Studies on inactivation of pathogenic microorganisms in culture media and in bovine semen by photosensitive agents.

The application of three photosensitive agents for disinfection of bovine semen was investigated. Bovine microbial pathogens suspended in tissue culture medium and/or PBS and also added to bovine semen were exposed to the photosensitive agents followed by irradiation. Hematoporphyrin, hematoporphyrin derivative and thiopyronine were effective against bovine herpes virus-1, bovine viral diarrhoea virus, Mycoplasma bovigenitalium, Mycoplasma canadense, and Ureaplasma diversum in culture media. In addition, thiopyronine was effective against Leptospira pomona. Similar treatments were not effective against Leptospira hardjo, Mycoplasma bovis, or Campylobacter fetus subsp. venerealis. When microorganisms were added to bovine semen, only bovine herpes virus-1 was controlled by the photosensitive agents used at concentrations which did not appear harmful to sperm cells.

Inactivation of bovine herpesvirus-1 and bovine viral diarrhea virus in association with preimplantation bovine embryos using photosensitive agents.

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 (He/Ne) 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 10(6) TCID50/ml) and bovine viral diarrhea virus (BVDV; titre 10(6) TCID50/ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 10(6) TCID50/ml) or BVDV (titre 10(6) 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 microg/ml) in combination with He Ne light, or by HP or HPD (10 microg/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 microg/ml) followed by He Ne 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 microg/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 microg of HPD followed by He Ne light, or 10 microg/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.

Transitory acidification of semen as a potential method for the inactivation of some pathogenic microorganisms. Effect on fertilization and development of ova in superovulated heifers.

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.

Investigation of some antimicrobial procedures on the in vitro development of early murine embryos aimed toward developing methods for the disinfection of mammalian embryos prior to transfer.

Eight-cell, zona pellucida-intact mouse embryos were exposed to the following substances or procedures that have been reported to have germicidal effects to determine if the embryos would survive and develop under in vitro conditions: the photosensitive substances hematoporphyrin, hematoporphyrin derivative, 8-methoxypsoralen, 4,5',8-trimethylpsoralen, and thiopyronine; the enzymes lipase (0.5%), phospholipase C (2 U/ml), chymotrypsin (0.5%), and trypsin (0.5%); pH 5.0; and helium/neon laser light, visible light, ultraviolet A light, and ultraviolet C light. Under the conditions used, embryos were not adversely affected by hematoporphyrin and/or helium/neon laser light; methoxypsoralen and/or ultraviolet A light; lipase; trypsin; pH 5.0 for 20 min; and visible light. Variable results were obtained from hematoporphyrin derivative with laser light. Thiopyronine, trimethylpsoralen in combination with ultraviolet A light, and ultraviolet C light killed embryos, and chymotrypsin and phospholipase C were harmful at 10- and 15-min exposure times, respectively.

Isolation of Mycoplasma bovis from intact and microinjected preimplantation bovine embryos washed or treated with trypsin or antibiotics.

Incubation of day 7 bovine embryos with 10(4) or 10(6) CFU/ml of Mycoplasma bovis (M. bovis) or microinjection of M. bovis into the cells of day 7 embryos did not influence embryonic development. M. bovis was recovered from all embryos washed 10 times by a standard pipetting method or vortexed and pipeted 10 times. M. bovis was also recovered from zonae pellucidae removed and washed from microinjected embryos. Neither treatment with trypsin nor exposure of embryos to combinations of penicillin, streptomycin, lincomycin and spectinomycin, or gentamicin, tylosin, lincomycin, and spectinomycin, inactivated M. bovis.

Inactivation of bovine herpesvirus-1 (BHV-I) from in vitro infected bovine semen.

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.

Nontransmission of bluetongue virus by embryos from bluetongue virus-infected sheep.

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.

Effect in vitro of bovine viral diarrhea virus on bovine embryos with the zona pellucida intact, damaged and removed.

