M.T. Ysebaert

Relationship between timing of development, morula morphology, and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos.

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast‐cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast‐cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro‐produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997.

Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media

Data from other laboratories have shown that speed of bovine blastocyst development is higher when Ménézo B2 is used for coculture compared to TCM199. It was our purpose to investigate whether this early blastocyst formation was also indicative of embryo quality by studying the allocation of inner cells in embryos generated by B2‐coculture and by TCM199‐coculture. For this purpose, a differential staining technique was used. General embryo development was similar for TCM199‐ and B2‐embryos expressed as rate of cleavage at day 3 and morula‐blastocyst formation at day 8 (P > 0.05), but significantly different when expressed as number of eight‐cell stages at day 3 and expanded or hatched blastocysts at day 8 (P < 0.01). B2‐embryos cultured until day 5, 6, and 7 post insemination, had total cell numbers of 24, 65, and 109 respectively, which was significantly higher than the cell number of TCM199 embryos cultured over the same time period (18, 41, and 71 respectively, P < 0.001). Morphological differentiation was significantly more advanced for B2‐embryos at day 7 and 8 (P < 0.0001 and P < 0.001, respectively). First presumptive inner cells appeared in eight‐ to 16‐cell stages at day 3. Because the determination of inner cells by differential staining is depending upon the presence of functional tight junctions, we concluded that the establishment of the tight junction seal in B2‐embryos differed from that in TCM199‐embryos: Inner cells appeared 0.56 cell cycle later in B2‐embryos (P < 0.001) and a larger variation existed in the number of ICM‐cells in B2‐blastocysts (P < 0.001). The higher total cell number of B2‐expanded blastocysts was mainly acquired by trophectoderm growth (P < 0.06). These data indicate that the apparent better quality of B2‐embryos (faster cleavage, earlier blastocyst formation) is not reflected in a reliable number of inner cells of B2‐blastocysts.

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.

Effects of aspiration vacuum and needle diameter on cumulus oocyte complex morphology and developmental capacity of bovine oocytes

The effects of aspiration vacuum and needle diameter on the morphology of the cumulusoocyte-complex (COC) and developmental capacity of the oocyte after IVF was studied in 2 experiments using a disposable ovum pick-up needle guidance system whose construction permits its use in vitro. In Experiment 1, the relationship was determined between the aspiration vacuum, expressed in millimetre of mercury, and the actual amount of water aspirated by the system, expressed in millilitre per minute. In Experiment 2, five different levels of aspiration vacuum for 3 different needle diameters (18g, 19g and 21g) were tested in slaughterhouse ovaries. The cumulus-oocyte complexes (COCs) were divided into 3 categories: 1) oocytes with a compact cumulus, 2) oocytes with an expanded cumulus and 3) naked oocytes. The results show that a change of needle diameter can triple the amount of fluid actually aspirated. The highest oocyte recovery rates are obtained when using the thickest needle (18-g), regardless of the aspiration vacuum. On the average, for all needle types, more oocytes are recovered at the highest aspiration vacuum. For all needle diameters, the proportion of oocytes surrounded by a compact cumulus decreases progressively as the vacuum increases. Regardless of the vacuum applied, thinner needles result in a higher proportion of recovered COCs with a compact cumulus. At a high aspiration vacuum, naked oocytes become predominant regardless of the needle diameter. The prevalence of blastocysts, expressed in proportion to the recovered COCs, decreases as the aspiration vacuum increases, being especially noticeable between 70 and 130 mm Hg.

Cell allocation and chromosomal complement of parthenogenetic and IVF bovine embryos

Considerable concerns exist regarding the quality of parthenogenetically activated embryos in terms of sufficient numbers of cells comprising the inner cell mass (ICM) and trophectoderm (TE) and the ploidy. Therefore, these two parameters were used to assess the quality of embryos derived from parthenogenetic activation by using calcium ionophore A23187 (CaI) followed by either 6‐dimethylaminopurine (6‐DMAP, 3.5 hr or 6.5 hr) or cycloheximide (CHX) plus cytochalasin D (CD). The conventional in vitro (IVF) produced embryos served as a control. Double staining of the parthenogenetic blastocysts showed that the total cell number (TC) of embryos from the 6‐DMAP 3.5 hr (87.0 ± 5.3) and CHX+CD (79.0 ± 6.1) groups was not different (P > 0.05), but was lower than that of control embryos (116.0 ± 5.8, P < 0.001). The mean ratios of inner cell mass (ICM) and trophectoderm (TE) cells in the 6‐DMAP 3.5 hr group (0.57 ± 0.04) and the control IVF group (0.50 ± 0.02) did not differ significantly. Both were higher than those of the CHX+CD group (0.36 ± 0.02; P < 0.05). Further analysis of chromosomal compositions of developing stage embryos at day four after IVF or parthenogenetic activation demonstrated that prolonged treatment with 6‐DMAP for 6.5 hr resulted in a significantly lower percentage of diploid embryos and a significantly higher percentage of abnormal ploidy embryos compared to treatment with 6‐DMAP for 3.5 hr or with CHX and IVF. In conclusion, parthenogenetic activation of bovine oocytes with CaI followed by 6‐DMAP for 3.5 hr could produce better quality embryos in terms of total cell numbers, the number of cells allocated to the ICM, and the ploidy of embryos. Mol. Reprod. Dev. 54:57–62, 1999.

Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos

Abstract Grade 1 in vitro produced bovine day 7 embryos at the compact morula-early blastocyst, blastocyst and expanded blastocyst stage were selected for cryopreservation. From 7 replicates, 572 embryos were randomly divided into two groups. One group was cryopreserved by controlled freezing after 20 minutes equilibration in 10% v/v glycerol and slow cooling (0.3 °C / minute) from −7 °C to −30 °C. The other group was vitrified after 3 minutes exposure to 20% v/v ethylene glycol and 30 to 45 seconds exposure to a vitrification solution consisting of 40% v/v ethylene glycol, 18% w/v ficoll and 10.26% w/v sucrose. Embryos from both groups were thawed in a water bath at 20±1 °C. Frozen-thawed embryos were diluted in three successive solutions consisting of 10.26% w/v sucrose and 6.6%, 3.3% and 0% glycerol, respectively, during 5 minutes for each step. Vitrified-warmed embryos were diluted in a 8.5% w/v sucrose solution during 5 minutes. All diluted embryos were cultured in Menezo-B2 medium supplemented with bovine oviduct epithelial cells. The survival rate of all developmental stages of embryos was significantly higher after vitrification than after controlled freezing (P

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.

Salvage of oocytes from sterile genetically valuable cows, resulting in the birth of a calf

The objective of this study was to determine the possible application of in vitro fertilization techniques for calf production in incurable sterility patients or in involuntary cull cows. Ten sterile or fatally injured dams of high genetic value were submitted to in vitro fertilization procedures. Ovaries were recovered after slaughter in eight animals or after ovariectomy in two cows. A total of 261 oocytes were obtained and matured in vitro. Frozen-thawed sperm from eight different bulls of high breeding value (selected to match with one particular cow) was separated with Percoll and used for in vitro fertilization in the presence of heparin. Seventy four embryos started to cleave and 7 days post-insemination, 13 transferable embryos were obtained. Eleven embryos were transferred fresh into nine recipients and produced two pregnancies. One pregnant recipient died of hydro-allantois, the other delivered a live bull calf 288 days after in vitro fertilization.