Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

Bovine herpesvirus 1 (BHV1) is an important bovine pathogen, responsible for respiratory diseases and reproductive problems. This study investigated the penetration capacity of BHV1 into oocytes after co-incubation for either 1 h or 24 h. Immunofluorescence assays in cumulus-oocyte complexes (COCs) and denuded oocytes (without the presence of cumulus cells) were performed and evaluated using confocal laser scanning microscopy. Blood samples and ovaries from BHV1 seronegative cows were used. The oocytes recovered were divided into two groups. Group I comprised COCs (n = 312) and denuded oocytes (n = 296), which were experimentally infected with BHV1 and incubated for 1 h at 38.5°C and 5% CO2. Group II comprised COCs (n = 425) and denuded oocytes (n = 405), which were co-incubated with BHV1 under the same conditions for 24 h. The negative control of these two groups was respectively subjected to the same protocol, except for exposure to BHV1. To our knowledge, this study provides the first evidence of BHV1 detection within COCs and denuded oocytes exhibiting intact zona pellucida when co-incubated with the virus for 24 h. Immunolocalization also confirmed the presence of BHV1 in the cytoplasm of the cumulus cells of all COCs exposed to the virus after both incubation periods. In conclusion, detection of BHV1 inside oocytes has a great meaning for the field of animal reproduction. The detection of BHV1 in different layers of cumulus cells also demonstrates that these cells are sources of viral infection.

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) TCID(50)/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.

Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle.

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (105 and 106/mL) and oocytes were incubated with 106 TCID50/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (105/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 106/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (105/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (106 sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF.

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.

Susceptibility of in vivo- and in vitro-produced porcine embryos to classical swine fever virus.

The objective of this study was to investigate the susceptibility of in vivo- and in vitro-produced (IVP) porcine embryos to classical swine fever virus (CSFV). IVP zona pellucida (ZP)-intact porcine embryos (n = 721) were co-cultured with CSFV for 120 h. After washing according to the International Embryo Transfer Society guidelines (without trypsin) and transferring embryos to CSFV-susceptible porcine kidney cells (PK15 cell line), no virus was isolated. However, when 88 IVP ZP-intact porcine embryos were co-cultured with CSFV for only 48 h before being transferred to PK15 cells, virus was isolated in three of six replicates. Similarly, 603 in vivo-produced porcine embryos were co-cultured with CSFV for 120 h. Subsequently, CSFV was isolated in eight of 50 groups (16%) and the ability of these to form a blastocyst was significantly reduced when compared with the control group (68.2 +/- 19.9% vs 81.9 +/- 9.7%; p < or = 0.001). In contrast, the development of CSFV-exposed IVP porcine embryos was not affected when compared with control embryos (19.1 +/- 10.8% vs 18.9 +/- 10.6%; p > or = 0.05). After removal of the ZP of IVP embryos and subsequent co-culture with CSFV, the virus was isolated from all groups of embryos. These data suggest that virus replication had occurred in the embryonic cells. In conclusion, data indicate that in vivo- and in vitro-produced ZP-intact porcine embryos differ in their susceptibility to CSFV. Hatched or micro-manipulated embryos may increase the risk of transmission of CSFV by embryo transfer, which has to be confirmed by in vivo tests under isolation conditions.

PCR-detected hepatitis C virus RNA associated with human zona-intact oocytes collected from infected women for ART.

BACKGROUND: The aim of this study was to investigate the susceptibility of human oocytes from hepatitis C virus (HCV) RNA-positive women to HCV contamination during assisted reproductive technology (ART). METHODS: A reverse transcriptase-PCR assay was used to test for the presence of HCV RNA associated with 24 unfertilized oocytes 48 h after follicular fluid aspiration in 10 IVF attempts (seven conventional IVF and three ICSI). Negative and positive controls (10 unfertilized oocytes from HCV-negative women and 20 unfertilized oocytes artificially contaminated with HCV RNA-positive plasma; HCV RNA was also quantified in plasma and follicular fluid) were included. RESULTS: HCV RNA was associated with 17/24 (70.8%) oocytes (6/7 after ICSI and 11/17 after conventional IVF) and was found in 19/20 (95%) follicular fluid samples. A weak correlation was found between plasma and follicular fluid HCV RNA loads (r = 0.73, P < 0.001). CONCLUSIONS: HCV associated with unfertilized oocytes surrounded by their intact zona pellucida from anti-HCV antibody-positive and viraemic women undergoing ART raises questions concerning the safe management of medically assisted procreation for these women and good practice of oocyte/embryo cryopreservation and donation.

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.

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.