Hematoporphyrin (HP), hematoporphyrin derivative (HPD), and thiopyronine (TP) are photosensitive agents (PSA) that have a germicidal effect when they are activated by light: helium neon laser (HeNe) light (HP, HPD), white light (HP, HPD), and yellow-green light (TP). Experiments were conducted with appropriate controls to determine the effect of photosensitive agents a) for inactivating bovine herpesvirus-1 (BHV-1; titre 106 TCID50 /ml) and bovine viral diarrhea virus (BVDV; titre 106 TCID50 /ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 106 TCID50 /ml) or BVDV (titre 106 TCID50 /ml); and c) on the in vitro development of embryos. Exposure to HP, HPD and TP followed by light irradiation inactivated BHV-1 and BVDV. Embryos exposed to BHV-I were disinfected by HP or HPD (5 μg/ml) in combination with HeNe light, or by HP or HPD (10 μg/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 μg/ml) followed by HeNe or white light irradiation. Exposure of embryos to light alone or to light and HP or HPD had no detrimental effect on their in vitro development; however, exposure of embryos to TP (5 μg/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 μg of HPD followed by HeNe light, or 10 μg/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.

Contents:Semen samples, collected from bulls pesistently infected with bovine viral diarrhea virus (BVDV) and containing BVDV (titer 105 ‐ 106TCID50/ml), were subjected to sperm separation procedures (washing, swim up, Percoll gradient, glass wool filtration, glass beads filtration) that are commonly used prior to in vitro fertilization (IVF) to determine if these procedures would yield spermatozoa free from BVDV. The final sperm pellets from frozen and fresh ejaculates were tested for the presence of BVDV by the immunoperoxidase technique; all tests were positive for BVDV in the range of 103 ‐ 104TCID50/ml). The study shows that when semen containing BVDV at the level of 105 ‐ 106TCID50/ml) is used for IVF, the virus is not completely removed by any of the simple physical methods commonly used to prepare sperm for IVF. Inhalt: Feldversuch das bovine Diarrhoe Virus (BVDV) aus Bullensperma durch Swim up oder andere Trennverfahren in Verbindung mit der In vitro Fertilisation zu entfernen Spermaproben, die von Bullen gewonnen wurden, welche dauerhaft mit dem Virus der bovinen Virus‐Diarrhoe (BVDV) infiziert waren und einen Titer zwischen 105 ‐ 106TCID50/ml enthielten, wurden verschiedenen Trennverfahren unterzogen (Waschen, swim up, Percoll gradient, Glaswollenfiltration, Glaskugelfiltration). Diese Verfahren werden üblicherweise für die Vorbereitung zur In vitro Befruchtung verwendet, und es sollte geprüft werden, ob diese Verfahren auch das Sperma von dem BVD‐Virus befreien können. Das endgültige Spermapellet von gefrorenen/aufgetauten und frischen Ejakulaten wurde in Gegenwart des BVD‐Virus und mit Hilfe der immunoperoxidase Technik getestet. Alle Testergebnisse waren positiv für BVD‐Virus in einem Bereich von 103 ‐ 104 TCID50/ml. Die Studie zeigt, daß Sperma, wenn es 105‐106TCID50/ml des Virus enhält, nicht vollständig mit den für die IVF üblichen, einfachen physikalischen Methoden von dem Virus befeit werden kann.

The fertilizing capacity of transitorily acidified semen was investigated with respect to using acidification as a method for destroying or inactivating acid labile pathogenic microorganisms in semen prior to freezing. Ejaculates diluted 1:1 with phosphate buffered saline (PBS) were acidified to pH 5.0 for 2 or 5 minutes before being returned to their original pH. They were then frozen by commercial methods and used to artificially inseminate superovulated heifers. A total 739 ova and embryos was collected from 119 inseminated donors. The fertilization rate was 88%, and 78% of the embryos were of transferable quality. Semen acidification had no apparent effect on the post-thaw percentage of intact acrosomes.

