Elizabeth L. Singh

The disease control potential of embryos

Abstract The mechanisms by which embryos could transmit infectious disease, and the research that has been carried out on the disease transmission potential of livestock embryos are described. Analysis of this data emphasizes the importance of the integrity of the zona pellucida and of proper washing of embryos to ensure infectious disease control. If handled properly, the disease transmission potential of embryos is much less than that of either semen or live animals.

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.

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.

Embryo transfer as a means of controlling the transmission of viral infections

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. IV. Non-transmission of infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus following trypsin treatment of exposed embryos

Abstract 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.

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.

The feasibility of sexing bovine morula stage embryos prior to embryo transfer.

Abstract Sex determination of bovine morula stage embryos was possible in only 33% of embryos manipulated when 15 to 17 cells were removed for analysis. These results, in contrast to those of Moustafa et al . (1) who reported a sexing rate of 63% for bovine morulae on the basis of analyzing 8 to 10 cells, cast doubt on the practicality of sexing embryos for transfer at this developmental stage. The question thus raised was investigated by analyzing the mitotic indices of bovine, rabbit and mouse embryos. The figures obtained were in agreement with other studies and indicated that only 50% of embryos could be expected to have one of ten aspirated cells in division, and that not every metaphase spread would be suitable for sexing. It was concluded that until methods of sex determination other than chromosomal analysis are developed, the sexing of morula stage embryos is not to be recommended. It is technically more complicated and much less successful than sexing later staged embryos.

Embryo transfer as a means of controlling viral infections. III non transmission of bluetongue virus from viremic cattle

Abstract Ten holstein heifers were made viremic by inoculation with type 17 or type 18 bluetongue virus (BTV), superovulated, bred artificially with semen from a BTV seronegative bull and slaughtered for the collection of 8-day embryos. A total of 28 embryos were transferred into 28 BTV seronegative recipients. Fourteen transfers resulted in pregnancies. None of the recipients developed BTV antibody during pregnancy or within 30 days of parturition. No antibody or virus was detected in the 14 calves at parturition (of which 4 were lost due to dystocia), in one recumbent calf at the time of euthanasia at 5 days of age or in the remaining 9 healthy calves at 30 days of age. This study suggests that BTV-free calves can be obtained from infected dams by embryo transfer.

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 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.