Alteration in ultrastructural morphology of bovine embryos following subzonal microinjection of bovine viral diarrhea virus (BVDV)

The aim of the present study was to evaluate the development and ultrastructure of preimplantation bovine embryos that were exposed to bovine viral diarrhea virus (BVDV) in vitro.The embryos were recovered from superovulated and fertilized Holstein-Friesian donor cows on day 6 of the estrous cycle. Compact morulae were microinjected with 20 pl of BVDV suspension (10(5.16) TCID(50)/ml viral stock diluted 1:4) under the zona pellucida (ZP), then washed in SOF medium and cultured for 24-48 h. Embryos were evaluated for developmental stages and then processed immunocytochemically for the presence of viral particles, using fluorescent anti-BVDV-FITC conjugate. Ultrastructure of cellular organelles was analysed by transmission electron microscopy (TEM).After microinjection of BVDV under the ZP, significantly more (p<0.001) embryos (83.33%) were arrested at the morula stage compared with the intact control (30.33%). Immunocytochemical analysis localized the BVDV-FITC signal inside the microinjected embryos. TEM revealed: (i) the presence of virus-like particles in the dilated endoplasmic reticulum and in cytoplasmic vacuoles of the trophoblast and embryoblast cells; (ii) the loss of microarchitecture: and (iii) abnormal disintegrated nuclei, which lacked reticular structure and the heterochromatin area. In all, the embryo nuclear structure was altered and the microarchitecture of the nucleolus had disappeared when compared with the nuclei from control embryos. Dilatation of the intercellular space and the loss of the intercellular gap junctions were often observed in bovine BVDV-exposed embryos.These findings provide evidence for the adverse effect of BVDV virus on the development of bovine embryos, which is related to irreversible changes in the ultrastructure of cell organelles.     

Trypsin treatment of bovine embryos after in vitro exposure to infectious bovine rhinotracheitis virus or bovine herpesvirus-4

The objectives of this study were to evaluate the efficacy of trypsin treatment for the removal/inactivation of infectious bovine rhinotracheitis virus (IBRV) adhering to zona pellucida-intact (ZP-I) bovine embryos and to determine if bovine herpesvirus-4 (BHV-4) adheres to ZP-I bovine embryos. When adherence of BHV-4 was demonstrated, an additional objective was to determine whether trypsin treatment removes or inactivates this virus. A total of 139 ZP-I embryos was collected from superovulated donor cows at 7 d after estrus. Embryos were exposed to 10(6) to 10(7) plaque-forming units (pfu) of either IBRV or BHV-4 for 1 to 2 h. Subsequently, approximately equal numbers of embryos exposed to each virus were either washed 12 times and the washes and embryos examined for the presence of infectious virus, or they were treated with trypsin and the embryos examined for the presence of infectious virus. Although the fourth wash was the last positive wash, an average of 18 pfu of virus was detected from each of six groups (a total of 24 embryos) after exposure to IBRV and washing. Infectious bovine rhinotracheitis virus was not isolated from any of nine trypsin-treated groups (a total of 43 embryos). The seventh wash was the last positive wash for any group after exposure to BHV-4, yet an average of 2 pfu of virus was detected from each of six groups (a total of 29 embryos) after washing. No BHV-4 was isolated from any of eight trypsin-treated groups (a total of 43 embryos). The study confirmed previous reports that IBRV adheres to the bovine ZP after in vitro exposure and that trypsin treatment is effective in keeping ZP-I embryos free of this virus. Adherence of BHV-4 to ZP-I bovine embryos was demonstrated for the first time. Trypsin treatment was also effective in removing this herpesvirus.     

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.     

