Susceptibility of pig embryos to porcine circovirus type 2 infection

The aim of the present study was to determine if porcine circovirus type 2 (PCV2) is able to infect embryonic cells of in vivo produced porcine embryos with and without zona pellucida (ZP). ZP-intact and ZP-free morulae (6-day post-insemination) and early blastocysts (7-day post-insemination), and hatched blastocysts (8-day post-insemination) were exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15). At 48 h post-incubation, the percentage of infected embryos and the percentage of viral antigen-positive cells per embryo were determined by indirect immunofluorescence (IF). Significantly different percentages of infected embryos were detected: 15% for ZP-free morulae, 50% for ZP-free early blastocysts and 100% for hatched blastocysts. The percentage of cells that expressed viral antigens was similar for the three stages of development. PCV2 exposure did not affect the in vitro development of the embryos during the 48 h study period. All ZP-intact embryos remained negative for viral antigens. In an additional experiment the diameter of the channels in the porcine ZP was determined. After incubation of early blastocysts with fluorescent microspheres of three different sizes, beads with a diameter of 20 nm and beads with a diameter of 26 nm crossed the zona whereas beads with a diameter of 200 nm did not. In conclusion, it can be stated that PCV2 is able to replicate in in vivo produced ZP-free morulae and blastocysts and that the susceptibility increases during development. The ZP forms a barrier to PCV2 infection, but based on the size of the channels in the ZP the possibility that PCV2 particles cross the ZP cannot be excluded.     

Early embryonic cells from in vivo-produced goat embryos transmit the caprine arthritis-encephalitis virus (CAEV)

The aim of this study was to investigate whether cells of early goat embryos isolated from in vivo-fertilized goats interact with the caprine arthritis-encephalitis virus (CAEV) in vitro and whether the embryonic zona pellucida (ZP) protects early embryo cells from CAEV infection. ZP-free and ZP-intact 8-16 cell embryos were inoculated for 2 h with CAEVat the 10(4) tissue culture infectious dose 50 (TCID50)/ml. Infected embryos were incubated for 72 h over feeder monolayer containing caprine oviduct epithelial cells (COECs) and CAEV indicator goat synovial membrane (GSM) cells. Noninoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls. Six days postinoculation, infectious virus assay of the wash fluids of inoculated early goat embryos showed typical CAEV-induced cytopathic effects (CPE) on indicator GSM monolayers, with fluids of the first two washes only. The mixed cell monolayer (COEC + GSM) used as feeder cells for CAEV inoculated ZP-free embryos showed CPE. In contrast, none of the feeder monolayers, used for culture of CAEV inoculated ZP-intact embryos or the noninoculated controls, developed any CPE. CAEV exposure apparently did not interfere with development of ZP-free embryos in vitro during the 72 h study period when compared with untreated controls (34.6 and 36% blastocysts, respectively, P > 0.05). From these results one can conclude that the transmission of infectious molecularly cloned CAEV-pBSCA (plasmid binding site CAEV) by embryonic cells from in vivo-produced embryos at the 8-16 cell stages is possible with ZP-free embryos. The absence of interactions between ZP-intact embryos and CAEV in vitro suggests that the ZP is an efficient protective embryo barrier.     

Effect of bovine herpesvirus-1 or bovine viral diarrhea virus on development of in vitro-produced bovine embryos.

In previous experiments, zona pellucida (ZP)-intact in vitro-produced (IVP) embryos incubated for 1 hr with 10(6.3) TCID(50)/ml bovine herpes virus-1 (BHV-1), 10(5.3) TCID(50)/ml cytopathic (CP) bovine viral diarrhea virus (BVDV) or 10(5.3) TCID(50)/ml noncytopathic (NCP) BVDV showed no signs of virus replication or embryonic degeneration. The aims of the present study were to investigate whether a prolonged presence (24 hr or 8 days) of 10(6.3) TCID(50)/ml BHV-1 or 10(5.3) TCID(50)/ml BVDV in an in vitro embryo production system affected the rate of cleavage and embryonic development of ZP-intact embryos, and to point out eventual causes of adverse effects. When virus was present in each step of an IVP system, significantly lower rates of cleavage and blastocyst formation of virus-exposed embryos were observed, in comparison with control embryos (P < 0.01). When embryos were only exposed to virus during the in vitro fertilization (IVF), the rates of cleavage and blastocyst formation were significantly affected. The introduction of BHV-1 or BVDV during in vitro maturation (IVM) or in vitro culture (IVC) resulted only in significantly lower rates of blastocyst (P < 0.01). In all experiments, virus replication was not detected in the embryonic cells. On the other hand, virus replication was clearly demonstrated in oviductal cells in the co-culture system, resulting in a degeneration of these cells. In an additional experiment, synthetic oviduct fluid (SOF) without somatic cells was used as an alternative culture system. Even when SOF-embryos were exposed to 10(6.3) TCID(50)/ml BHV-1 or 10(5.3) TCID(50)/ml CP, and NCP BVDV, the rates of blastocyst formation of the BHV-1-, CP-, and NCP BVDV-exposed embryos were not different from the unexposed control embryos, 23%, 24%, and 24%, respectively, vs. 27%. Taken together, it can be concluded that the virus-induced adverse effects on embryonic development in conventional co-cultures were due to changes in the embryonic environment caused by infection of oviductal cells.     

