Embryo transfer as a means of controlling the transmission of viral infections. XV. Failure to transmit bluetongue virus through the transfer of embryos from viremic sheep donors.

The first experiment involved in vitro exposure of clean embryos to bluetongue virus (BTV) while three subsequent experiments involved the collection of embryos from BTV-infected donor ewes and their transfer to disease-free recipients. In Experiment I, 22 embryos/ova were exposed to BTV type 11 (BTV-11) for 1 h, washed 10 times in PBS and assayed in pairs for BTV. All 11 samples were positive for BTV in the 11-d-old embryonated chicken egg (ECE) assay system and 5/11 samples were positive in baby hamster kidney-21 (BHK-21) cells. In Experiment II, 5 donors were infected with BTV type 10 (BTV-10). All embryos were washed 10 times prior to assay or transfer. Thirty-three embryos/ova were assayed in groups of 2 or 3 and none yielded virus in ECE. Two BTV-seronegative recipients each received 6 embryos and a total of 3 lambs free of BTV antibodies were delivered. In Experiments III and IV, a total of 9 donors were infected with BTV-11. All embryos were washed 10 times prior to assay or transfer. Seventy-four embryos/ova were assayed in groups of 2 or 3 and none yielded virus in ECE, while for each experiment, 6 embryos were transferred into 2 BTV-seronegative recipients. The four recipients and their 3 lambs and 2 aborted fetuses were also seronegative for BTV.

Embryo transfer as a means of controlling the transmission of viral infections. XIII. Failure to transmit foot-and-mouth disease virus through the transfer of embryos from viremic donors.

A total of 436 embryos/unfertilized ova was collected from 30 foot-and-mouth disease (FMD) viremic cattle; 106 of these embryos/ova were from eight donors that had FMD virus in their reproductive tracts. The 436 embryos/ova were washed and then either assayed in cell culture or intradermally in steer tongues or transferred to recipients. Foot-and-mouth infectivity was not found to be associated with any of the embryos/ova assayed in cell culture or intradermally. The 149 embryos transferred produced two abortions, five sets of twins born prematurely, and 15 normal calves. All of the recipients and all of the calves remained FMD-seronegative.

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.

Embryo transfer as a means of controlling the transmission of viral infections. XI. The in vitro exposure of bovine and porcine embryos to vesicular stomatitis virus.

Infectious virus was isolated from both porcine and bovine zona pellucida-intact embryos that had been exposed to the Indiana strain of vesicular stomatitis virus (VSV) and then washed. The amount of virus isolated from embryos depended on their initial exposure level. Porcine embryos always retained more virus than bovine embryos. When embryos were cultured for 24 h after viral exposure and washing, the number of embryos carrying VSV and the amount of virus on each of the embryos was reduced. Trypsin (0.25%) was also found to be effective in inactivating/removing the VSV from embryos, suggesting that most, if not all, of the virus was bound to the zona pellucida.

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. IX. The in vitro exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus.

When zona pellucida-intact porcine embryos were exposed to 10(7) plaque-forming units (pfu)/ml of swine vesicular disease virus (SVDV) and then washed, infectious virus could be isolated from all of the embryos. Culturing the embryos for 24 or 48 h or treating the embryos with pronase, trypsin, or antiserum after virus exposure and washing reduced the number of embryos carrying virus and lessened the amount of virus on each of the embryos. None of the treatments, however, was capable of disinfecting every embryo.

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.

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.

Embryo transfer as a means of controlling the transmission of viral infections. II. The in vitro exposure of preimplantation bovine embryos to infectious bovine rhinotracheitis virus.

Bovine embryos, at the 16-cell to the blastocyst stage of development, were exposed to infectious bovine rhinotracheitis virus (IBRV) for either one or 24 hours. These embryos were then washed and incubated for 24 or 48 hours before being assayed for IBRV. Under these conditions, infectious virus at the level of 0-10(2.2) TCID(50)/ml was isolated from 57-64% of the embryos exposed to IBRV. Trypsin and IBRV-antiserum were found to be capable of removing and/or inactivating the IBRV from exposed embryos. Both the low level of the virus isolated from these embryos and the susceptibility of this virus to trypsin and antiserum suggests that IBRV attaches to the zona pellucida of embryos and cannot penetrate this structure to gain access to the embryonic cells. IBRV was found to have no effect on embryonic development in vitro . In addition, thirty-one eggs/embryos isolated from donors that were seropositive to IBRV were found to be uninfected with this virus.