Prevention of bovine herpesvirus-1 transmission by the transfer of embryos disinfected with recombinant bovine trypsin

This study deals with the potential for the introduction of infectious agents through the use of animal-derived products. The efficacy of a recombinant bovine trypsin (RBTr) as a replacement for porcine pancreatic trypsin and a disinfectant for bovine herpesvirus-1 (BHV-1)–infected embryos was investigated according to the sanitary guidelines of the International Embryo Transfer Society. Treatment of in vivo and in vitro fertilized embryos contaminated with BHV-1 (10⁵ TCID50/mL) in the presence of RBTr (525 U/mL) for 120 s, effectively removed the infectious virus compared with untreated and washed embryos (P < 0.05). Transfer of in vivo fertilized and disinfected embryos to BHV-1 seronegative recipients (n = 24) resulted in 14 pregnancies and 11 calves born free of BHV-1. In contrast, transfer of unwashed or undisinfected embryos to four recipients resulted in seroconversion and no pregnancies at term. It was concluded that the use of RBTr could be considered as an alternative method of rendering embryos free of BHV-1 and thus reduce the potential risk of disease transmission to embryo recipients and offspring.     

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.     

Bovine herpesvirus-1 associated with single, trypsin-treated embryos was not infective for uterine tubal cells

It has been reported that bovine herpesvirus-1 (BHV-1) remains associated with in vitro-produced (IVP) bovine embryos after exposure to the virus and either washing or trypsin treatment. However, it is not known if the quantity of virus associated with an exposed IVP embryo is likely to infect a recipient cow after transfer. The specific objective of this study was to determine if IVP embryos that were exposed to BHV-1 would infect uterine tubal cells (UTC) in a co-culture system. In vitro-produced Day 7 embryos were exposed to BHV-1 and then washed or trypsin treated according to the IETS guidelines. These embryos were then co-cultured individually or in groups with UTC in microdrops of tissue culture medium 199 (TCM 199) supplemented with 10% equine serum. Following co-culture for 48 h, virus isolation was attempted on the embryos and the UTC from each drop. Virus was detected in washed individual embryos, groups of washed embryos, groups of trypsin-treated embryos and the UTC co-cultured with each of these treatments. However, BHV-1 was not detected in the individual, trypsin-treated embryos or the UTC co-cultured with them. It is concluded that trypsin treatment might effectively prevent infection of recipients if individual, Day 7, exposed embryos were transferred into the uterus.     

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.     

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.     

Efficacy of a recombinant trypsin product against bovine herpesvirus 1 associated with in vivo- and in vitro-derived bovine embryos

Although porcine-origin trypsin will effectively remove bovine herpesvirus 1 (BHV-1) associated with in vivo-derived embryos, TrypLE, a recombinant trypsin-like protease, has not been evaluated. In Experiment 1, 17 groups of 10 in vivo-derived embryos were exposed to BHV-1, treated with TrypLE Express or TrypLE Select (10x concentration) for varying intervals, and assayed as 2 groups of 5 embryos. TrypLE Select treatment for 5 and 10 min (two and seven groups of five embryos, respectively) effectively inactivated BHV-1. In Experiment 2, 22 groups of 10 IVF embryos were treated and assayed. Treatment with TrypLE Select for 7 and 10 min (six groups of five embryos each) and with TrypLE Select diluted 1:2 for 10 min (seven groups of five embryos) was also effective. In Experiment 3, 17 groups of 10 IVF embryos were further evaluated with TrypLE Select undiluted and diluted 1:2 for 10 min. Treatment with the diluted product was effective (18 groups of five embryos), whereas the undiluted product was not completely effective (virus isolated from 2 of 16 groups). In Experiment 4, IVF embryos were treated as described in Experiment 3 and then cultured individually or as groups of five on uterine tubal cells (UTCs) for 48 h; 60% of UTC samples associated with groups of embryos and 35% of UTC associated with individual embryo samples were positive for BHV-1. Therefore, although TrypLE Select appeared to have promise for the treatment of in vivo-derived embryos, it cannot be recommended for treatment of in vitro-derived embryos.     

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.     