Can bluetongue virus (BTV) be transmitted via caprine embryo transfer?

The three objectives of this study were to investigate whether cells of early goat embryos isolated from in vivo fertilized goats interact with bluetongue virus (BTV) in vitro, whether the embryonic zona pellucida (ZP) protects early embryo cells from BTV infection, and whether the 10 wash cycles recommended by the International Embryo Transfer Society (IETS) for bovine embryos effectively decontaminates caprine embryos exposed to Bluetongue Virus (BTV) in vitro. Donor goats and bucks were individually screened and tested negative for the virus by RT-PCR detection of BTV RNA in circulating erythrocytes. ZP-free and ZP-intact 8-16 cell embryos were co-cultured for 36 h in an insert over a Vero cell monolayer infected with BTV. Embryos were washed 10 times in accordance with IETS recommendations for ruminant and porcine embryos, before being transferred to an insert on BTV indicator Vero cells for 6 h, to detect any cytopathic effects (CPE). They were then washed and cultured in B2 Ménézo for 24 h. Non-inoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls.The Vero cell monolayer used as feeder cells for BTV inoculated ZP-free and ZP-intact embryos showed cytopathic effects (CPE). BTV was found by RT-qPCR in the ten washes of exposed ZP-free and ZP-intact embryos. In the acellular medium, the early embryonic cells produced at least 10^2.5 TCID₅₀/ml. BTV RNA was detected in ZP-free and ZP-intact embryos using RT-qPCR.All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with BTV and that infection with this virus is productive. The washing procedure failed to remove BTV, which indicates that BTV could bind to the zona pellucida.     

The effect of high concentrations of cryoprotectants on the passage of bovine viral diarrhea virus through the zona pellucida of in vitro fertilized embryos.

The effect of high concentrations of cryoprotectants on the passage of bovine viral diarrhea virus (BVDV) through the zona pellucida (ZP) of intact bovine embryos during the pre-freezing step of cryopreservation was investigated in a series of experiments. In vitro fertilized (IVF) embryos at the blastocyst stage were exposed to 10(6) TCID50 BVDV (non-cytopathic NY-1 strain) in a 30% suspension of either ethylene glycol, glycerol, DMSO, or 2 M sucrose in physiological saline for 10 min at 20 degrees C. Subsequently, the embryos were washed free of residual unbound viral particles, and the ZP of some embryos were removed by micromanipulation. Groups of ZP-intact embryos, ZP-free embryonic cells and their respective ZP were then tested separately for the presence of virus. The infectious virus was detected in association with 81% (17/21) of samples containing non-micromanipulated ZP-intact embryos which were exposed to the virus and cryoprotectants and then washed 10 times and in 83% (43/53) of the samples containing only ZP from micromanipulated embryos (P > 0.05). The virus was not found in the samples containing the corresponding embryonic cells of embryos exposed previously to the virus and cryoprotectants. It was concluded that the transfer of embryos from the isotonic PBS solution into a highly hypertonic cryoprotectant solution did not cause the passage of BVDV through ZP and its entry to embryonic cells.     

