Geert Vanroose

Structural Aspects of the Zona Pellucida of In Vitro-Produced Bovine Embryos: A Scanning Electron and Confocal Laser Scanning Microscopic Study

Abstract 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 zonas 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 μm2 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 μm2) 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.

Embryonic mortality and embryo–pathogen interactions

Embryonic mortality (EM) has a substantial impact on the fertility of domestic animals. Most of the embryonic losses occur during the first days after fertilization and during the process of implantation. Causes of EM can be divided into infectious and non-infectious categories. Primary attention has often been given to infectious agents but non-infectious causes probably account for 70% or more of the cases of embryonic death. Infection of the embryonic environment can be caused by specific and non-specific uterine pathogens. Specific uterine infections are caused by a number of viruses, bacteria and protozoa that enter the uterus by the haematogenous route or via the vagina. Non-specific pathogens are mainly bacteria that enter the uterus by ascending infection. Uterine pathogens may cause EM by changing the uterine environment (endometritis) or by a direct cytolytic effect on the embryo. Non-infectious causes of EM such as chromosomal aberrations, external factors (e.g., high ambient temperature and nutritional factors) and maternal factors (e.g., hormonal imbalances and age) are multifactorial and difficult to diagnose.

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 106.3 TCID50/ml bovine herpes virus‐1 (BHV‐1), 105.3 TCID50/ml cytopathic (CP) bovine viral diarrhea virus (BVDV) or 105.3 TCID50/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 106.3 TCID50/ml BHV‐1 or 105.3 TCID50/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.

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.

Sucrose-induced shrinkage of in vitro produced bovine morulae: Effect on viability, morphology and ease of evaluation

Sucrose (0.3 M) was used to cause artificial compaction of the embryonic cell mass of in vitro produced bovine embryos to facilitate morphological evaluation. Embryos were produced using routine in vitro maturation (IVM) and fertilization (IVF) techniques. The time necessary to induce shrinkage in 0.3 M sucrose to 75% of the original volume of Day 5 morulae was found to be less than l min, and 95% of the volume was regained in PBS after 2.5 min. No detrimental effect was observed after a 5- to 10-min sucrose treatment on subsequent blastocyst formation at Days 6 and 7 (P > 0.05). Furthermore, no significant differences were observed in the total number of cells, or in the mitotic and pycnotic cell index of blastocysts in different treatment groups. Agreement among 7 evaluators grading 40 Day 6 embryos was examined using the kappa coefficient of agreement (kappa). Overall agreement among evaluators for classification of quality grade was poor (48.2 %, kappa = 0.31) for embryos evaluated in PBS, but the rate improved when the same embryos were scored in sucrose (62.5 %, kappa = 0.49). Evaluating less compact in vitro produced bovine morulae in sucrose increases agreement among evaluators, since embryos in sucrose mimick the appearance of in vivo produced embryos. Thus, we conclude that scoring in vitro produced embryos in sucrose improves agreement among evaluators.