Contents: A key factor in the hygienic application of embryo transfer (ET) and its associated technologies is effective risk management. This depends on knowing what the risks of infectious disease transmission are and how they can be reduced or removed. Risk assessment depends on a knowledge of the pathogenesis of the disease, the procedures used in ET and its associated technologies and the results from research into infectious disease transmission by embryos or through ET. Two approaches can be taken to ensure the safe health status of the embryo: 1) by determining that the donor animals (male and female) are free from specific diseases, or 2) by ensuring that the zona pellucida (ZP)‐intact embryo is collected in a sanitary manner and handled (washed, treated, evaluated, etc.) according to recommended procedures. The former approach is applicable to diseases on which no or insufficient research has been done to determine the risk of their being transmitted by embryos or where the results of research done indicate a risk of the disease being transmitted. The latter approach is a preferred option with diseases where results of research done indicate that the risk of their being transmitted by embryos collected from infected or recovered donors is negligible. Infectious diseases for which research results indicate negligible risk of transmission by ZP‐intact (ZP‐I) embryos collected from infected or recovered donors include: pseudorabies (Aujesrky's disease), hog cholera (swine fever), foot and mouth disease, and swine vesicular disease. Infectious diseases on which insufficient research has been done include: African swine fever, vesicular stomatitis, enterovirus disease, parvouirus disease and leptospirosis. A problem associated with this approach is ensuring that procedures shown to be effective under experimental conditions are properly carried out under field conditions: the use of nationally accredited embryo collection teams may solve this problem. Healthy recipients and good record keeping are also critical factors in the hygienic application of ET. The health status of an embryo collected from a specific disease‐free donor will not be adversely affected by damage to its ZP. Where research has shown that ZP‐I embryos that have been exposed to particular pathogens are not infected or contaminated after proper washing or washing and treatment, it can be assumed that the risks of transmission of the diseases caused by these pathogens will not be increased by deliberate (micromanipulation) or accidental damage to the ZP after the washing/treatment procedures have been carried out. The risks of infectious disease transmission when embryos are produced by in vitro fertilization or blastomere transplantation to mature oocytes, followed by culture, have yet to be determined.

Research on infectious disease transmission by embryos bears on the development of protocols for the health certification of embryos in international trade. Consequently, sound design, execution and analysis of studies and compatibility with current embryo transfer technology are particularly important. This paper reviews approaches to research on infectious disease transmission by embryos and draws attention to circumstances that could compromise the validity and interpretation of the results.

Frozen-thawed bovine semen, experimentally infected with bovine herpesvirus-1 (BHV-1) at levels of 10(3) TCID(50)/ml and 10(4) TCID(50)/ml, was treated with a 0.3% trypsin solution to determine the effect of trypsin on the virus and on fertilization using superovulated animals. Virus was not isolated from any trypsin-treated samples using a cell culture assay system. Nor did two calves develop antibodies to BHV-1 following inoculation with trypsin-treated semen pooled from six bulls. Nonsurgical flushing of eight heifers inseminated with trypsin-treated frozen-thawed semen yielded 28 transferable-quality embryos.

Donor sheep were infected either by bites of bluetongue virus (BTV)-infected (serotype 11, "Texas Station strain") Culicoides variipennis or by inoculation with 100,000 median chicken embryo intravascular lethal doses of BTV (serotype 11) from a suspension made from infected C variipennis. Fourteen embryos from 4 BTV-infected ewes bred by rams not infected with BTV were transferred to 8 BTV-seronegative recipient ewes, and 35 embryos and 4 unfertilized eggs from 14 BTV-infected ewes bred by BTV-infected rams were transferred to 19 BTV-seronegative recipient ewes. Eleven pregnancies and 12 lambs resulted. None of the recipients or lambs seroconverted, and BTV was not isolated from the pregnant recipient ewes or their lambs at slaughter 30 days after parturition.