Observations on the fertilization and development of preimplantation bovine embryos in vitro in the presence of Tritrichomonas foetus

Tritrichomonas foetus, a world-wide distributed parasitic protozoan is a cause of infertility and abortion. There is no documented information on the susceptibility of bovine embryos to the parasite. To determine the effect of T. foetus on fertilization and embryonic development of preimplantation bovine embryos, we added approximately 10(4)/ml or 10(6)/ml T. foetus (Belfast strain) to sperm cells and oocytes prior to in vitro fertilization (IVF) or to presumptive zygotes 24 h post-fertilization. Light and scanning electron microscopy (SEM) revealed that exposure of oocytes or embryos at any stage of development to T. foetus caused rapid adhesion of the trichomonads to the embryonic intact zona pellucida (ZP) and to trophoblastic cells of hatched blastocysts. Treatment of contaminated embryos with 0.25% trypsin for 3 min did not render them free from T. foetus. Motile parasites were not observed after 18 h incubation in IVF medium, or after 72 h in synthetic oviductal fluid (SOF) embryo culture medium. The percentages of cleaved zygotes, blastocysts and hatched embryos resulting from culture of experimental and uninfected control groups of embryos were not different (P > 0.05). Tritrichomonas foetus was not detected in embryonic cells of ZP-intact or hatched embryos when examined by transmission electron microscopy (TEM). In conclusion, T. foetus has no detrimental effect on the fertilization and development of IVF embryos and the potential risk of transmission of trichomonosis is unlikely, due to the limited survival of the parasite in IVF culture conditions.     

Development and viability of bovine preimplantation embryos after the in vitro infection with bovine herpesvirus-1 (BHV-1): immunocytochemical and ultrastructural studies

The aim of our study was to examine whether: (1) the exposure of bovine embryos to the BHV-1 virus in vitro can compromise their further development and alter the ultrastructural morphology of cellular organelles; (2) whether the zona pellucida (ZP) can be a barrier protecting embryos against infection; and (3) whether washing with trypsin after viral exposure can prevent virus penetration inside the embryo and subsequent virus-induced damages. The embryos were recovered from superovulated Holstein-Friesian donor cows on day 6 of the estrous cycle. Only compact morulas or early blastocysts were selected for experiments with virus incubation. We used the embryos either with intact ZP (either with or without trypsin washing) or embryos in which the ZP barrier was avoided by using the microinjection of a BHV-1 suspension under the ZP. ZP-intact embryos (n = 153) were exposed to BHV-1 at 10(6.16) TCID(50)/ml for 60 min, then washed in trypsin according to IETS guidelines and postincubated in synthetic oviduct fluid (SOF) medium for 48 h. Some of the embryos (n = 36) were microinjected with 20 pl of BHV-1 suspension under the ZP, the embryos were washed in SOF medium and cultured for 48 h. Embryo development was evaluated by morphological inspection, the presence of viral particles was determined both immunocytochemically, using fluorescent anti-IBR-FITC conjugate and by transmission electron microscopy (TEM) on the basis of the ultrastructure of the cellular organelles. It was found that BHV-1 exposure impairs embryo development to higher preimplantation stages independent of the presence of the ZP or the trypsin treatment step, as most of the embryos were arrested at the morula stage when compared with the control. Immunofluorescence analysis confirmed the presence of BHV-1 particles in about 75% of embryos that were passed through the trypsin treatment and in all the BHV-1-microinjected embryos. Ultrastructural analysis, using TEM, revealed the presence of virus-like particles inside the BHV-1-exposed embryos, where the trypsin washing step was omitted. Conversely, in trypsin-treated BHV-1-exposed embryos, TEM detected only the envelope-free virus-like particles adhered to pores of the ZP. The embryos that were microinjected with BHV-1 suspension showed the presence of BHV-1 particles, as well as ultrastructural alterations in cell organelles. Taken together these findings may suggest that BHV-1 infection compromises preimplantation development of bovine embryos in vitro and therefore the ZP may not be enough on its own to prevent virus-induced damage, unless it is not accompanied with trypsin washing.     

The infection of mouse preimplantation embryos to Sendai virus (parainfluenza I)

To provide information on the susceptibility of mouse embryos to Sendai virus, it was investigated if viral replication occurs in the preimplantation embryo at different stages of development, with or without the zona pellucida (ZP). Mice were induced to superovulate, and embryos were collected on Days 2, 3 and 4 after mating. The ZP was removed by digestion with 0.5% pronase. Embryos were exposed to Sendai virus, washed, and allowed to develop in fresh culture medium. The presence of viral antigen in the embryonic cells was examined by the fluorescent antibody test (FAT). Specific immunofluorescence was demonstrated in the ZP-free morula and ZP-intact blastocyst. However, viral antigen was not detected in the ZP-intact two-cell, four-cell, eight-cell or morula stage embryos. Infected embryos developed normally to expanded blastocysts. These findings show that mouse embryonic cells are permissive hosts to Sendai virus replication and that the ZP played the role of a barrier against the virus.     