Transfer of embryos from bovine leukaemia virus-infected cattle to uninfected recipients: preliminary results.

One hundred and fifty-one, day 6 or 7, embryos collected from cattle infected with bovine leukaemia virus (BLV) were transferred to uninfected recipients. Thirty-two pregnancies resulted. Two animals aborted at seven months. Three sets of twins and one single calf were still-born. The remaining 26 pregnancies produced 27 live calves which were raised to six months of age. All of the recipients, pregnant and non-pregnant, and all of the calves remained serologically negative for antibodies to BLV-glycoprotein antigen.

Embryo transfer as a means of controlling the transmission of viral infections. I. The in vitro exposure of preimplantation bovine embryos to akabane, bluetongue and bovine viral diarrhea viruses.

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.

Attempts to isolate bovine leukemia and bovine syncytial viruses from blood, uterine flush fluid, unfertilized ova and embryos from infected donor cattle.

Blood leucocytes, sediments of uterine flush fluid (UFF), eggs and embryos from 25 BLV-positive donor cows were tested for bovine leukemia (BLV) and bovine syncytial (BSV) viruses by cocultivation with fetal lamb spleen cells and by applying syncytium induction and immunofluorescence tests. BLV was diagnosed in 11/15 (73.3%) leucocyte and 4/25 (16.0%) UFF-sediment specimens as compared to BSV in 14/15 (93.3%) and 21/25 (84.0%) of the similar specimens and neither BLV or BSV were found in 26 eggs and 60 embryos collected from 20 of the 25 cows. Detection of BLV antigens by immunofluorescence was hampered by the competitive replication of both BLV and BSV and competitive growth in indicator cells and uterine cells. As BLV has not been observed in cells of UFF sediments, it was probably isolated from leucocytes present in the lumen of uterus or from blood seeping out from inapparent vessel damage during flushing. Isolation of BLV in UFF sediments gives additional evidence to the concept of a transplacental transmission by a not yet elucidated mechanism. The high rate of BSV recovery from cells of UFF sediments indicated that this virus is more wide-spread than previously shown and that it may play a role in causing disorders of the reproductive tract.

Embryo transfer: a discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents.

Studies on laboratory animals have shown that viruses vary as to whether or not they are transmissible by the gametes or are capable of passing through the zona pellucida and infecting the embryo.Methods of studying early embryos for the presence of infectious agents include electron microscopy, immunocytochemistry and cell cultivation.Determination that early bovine embryos do not become infected by certain agents might allow for easing of restrictions in the current import and export regulations for cattle embryos.Embryo transfer could be used as a means of controlling or eliminating disease in a herd or flock if the causal agent does not infect the early embryo via the gametes or by penetrating the zona pellucida.

Chromosomal analysis of 159 bovine embryos collected 12 to 18 days after estrus.

One hundred and fifty-nine embryos were analysed in conjunction with embryo transfer studies. Chromosome preparations were made from small biopsy fragments of trophoblast, from large portions of trophoblast and the inner cell mass, or from the entire embryo. Results obtained from fragments were similar to those obtained from large portions of trophoblast and inner cell mass. No structural chromosomal abnormalities were observed. Single triploid, diploid-triploid and diploid-hexaploid and 66 diploid-tetraploid embryos were found. The 41.5% incidence of diploid-tetraploid embryos was relatively high, and appeared to be associated with a donor factor. The transfer of 49 biopsied and analysed embryos to recipients resulted in 15 pregnancies, seven with diploid-tetraploid embryos having up to 25% of polyploid cells. The diploid-triploid and diploid-hexaploid embryos were among the transfers that did not result in pregnancy. The low incidence of embryos with chromosomal abnormalities, excluding the diploid-tetraploid embryos, may have been due to the embryos being analysed at 12 to 18 days of age rather than at an earlier age before death or degeneration could have occurred.