One hundred and eighty-five embryos were collected from 29 superovulated donors 6 to 8 d post estrus. The zona pellucida (ZP) of these embryos was either cracked, removed mechanically or removed with acidified Tyrode's solution, or left intact. Forty-eight of 103 (47%) ZP-cracked and ZP-free embryos, exposed for 24 h to infectious bovine rhinotracheitis virus (IBRV), survived. No significant difference was found in the embryonic survival of the ZP-cracked embryos exposed to IBRV and control embryos not exposed to IBRV. However, there was a significant (P < 0.001) difference in the survival of ZP-free embryos exposed to IBRV and ZP-free embryos not exposed to IBRV (30% vs 80%).

The approach of reverse transcription (RT) followed by the polymerase chain reaction (PCR) was used to amplify three different fragments of the bovine viral diarrhea virus (BVDV) genome. Two sets of primers framed two different regions within the gene coding for protein p80, the third set of primers was selected to amplify cDNA within the envelope glycoprotein (gp53) region. All three sequences could be detected in the homologous strain (NADL), whereas only some of the fragments could be amplified in heterologous strains of BVDV. RNA extracted from infected cells as well as RNA extracted from viral particles could be detected using RT-PCR. The detection limit was 10−1 – 10−2 TCID50 in ethidium bromide stained gels and could be further enhanced to 10−2 – 10−4 TCID50 by hybridization after Southern transfer. The speed and the sensitivity of this method might be of relevance for diagnostic purpose as well as for studies on epidemiology and pathogenesis of infection with BVD virus.

When 169 zona pellucida-intact bovine embryos were exposed to 106 pfu/ml of foot-and-mouth disease virus and then washed, no infectious virus was detected on any of the embryos. FMD viral infectivity was found, however, in association with 14 of 42 hatched (zona pellucida-free) bovine embryos and in a small number of zona pellucida-intact porcine embryos. The porcine embryos were assayed individually and in groups of 8 embryos. Four of the 124 individual embryos and 2 of the 9 groups of embryos carried the infectious virus.

Foot-and-mouth disease (FMD) viral infectivity detectable in cell cultures or by animal inoculation was not found to be associated with any of 48 washed zona pellucida-intact (ZPI) embryos collected from 8 cattle during the acute stages of disease. Similarly, infectivity was not found to be associated with any of 42 washed ZPI embryos collected from 3 cattle 21 d after infection with FMD.

The current, generally accepted approach to formulating health requirements for the international movement of embryos is to base them on the health status of the male and female donor animals. The alternative approach of basing them on the health status of the embryos themselves has been blocked by the lack of scientific information about the potential of the early embryo to transmit agents of infectious disease. Consequently, most research into infectious disease transmission by embryos has had the objec-tive of assessing the potential of the embryo to transmit infectious disease, at the stage of development at which it is transferred commercially, with the thought in mind that, for some diseases, it may be possible in the future to focus on the embryo rather than the donor when drawing up health requirements for import permits. Results from experiments involving the bovine leukemia virus, bluetongue virus, infectious bovine rhinotracheitis virus, foot and mouth disease virus and Brucella abortus are encouraging to the point where, with the exception of foot and mouth disease virus, they could and should be put to the test in field studies. Research on several other bovine pathogens is underway, but the studies are not sufficiently advanced for a judgement to be made on the potential of embryos to transmit them. There is evidence that the research done is starting to have a positive effect through the relaxation of some health requirements for the international movement of embryos. Resume

Four Holstein heifers were superovulated and inseminated with infectious semen from a bull experimentally infected with type 17 bluetongue virus (BTV). A total of 20 embryos were collected at donor slaughter and transferred to 16 recipients. Ten recipients became pregnant of which one subsequently aborted, one gave birth to twins which died at birth, one was killed at term because of dystocia, and 7 gave birth to live calves one of which died perinatally. All animals were tested for BTV antibodies at the time of slaughter which was at least 30 days post partum for surviving heifers and calves. Two of the four donor heifers were retrospectively determined to have been infected by the semen (viremia demonstrated) and their embryos accounted for 9 of the 10 pregnancies including the six surviving calves. None of the recipients or calves developed BTV antibody by the termination of the experiment. This study suggests that BTV-free calves can be readily obtained from the use of BTV-positive semen.