In vitro fertilization and culture of ova from heifers infected with bovine herpesvirus-1 (BHV-1)

Oocytes collected from heifers infected experimentally with bovine herpesvirus-1 (BHV-1, 10(8) TCID(50)/ml) and from dexamethasone-treated (stressed) BHV-1 seropositive animals were matured, fertilized and co-cultured in vitro for 7 d prior to being tested for the presence of the virus. Nineteen of the 21 infected donors yielded embryos and follicular fluids that were BHV-1 positive. Oviductal cells (17 21 ) and uterine fluids (14 21 ) were also positive. Titers for the positive samples ranged 10(1.6)-10(9.6) TCID(50)/ml. The cleavage rate and the proportion of blastocysts that developed from oocytes of BHV-1 infected animals were 26% (n=361) and 6% compared with 56% (n=112) and 26% for uninfected control donors (P<0.05). In contrast, embryos produced from dexamethasone-treated animals tested negative for BHV-1 and yielded 11% blastocysts as compared with 25% for the control group. The results indicate that transferable-stage embryos can be produced by IVF from infected BHV-1 animals and that such embryos are associated with the virus, and might have potential for disease transmission.     

Culture of 5-day horse embryos in microdroplets for 10 to 20 days

Embryos were recovered from the uteri of mares 5 d after ovulation. Six embryos, all morulae, were placed singly in 200-ul droplets of Ham's F-12 with 10% fetal calf serum and cultured at 37 degrees C in a 5% CO(2) atmosphere. The embryos expanded to form blastocysts by the third day of culture. The blastocysts hatched from their zona pellucida, rather than the zona thinning and flaking off, as occurs in vivo. Hatching from the zona pellucida began on the third day of culture and was complete in five of six embryos by the sixth day. The embryonic capsule, normally present in equine embryos after Day 6, was not seen in the cultured embryos. The blastocysts continued to expand until 15 to 17 d of age (10 to 12 d in culture), reaching an average diameter (+/- SD) of 2052 +/- 290 um, after which time they either collapsed or contracted. These results demonstrate that equine embryos can be maintained in long-term culture in vitro, exhibiting continued growth and expansion in the absence of the embryonic capsule.     

Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report

A method for producing identical twin calves is described in which Day 7 frozen-thawed bovine embryos in 12.5% sucrose solution were bisected using a fine microsurgical blade. The resulting bisected embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/-1 d), two bisected embryos per recipient. The pregnancy rate when both halves remained in the same zona pellucida was 50% (5 10 ); the pregnancy rate was 1 5 for morulae and 4 5 for blastocysts. The pregnancy rate for unfrozen morulae bisected in PBS and transferred without zona pellucida was 27% (4 15 ). The in vitro survival rate of embryos bisected in 12.5% sucrose when both halves remained in the original zona pellucida was 82% (18 22 ), which was higher than when embryos were bisected in PBS (53%, 9 17 ).     

Transmission of mouse minute virus (MMV) but not mouse hepatitis virus (MHV) following embryo transfer with experimentally exposed in vivo-derived embryos

The present study investigated the presence and location of fluorescent microspheres having the size of mouse hepatitis virus (MHV) and of mouse minute virus (MMV) in the zona pellucida (ZP) of in vivo-produced murine embryos, the transmission of these viruses by embryos during embryo transfer, and the time of seroconversion of recipients and pups. To this end, fertilized oocytes and morulae were exposed to different concentrations of MMVp for 16 h, while 2-cell embryos and blastocysts were coincubated for 1 h. In addition, morulae were exposed to MHV-A59 for 16 h. One group of embryos was washed, and the remaining embryos remained unwashed before embryo transfer. Serological analyses were performed by means of ELISA to detect antibodies to MHV or MMV in recipients and in progeny on Days 14, 21, 28, 42, and 63 and on Days 42, 63, 84, 112, 133, and 154, respectively, after embryo transfer. Coincubation with a minimum of 10(5)/ml of fluorescent microspheres showed that particles with a diameter of 20 nm but not 100 nm crossed the ZP of murine blastocysts. Washing generally led to a 10-fold to 100-fold reduction of MMVp. Washed MMV-exposed but not MHV-exposed embryos led to the production of antibodies independent of embryonic stage and time of virus exposure. Recipients receiving embryos exposed to a minimum of 10(7) mean tissue culture infective dose (TCID(50))/ml of MHV-A59 and 10(2) TCID(50)/ml of MMVp seroconverted by Day 42 after embryo transfer. The results indicate that MMV but not MHV can be transmitted to recipients even after washing embryos 10 times before embryo transfer.     