Interaction of Mycoplasma bovis and Mycoplasma bovigenitalium with preimplantation bovine embryos

In vitro exposures of bovine embryos to Mycoplasma bovis and Mycoplasma bovigenitalium were conducted to determine if these organisms adhered to the zona pellucida-intact (ZP-I) bovine embryo, and standard procedures for washing and treating embryos were evaluated to determine their effectiveness for removing or killing mycoplasmas. Mycoplasma bovis and M. bovigenitalium were isolated from 19 of 19 and 24 of 24 ZP-I embryos, respectively, after in vitro exposure and subsequent washing, thus demonstrating adherence of the two species of Mycoplasma to the ZP. Additionally, M. bovis was isolated from 20 of 20 and 23 of 23 embryos, while M. bovigenitalium was isolated from 25 of 25 and 22 of 22 embryos after antibiotic and trypsin treatment, respectively. It was concluded that neither of the standard procedures currently used for cleansing embryos should be relied upon for insuring freedom from mycoplasmas.     

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.     

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.     

Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction

We present a polymerase chain reaction (PCR)-based procedure for rapid bovine embryo sexing and classifying embryos for the presence of exogenous DNA. Fourteen bovine blastocysts microinjected with gene construct DNA at the pronuclear stage were divided into quarters and subjected to amplification with construct-specific and sex gene-specific (ZFY/ZFX) primers in the same initial PCR reaction. Blastocysts carrying microinjected construct DNA could be identified by the presence of construct-specific PCR product in approximately 4 h. Approximately half of the microinjected and two of 16 non-microinjected blastocysts typed PCR-positive for the construct DNA. Owing to erroneous amplifications in the two non-microinjected control blastocysts, and the inability of the system to distinguish integrated from non-integrated copies of the microinjected construct, the number of construct-positive blastocysts determined in our assay most likely overestimates the number of true transgenic embryos. Nevertheless, using this assay, we were able to determine that approximately half of the microinjected embryos were negative for the transgene construct and thus could be eliminated from transfer to a recipient cow. Embryo sexing was achieved in less than 6 h by restriction fragment length polymorphism analysis of nestedZFY/ZFXPCR products reamplified from initial PCR reactions. In 11/14 microinjected blastocysts all sections assayed unambiguously as the same sex. In one embryo, only one section was analysed, while two other blastocysts whowed some discrepancies of sexing results between the sections analysed. The approach employed here to determine the sex and presence of microinjected construct DNA in bovine preimplantation embryos is rapid, accurate among different sections of an embryo and can be used to increase the efficiency of current transgenic cattle production procedures.     

Isolation of bovine herpesvirus-1 (BHV-1) and bovine viral diarrhea virus (BVDV) in association with the in vitro production of bovine embryos

The purpose of this study was to determine whether oocytes obtained from bovine ovaries collected at commercial abattoirs for use in in vitro fertilization programs would be contaminated with bovine herpesvirus-1 (BHV-1) and/or bovine viral diarrhea virus (BVDV). In total, of 85 samples tested containing 759 embryos produced by in vitro fertilization, 2 (2.4%) were positive for BHV-1 while none were positive for BVDV. The follicular fluid collected during oocyte aspiration tested positive in 11.8% for BVH-1 and in 4.7% for BVDV. Oviductal cells used to co-culture zygotes/embryos tested positive for BHV-1 and BVDV in 6.2% and 1.2% samples respectively.     

Inhibition of antigen-induced and interleukin-2-induced proliferation of bovine peripheral blood leukocytes by inactivated bovine herpes virus 1.

The mechanism by which bovine herpesvirus 1 (BHV-1) predisposes cattle to bacterial pneumonia was investigated by using an in vitro system to demonstrate immunosuppression. At a multiplicity of infection of 0.001, live or inactivated BHV-1 induced a 50% inhibition of the proliferative response of peripheral blood mononuclear leukocytes to antigen (vaccinia virus in vaccinia virus-immunized cattle which were BHV-1 negative) or interleukin-2. At this same multiplicity of infection, the mitogen-induced proliferation of peripheral blood mononuclear leukocytes was unaffected. This inhibition of antigen and interleukin-2-induced proliferative responses could not be reversed by the addition of excess amounts of interleukin-2 and could not be prevented by the addition of indomethacin to block prostaglandin production. Antibodies to BHV-1, especially those specific for glycoproteins gI and gIV, were able to block the inhibitory effect of BHV-1 in these in vitro assays. These results showed that antibody to BHV-1 blocks the immunosuppressive effect of the virus in vitro and suggested that an appropriate antibody response to BHV-1 could protect cattle from virus-induced immunosuppression leading to secondary bacterial pneumonia.     

