonoformic acid inhibited 4 logs of BHV-1. Subsequently, phosphonoformic acid (200 and 400 microg/ml) added to both in vitro fertilization and culture medium resulted in a decrease in the proportion of developed blastocysts, and the number of cells per blastocyst was lower in the treated embryos.

Seroconversion of calves following intravenous injection with embryos exposed to bovine viral diarrhea virus (BVDV) in vitro.

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.

Normal calves produced after transfer of in vitro fertilized embryos cultured with an antiviral compound.

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.

Effects of aromatic cationic molecules on bovine viral diarrhea virus and embryonic development.

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.

Bovine viral diarrhea virus (BVDV) in cell lines used for somatic cell cloning.

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.

Prevention and elimination of bovine viral diarrhea virus infections in fetal fibroblast cells.

Noncytopathic infections with bovine viral diarrhea virus (BVDV) can compromise research and commercial use of cultured cells. The purpose of this research was to evaluate the ability of aromatic cationic compounds to prevent or treat BVDV infections in fetal fibroblast cell lines that are used in somatic cell nuclear transfer. To evaluate preventative use of compounds, 10 cell lines were inoculated with BVDV in the absence or presence of 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606), 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772), or 2-(1-methyl-2-benzimidazolyl)-5-[4'-(2-imidazolino)-2'-methylphenyl]furan dihydrochloride (DB824).

Post hatching development: a novel system for extended in vitro culture of bovine embryos.

Although acceptable rates of blastocyst formation are achieved with in vitro production of bovine embryos, several problems still compromise the subsequent development of the fetus and newborn, especially in embryos originating from somatic cell nuclear transfer. Routinely, the potential development of a bovine conceptus is predicted either on blastocyst quality or on various parameters related to the embryonic-fetal development in a foster mother. These methods are either imprecise or costly, highlighting the need for more reliable and practical methods to evaluate early embryonic development and differentiation.

Infectivity of bovine viral diarrhea virus associated with in vivo-derived bovine embryos.

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.

Different strains of noncytopathic bovine viral diarrhea virus (BVDV) vary in their affinity for in vivo-derived bovine embryos.

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.

Biosecurity issues associated with current and emerging embryo technologies.

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.

Bovine herpesvirus-1 associated with single, trypsin-treated embryos was not infective for uterine tubal cells.

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.