Susceptibility of zona-intact and zona-free in vitro-produced bovine embryos at different stages of development to infection with bovine herpesvirus-1

The aim of the present study was to determine if BHV-1 is able to replicate within in vitro produced embryos and to investigate the degree to which the zona pellucida (ZP) is able to protect in vitro produced embryos against infection with BHV-1. Both ZP-intact and ZP-free matured oocytes, zygotes (1 d post insemination; 1dpi), 8-cell stage embryos (3 dpi), morulae (6 dpi) were incubated for 1 h in 1 ml of MEM containing 10(7.7) TCID(50)/ml BHV-1 (Cooper strain). Three titers (10(5.7), 10(6.7) and 10(7.7) TCID(50)/ml) of the Cooper strain were used for incubation of hatched blastocysts (9 dpi). Bovine embryonic lung cells (BEL) on microcarriers were inoculated following the same protocol as for the embryos. At 0, 12, 24, 36 and 48 h post inoculation (hpi), groups of embryos and BEL cells were collected for virus titration and for the determination of the percentage of viral antigen positive cells by immunofluorescence. For the 3 developmental stages in ZP-free embryos, similar maximal intracellular virus progeny titers were obtained at 24 to 48 hpi ranging from 10(1.32) to 10(1.43) TCID(50)/ 100 embryonic cells. The intracellular virus titer in the BEL cells peaked at 10(3.08) TCID(50)/ 100 BEL cells. The percentage of cells which expressed viral antigens was 13% in ZP-free hatched blastocysts, 17% in ZP-free morulae and 100% in BEL cells. In ZP-intact embryos, no replication of BHV-1 was detected. These results clearly show that only after removal of the zona pellucida, BHV-1 is able to replicate within the in vitro produced embryos, with only a subset of embryonic cells being fully susceptible.     

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.     

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.     

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.     

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.     

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.     

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.     

Damage of zona pellucida reduces the developmental potential and quality of porcine circovirus type 2–infected oocytes after parthenogenetic activation

The present aimed to study if porcine circovirus type 2 (PCV2), which adhered to zona pellucida (ZP), was able to enter mature porcine oocytes with intact and damaged ZP. Four groups, including uninfected ZP-intact oocytes (UOZI), uninfected ZP-damaged oocytes (UOZD), PCV2-infected ZP-intact oocytes (POZI), and PCV2-infected ZP-damaged oocytes (POZD) were studied. The oocytes were incubated with 1 mL minimum essential medium, containing 3.1 × 10⁸ copies of PCV2 DNA for 1 hour. Mechanical procedure of the insertion by microneedle induced injuries to the ZP of porcine oocytes. At the blastocyst stage, the percentage of PCV2-infected embryos and the ratio of viral antigen-positive cells per embryo were determined by indirect immunofluorescence. To assess the effect of ZP injury on the developmental competence and quality of porcine PCV2-infected oocytes after parthenogenetic activation, blastocyst formation rates and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining were analyzed. Moreover, real-time polymerase chain reaction was used to evaluate the expression of genes related to apoptosis and pluripotency at different developmental stages. The results of indirect immunofluorescence showed that only POZD group presented PCV2-infected embryos and viral-positive cells. The blastocyst rate of POZD group dropped down to approximately half of POZI group's (7.1 ± 1.5 vs. 14.5 ± 3.3). At the blastocyst stage, ZP injury increased apoptotic index of PCV2-infected embryos. The relative expression levels of Caspase 3 were higher in POZD group than the ones in POZI group at the two- and four-cell stages (not statistically significant). Compared with the one in POZI group, the ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of the ability to resist apoptosis, was lower in POZD group at the one-cell stage, but higher at the two- and four-cell stages. Expression levels of Oct4 and Nanog associated with pluripotency were lower in POZD group than the ones in POZI group at the morula stage (not statistically significant). Noteworthily, the expression of Nanog was significantly lower in POZD group versus POZI group (P < 0.05), whereas relative expression of Oct4 was significantly higher in the former at the blastocyst stage (P < 0.01). In conclusion, PCV2, which attached to ZP, was able to enter mature porcine oocytes with damaged ZP and subsequently reduced the developmental competence and quality of the oocytes after parthenogenetic activation.     