Maedi-Visna virus was detected in association with virally exposed IVF-produced early ewes embryos

The objective of this study was to determine whether MVV can be transmitted by ovine embryos produced in vitro and whether the zona pellucida (ZP) provides any protection against MVV infection.Zona pellucida (ZP)-intact and ZP-free embryos, produced in vitro, at the 8-16 cell stage, were cocultured for 72h in an insert over an ovine oviduct epithelial cell (OOEC)-goat synovial membrane (GSM) cell monolayer that had been previously infected with MVV (K1514 strain). The embryos were then washed and transferred to either direct contact or an insert over a fresh GSM cell monolayer for 6 h. The presence of MVV was detected using RT-PCR on the ten washing fluids and by the observation of typical cytopathic effects (CPE) in the GSM cell monolayer, which was cultured for 6 weeks.This experiment was repeated 4 times with the same results: MVV viral RNA was detected using RT-PCR in the first three washing media, while subsequent baths were always negative. Specific cytopathic effects of MVV infection and MVV-proviral DNA were detected in GSM cells that were used as a viral indicator and cocultured in direct contact or as an insert with MVV-exposed ZP-free embryos. However, no signs of MVV infection were detected in cells that were cocultured with exposed ZP-intact or non-exposed embryos.This study clearly demonstrates that (i) in vitro, ZP-free, early ovine embryos, which had been exposed to 10³ TCID₅₀/m MVV in vitro, are capable of transmitting the virus to susceptible GSM target cells, and that (ii) the IETS recommendations for handling in vivo produced bovine embryos (use of ZP-intact embryos without adherent material and performing ten washes) are effective for the elimination of in vitro MVV infection from in vitro produced ovine embryos. The absence of interaction between ZP-intact embryos and MVV suggests that the in vitro produced embryo zona pellucida provides an effective protective barrier.     

Risk of Coxiella burnetii transmission via embryo transfer using in vitro early bovine embryos

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main source of infection for humans. The objectives of this study were to determine (1) whether C. burnetii would adhere to the intact zona pellucida (ZP-intact) of early in vitro–produced bovine embryos; (2) whether the bacteria would adhere to or infect the embryos (ZP-free) after in vitro infection; and (3) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. One hundred and sixty, eight- to 16-cell bovine embryos produced in vitro, were randomly divided into 16 batches of 10 embryos. Twelve batches (eight ZP-intact and four ZP-free) were incubated in a medium containing C. burnetii CbB1 (Infectiologie Animale et Santé Publique, Institut National de Recherche Agronomique Tours, France). After 18 hours of incubation at 37 °C and 5% CO₂ in air, the embryos were washed in 10 successive baths of a PBS and 5% fetal calf serum solution in accordance with the IETS guidelines. In parallel, four batches (two ZP-intact and two ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. Ten washing fluids from each batch were collected and centrifuged for 1 hour at 13,000× g. The embryos and wash pellets were tested using conventional polymerase chain reaction. C. burnetii DNA was found in all ZP-intact and ZP-Free embryos after 10 successive washes. It was also detected in the first four washing fluids for ZP-intact embryos and in the 10th wash fluid for two of the four batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adheres to and/or penetrates the early embryonic cells and the ZP of in vitro bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP and to verify if similarly results are obtained with in vivo–derived embryos.     

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) TCID(50) /ml) and bovine viral diarrhea virus (BVDV; titre 10(6) TCID(50) /ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 10(6) TCID(50) /ml) or BVDV (titre 10(6) TCID(50) /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 mug/ml) in combination with He Ne light, or by HP or HPD (10 mug/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 mug/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 mug/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 mug of HPD followed by He Ne light, or 10 mug/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.     

Can Coxiella burnetii be transmitted by embryo transfer in goats?

The detection of significant bacterial loads of Coxiella burnetii in flushing media and tissue samples from the genital tracts of nonpregnant goats represents a risk factor for in utero infection and transmission during embryo transfer. The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo–fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and polymerase chain reaction, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9-9, 11-11, and 4-4 in replicates 1, 2, and 3, respectively) were placed in 1 mL minimum essential medium containing 10⁹C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37 °C and 5% CO₂, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3-3, 5-5, and 2-2 in replicates 1, 2, and 3, respectively) were subjected to the same procedures, but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 hour at 13,000 × g. The washed embryos and pellets were tested by polymerase chain reaction. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first five washing fluids for ZP-intact embryos and in the first eight washing fluids for ZP-free embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly indicate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to adhere to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from 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%).     

Structural aspects of the zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 microm(2) of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 microm(2)) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40-50 nm) and bovine herpesvirus-1 (BHV-1; 180-200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.     

Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo

We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.     

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.