The in vitro effect of bovine viral diarrhea virus (BVDV) on the survival of day 7 to day 7.5 bovine embryos collected from superovulated donors was studied. Fifty-four experimental embryos with the zona pellucida (ZP) intact, damaged or removed were exposed to 1 X 10(4) TCD50/ml of the NADL cytopathic strain of BVDC at 37 degrees C for 24 hrs and compared to 36 control embryos that were cultured for 24 hr. Seven embryos with the ZP-removed were similarly exposed for 48 hrs and compared to five control embryos. The overall survival rate was 68% for embryos exposed to BVDV for 24 hrs and 77% for embryos not exposed (P greater than 0.05). Extended exposure of the embryos with the ZP removed to virus for 48 hrs did not affect their survival rate compared to controls. Damage to the ZP by cracking or total removal of the ZP by micromanipulation or acidic Tyrode's solution had no effect on subsequent embryonic survival in the presence of BVDV. It was concluded that exposure to BVDV in vitro is not cytopathic for morula and blastocyst stage bovine embryos over a 48 hr period, even when they are not protected by the ZP.

The in vitro exposure to bovine rhinotracheitis virus of zona pellucida-micromanipulated bovine embryos with the zona pellucida damaged or removed.

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%).

Embryo transfer as a means of controlling the transmission of viral infections. X. The in vivo exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus.

Two experiments involving the transfer of embryos from donors infected with swine vesicular disease virus (SVDV) to "clean" recipients were carried out. In Experiment 1, 47 embryos were collected from 4 SVDV-infected donors and transferred to 2 recipients that subsequently produced 10 piglets. All of the recipients and piglets remained seronegative for SVDV. In addition to the transfers, 10 embryos and 58 unfertilized eggs from the infected donors were assayed in vitro and found to be negative for SVDV infectivity. A fifth donor was also inoculated with SVDV in this experiment, but it could not be demonstrated that infection had occurred. This SVDV-exposed donor provided two embryos for transfer and one embryo and two unfertilized eggs for in vitro assay. In Experiment 2, 158 embryos from 9 infected donors were transferred to 7 recipients, resulting in 12 piglets. A total of 7 embryos and 37 unfertilized eggs were assayed in vitro. The recipients, piglets, and embryos/eggs were all negative for SVDV infectivity. Although a final conclusion on the safety of using embryo transfer for the control of swine vesicular disease (SVD) is not possible, the results obtained justify additional studies.

Embryo transfer as a means of controlling the transmission of viral infections. VII. The in vitro exposure of bovine and porcine embryos to foot-and-mouth disease virus.

When 169 zona pellucida-intact bovine embryos were exposed to 10(6) 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.

Embryo transfer as a means of controlling the transmission of viral infections. VIII. Failure to detect foot-and-mouth disease viral infectivity associated with embryos collected from infected donor cattle.

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 persistent infection of a canine thymus cell line by equine infectious anaemia virus and preliminary data on the production of viral antigens.

Equine infectious anaemia virus (EIAV) was adapted to the Cf2Th cell line, a heterologous malignant line from canine thymus. A persistent infection was monitored for 100 serial passages by demonstrating the presence of virus and viral antigens at each 10th passage by electron-microscopy, immunodiffusion and immunofluorescence. Chromosome analysis of EIAV-infected cells indicated they had a karyotype resembling the control cells of similar passage history. Virus-infected cells, grown in roller cultures for 65 days without subculturing, continuously produced viral antigens into supernatant fluids which were harvested every 3-4 days. Antigen peaks were observed at approximately 12-day intervals. Immunoprecipitin lines of identity were demonstrated between ether-extracted antigens from virus-infected canine cell line and known positive EIAV antigen extracted from infected equine spleen and a commercial source. Replication of a non-oncogenic retrovirus (EIAV) resulted in the continuous release of viral antigens from a persistently infected and infinite cell line.

PROCEEDINGS - Embryo Transfer in the Canadian Cattle Industry Status of Disease Transmission Studies and thier Relationship to the International Movement of Bovine Embryos.

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 objective 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.

Embryo transfer as a means of controlling viral infections. VI. Bluetongue virus-free calves from infectious semen.

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.

Embryo transfer in relation to bovine leukemia virus control and eradication.

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.

Effect of separating bull semen into X and Y chromosome-bearing fractions on the sex ratio of resulting embryos.

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.