Evaluation of bluetongue virus (BTV) decontamination techniques for caprine embryos produced in vivo

The objective of this study was to investigate methods of decontaminating early goat embryos that had been infected in vitro with bluetongue virus (BTV). Embryos were isolated from in vivo-fertilized BTV-free goats. Zona pellucida (ZP)-intact 8 to 16 cell embryos were cocultured for 36 h in an insert over a Vero cell monolayer infected with BTV serotype 8. The embryos were then treated with one of five different washing procedures. The treatment standard (TS) comprised phosphate-buffered saline (PBS) + 0.4% BSA (five times over for 10 s), Hank's +0.25% trypsin (twice for 45 s), and then PBS + 0.4% BSA again (five times for 10 s). The four other washing procedures all included the same first and last washing steps with PBS but without BSA (five times for 10 s) and with PBS + 0.4% BSA (five times for 10 s), respectively. The intermediate step varied for each washing procedure. Treatment 1 (T1): 0.25% trypsin (twice for 45 s). Treatment 2 (T2): 0.25% trypsin (twice for 60 s). Treatment 3 (T3): 0.5% trypsin (twice for 45 s). Treatment 4 (T4): 1% hyaluronidase (once for 5 min). After washing, the embryos were transferred and cocultured with BTV indicator Vero cell monolayers for 6 h, to detect any cytopathic effects (CPE). The effectiveness of the different washing techniques in removing the virus was evaluated by RT-qPCR analysis. The TS, T1, T3, and T4 trypsin or hyaluronidase treatments did not eliminate BTV; Treatment 2 eliminated the virus from in vitro infected goat embryos.     

Effect of cryopreservation by slow cooling and vitrification on viral contamination of IVF embryos experimentally exposed to bovine viral diarrhea virus and bovine herpesvirus-1

The objective was to determine the effect of cryopreservation by conventional slow controlled cooling (0.5 °C/min) and by vitrification on the presence of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) infectivity associated with frozen-thawed Day 7 bovine embryos. In this study, Day 7 embryos generated by in vitro fertilization (IVF) were exposed in vitro for 1.5 h to BVDV (N = 393) and BHV-1 (N = 242) and subsequently tested before and after cryopreservation for the presence of infectivity. Exposure of embryos to viral agents resulted in 72% of them infected prior to cryopreservation. Stepwise exposure of embryos to cryoprotectants, as well as their removal, substantially reduced the proportion of contaminated embryos (46% vs. 72%, P < 0.05). Overall, both freezing methods reduced the percentage of infectious embryos compared with that of embryos similarly exposed to viruses but not cryopreserved (31% vs. 72%, respectively; P < 0.001). The percentage of embryos with infectious viruses was not significantly higher after vitrification than after slow cooling (38% vs. 22%). In addition, after cryopreservation, a higher percentage (P < 0.002) of embryos exposed to BHV-1 (42%) remained infectious than did embryos exposed to BVDV (24%). In conclusion, cryopreservation reduced the proportion of infected embryos but did not render all of them free from infectious pathogens.     

Ultrastructural study of cat zona pellucida during oocyte maturation and fertilization

The mammalian zona pellucida (ZP) is an extracellular glycoprotein structure formed around growing oocytes, ovulated eggs and preimplantation embryos. The specific functions of ZP are highly determined by its morphological structure. Studies on cat oocytes during maturation and after fertilization were undertaken, using routine transmission (TEM) and scanning electron microscopy (SEM). Two basic ZP layers – outer with rough spongy appearance and inner with smaller fenestrations and smooth fibrous network – were visible. Deposits, secreted by oviductal cells formed new layer, the so called oviductal ZP. After fertilization outer ZP showed rougher meshed network due to fusion between filaments as a consequence from sperm penetration while the inner was smoother with melted appearance. The presented data on the SEM and TEM characteristics of cat oocytes, together with our previous studies on carbohydrate distribution suggest that during oocyte maturation and fertilization ZP undergoes structural and functional rearrangements related to sperm binding and penetration.