Two hundred and seven, zona pellucida-intact bovine embryos were collected from bovine leukemia virus-infected donors, washed, and transferred to uninfected recipients: 111 of these embryos were sired by bovine leukemia virus-infected bulls. Fifty live calves were obtained from the 57 pregnancies resulting from the transfers. None of the recipients or calves developed antibodies to bovine leukemia virus. Nine zona-intact ova, 12 zona-intact morulae and 15 hatched blastocysts, exposed "in vitro" to bovine leukemia virus, washed and then tested for bovine leukemia virus were negative. Twenty-seven, zona-intact embryos and 14 hatched embryos were similarly exposed and washed prior to being transferred in groups to two uninfected recipients: no pregnancies resulted, nor did the recipients develop antibodies to bovine leukemia virus up to 120 days posttransfer. The conclusion from these and other bovine leukemia virus studies is that zona-intact embryos can be transferred from bovine leukemia virus-infected donors, including those bred by bovine leukemia virus-infected bulls, without risk of transmitting bovine leukemia virus, providing that they are properly washed prior to transfer.

African swine fever virus (ASFV) was detected on or in zona pellucida-intact porcine embryos that had been exposed to 106.6 hemadsorption dose 50%/ml (HAdD50/ml) of ASFV for 18 hours, washed and then cultured. Ninety-five percent of the embryos retained infectious virus after washing. Treating the embryos with papain, EDTA or ficin had no effect on the retained virus, whereas treating them with trypsin or pronase reduced the number of embryos carrying detectable virus (30% instead of 95%) and lowered the amount of virus on the embryos. It has not yet been determined whether ASFV enters the embryonic cells but the evidence suggests that most of the virus, and possibly all of it, is bound to the zona pellucida.

Seventy-six, day 12 to day 15 bovine embryos, collected from 14 donors which had been inseminated with either X or Y chromosome-bearing spermatozoa fractions of semen separated by a thermal convection counterstreaming sedimentation and forced convection galvanization process, were processed for sexing by chromosomal analysis. Fifty-seven embryos were sexed; 20 from Y chromosome-bearing and 37 from X chromosome-bearing fractions of semen. Statistical analysis of the sexing data indicated that there was no significant difference in the male: female ratio for donors receiving male fractions compared to those receiving female fractions. The Y chromosome-bearing fractions produced a male: female ratio that was indistinguishable from the expected 1:1 ratio. However, the X chromosome-bearing fractions of semen produced a highly significant deviation from the expected 1:1 ratio towards the male.

Twenty-two superovulated IBRV-seronegative donors were infected intranasally, intravaginally or per uterus with IBRV prior to being inseminated with semen from seronegative bulls. All of the donors became infected and shed IBRV. Sixty-three embryos, collected non-surgically 7 and 8 days after estrus, were washed, treated with trypsin for 1 minute and transferred to 49 synchronized IBRV-seronegative recipients. Twenty-one pregnancies resulted. Three months later, 11 of the 22 donors were treated with dexamethasone to induce virus shedding. This drug inhibited ovulation, however, and only one embryo, which resulted in a pregnancy when transferred, was collected. Of the 22 pregnancies, one was accidently aborted, a second spontaneously aborted, one calf and two sets of twins were stillborn and 20 live calves were delivered. All recipients and calves remained serologically negative for antibodies to IBRV. Virus isolation and histopathology on both abortuses, the stillborn calves, and three calves slaughtered at three months of age were negative for IBRV.

As part of a program to study the feasibility of using embryo transfer to control disease, initial experiments were undertaken to determine the virus susceptibility of early embryos. Two hundred and ninety-three preimplantation bovine embryos (16-cell to blastocyst stage) were exposed to either akabane virus (AV), bluetongue virus (BTV) or bovine viral diarrhea virus (BVDV). Two hundred and thirty-seven of these embryos were then cultured for 24-48 hours in order to determine whether the virus had any effect on embryonic development and to allow viral replication to occur. No infectious virus was isolated from any of the embryos and the in vitro development of virus exposed embryos proceeded normally. In addition, twenty-nine eggs/embryos isolated from donors that were seropositive to BVDV were found to be uninfected with this virus.