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.     

Developmental capacity,size and number of nuclei in pig embryos cultured in vitro

Twenty-five surgical embryo recoveries were made from 17 postpuberal gilts 3 to 6 days after mating. A total of 242 eggs was recovered. Recovery rate was 87.5%, fertilization rate was 97.5%, and 98.7% of the fertilized eggs were morphologically intact. The embryos were cultured in vitro in Krebs-Ringer-Bicarbonate (KRB) with 10% heat inactivated lamb serum for 72 or 96 h at +37°C in a humidified 5% CO₂ atmosphere. Of the cultured four-cell embryos 26.6% developed to expanded blastocysts, 16.7% to hatching blastocysts and 5.0% to hatched blastocysts. Of the eight-cell embryos 52.6% developed to hatching blastocysts, 10.5% to hatched blastocysts. When recovered as morulae, the percentage of hatching blastocysts subsequently obtained was 25.8% and 33.9% hatched. A total of 75.0% of the cultured early blastocysts were in the process of hatching (30.6%) or had hatched (44.4%). Significant differences in overall embryo diameter were determined between morulae (156.5 ± 3.94 μm) and early blastocysts (156.9 ± 3.72 μm) versus expanded (197.6 ± 12.57 μm), hatching (207.4 ± 15.86 μm) or hatched (270.0 ± 36.67 μm) blastocysts. The zona pellucida of expanded blastocysts was significantly thinner (5.5 ± 1.59 μm) than that of morulae (12.0 ± 1.01 μm). The number of nuclei was significantly higher for hatching (151 ± 49.8) and hatched (130 ± 17.9) blastocysts cultured as early blastocysts as compared to those cultured from the four-cell stage (88 ± 12.7 and 69 ± 3.6 respectively). Hatching blastocysts that had developed from early blastocysts also had significantly more nuclei than those cultured as eight-cell embryos (99 ± 32.5) or morulae (91 ± 21.2).By the culture method used in this study, a high percentage of pig embryos was capable of developing.     

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida

In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepes-buffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida.     

Development of zona-free hamster ova to blastocysts in vitro

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 microL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst.     

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.     

In vitro and in vivo development of mouse morulae encapsulated in 2% sodium alginate or 0.1% poly-l-lysine

In Experiment 1, development of zona pellucida-intact (ZPI) morulae was measured every 24 hours for 120 hours after encapsulation in 2% sodium alginate (ALG) or 0.1% poly-L-lysine (PLL). Encapsulation significantly reduced development to hatched blastocysts at 48 and 72 hours. Developmental stages and diameters of ZPI and zona pellucida-free (ZPF) unencapsulated and encapsulated morulae were measured every 24 hours for 72 hours in Exeriment 2. At 72 hours, the percentage of ZPI embryos developing to expanded blastocysts, their diameters and their nuclear counts were not different from each other or from ZPF embryos. In Experiment 3, ZPI morulae encapsulated in ALG or PLL were transferred into recipients. Five of six recipients that received unencapsulated embryos (n=71) delivered 16 live pups. None of the recipients of encapsulated embryos delivered offspring; therefore, a final experiment was performed to examine fetal development on Day 10 of gestation. The percentage of pregnant recipients was similar for all 3 treatments: unencapsulated (71.4%), ALG (87.5%) and PLL (87.5%). However, the presence of viable fetuses was higher for unencapsulated embryos (42.1%) than for ALG (17%) and PLL (14.6%) embryos. These results suggest that encapsulation did not detrimentally affect embryonic size or cellular development in vitro; however, mortality occurred in vivo due to an asynchronous condition between the uterine environment and the embryos.     

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.     

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.     

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.     

Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration

The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20–22°C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4°C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22°C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zona dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS + albumin, TCM199 and TCM199 + calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20–22°C, the embryo survival rate decreased (PBS + albumin) or no embryo survived (TCM199 + calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.     

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.     

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.