No 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 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.

Preimplantation development in the bovine embryo was examined by relating the occurrence of three morphogenetic processes (compaction, blastulation, and hatching) to the timing of allocation of embryonic cells to the inner cell mass (ICM) or to the trophectoderm (TE). Embryos were collected from 26 cows between Days 4 and 9 postovulation. Compaction started 5 days postovulation at the 32-cell stage. Morulae remained firmly compact until the seventh cell cycle was almost completed. Blastocyst formation started between the 64- and 128-cell stage at Days 6, 7, and 8 postovulation. Hatching was predominant at Day 9 postovulation. ICM and TE cells could successfully be distinguished by differential staining in 107 of 142 embryos (75%). Inner cells could first be detected in 20% of 16-cell embryos. Unexpectedly, it was found that inner cell allocation and compaction were independent processes, since 31% of compacted morulae displayed no ICM. Beyond the 50-cell stage, in vivo compact morulae displayed at least 10 ICM cells, whereas blastocysts with a minimum total cell number of 65 cells displayed at least 23 ICM cells. It can be concluded that the slow in vivo transition from the morula to the blastocyst stage allows sufficient time for allocation of inner cells to the ICM of the embryo.

The aim of the present study was to determine whether or not cytopathic (CP) and noncytopathic (NCP) bovine viral diarrhea virus (BVDV) are able to replicate within in vitro-produced embryos and to investigate whether inoculation of embryos with BVDV affects their normal development. Zona pellucida (ZP)-free oocytes, zygotes, 8-cell-stage embryos, morulae, and hatched blastocysts (HB) were incubated for 1 h in 1 ml of Minimal Essential Medium containing 10(6.00) tissue culture infectious dose (TCID)50/ml NCP BVDV isolate 22,146 or 10(6.25) TCID50/ml CP BVDV strain Oregon C24V. At 0, 12, 24, 36, 48, 60, and 72 h postinoculation (hpi), groups of embryos were collected for virus titration. A small amount of newly produced virus was detected in 8-cell embryos at 60 hpi (10(1.8) TCID50/100 cells), but only for CP BVDV. For ZP-free morulae and HB, maximal intracellular virus titers were, respectively, 10(1.47) and 10(2.33) TCID50/100 cells at 48 hpi for the CP biotype and 10(0.64) and 10(0.84) TCID50/100 cells at 72 hpi for the NCP biotype. Only an infection with CP BVDV had a significant inhibitory effect on further development of ZP-free morulae. It can be concluded that ZP-free in vitro-produced embryos are permissive to an infection with BVDV, with increasing susceptibility of the embryos in accordance with their developmental stage. In contrast to observations in ZP-free in vitro-produced embryos, no virus replication or signs of embryonic degeneration were detected in ZP-intact in vitro-derived embryos.

Techniques for in vitro production of embryos have been developed world-wide in different species, with promising results in human and ruminants. Thousands of human IVF-babies have been born during the last 20 years and thousands of in vitro-produced calves have been born since the late 1980s. With current methods for bovine in vitro fertilization, about 30-40% of in vitro-fertilized bovine oocytes develop further to the blastocyst stage and can be used for transfer. A proper evaluation of blastocyst quality remains however, an important challenge for every researcher involved in embryology and for every clinician who wants to select the best embryos for transfer. This review attempts to summarize the different methods available for estimation of blastocyst quality with a special emphasis upon differential staining.