258 APOPTOSIS IN FAST- AND SLOW-CLEAVING BOVINE EMBRYOS IN VITRO
L. Vandaele A , B. Mateusen A , D. Maes A and A. Van Soom AADepartment of Reproduction, Obstretics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 9320 Ghent, Belgium. Email: leen.vandaele@Ugent.be
Reproduction, Fertility and Development 17(2) 258-258 https://doi.org/10.1071/RDv17n2Ab258
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
Fast-cleaving embryos have significantly higher chances of reaching advanced developmental stages, and hatched blastocysts developed from fast-cleaving embryos have higher total cell and inner cell mass cell numbers. Few data are available on the prevalence of apoptosis in blastocysts resulting from fast-cleaving bovine embryos. Therefore, it was the aim of this study to search for a correlation between this early quality marker (timing of first cleavage) and a later quality marker (apoptosis in blastocyst stage). A total of 836 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes were denuded 24 h after fertilization and cultured in 50 μL droplets of modified SOF medium without serum at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 30 h, 36 h and 48 h post-fertilization (PF), zygotes that had developed to the 2-cell stage or beyond were placed in new droplets and cultured in separate groups. In each of the 3 replicates a control group of 100 presumed zygotes was cultured in SOF medium without manipulation. After 24 h of culture, the SOF medium was supplemented with fetal calf serum (FCS) up to 10%. Blastocysts were evaluated at Days 7 and 8, and fixed in 4% paraformaldehyde. After TUNEL staining, total cell number and TUNEL-positive cells were counted for each group. An univariate analysis of variance was used with % apoptotic cells as the dependent variable, timing of first cleavage as the fixed factor, and replicate as the random factor (mixed model ANOVA). The mean cumulative blastocyst yields in the control group were 20.9% and 27.0% at Days 7 and 8, respectively, which were not different from the total yields from manipulated embryos (Table 1). The cumulative cleavage rates for manipulated embryos were 44.9, 65.1, and 85.7% at 30, 36, and 48 h PF, respectively. The blastocyst yields at Days 7 and 8 PF were declining for embryos which cleaved later, with a distinct drop (P < 0.05) between the 36-h and 48-h groups. The percentage of apoptotic cells in Day 7 blastocysts was significantly lower in the 30-h compared with the 36-h and 48-h groups (P < 0.05). Within the 30-h and 36-h groups, Day 8 blastocysts had significantly more apoptotic cells than Day 7 blastocysts (P < 0.05), but no differences could be detected between groups at day 8. In conclusion, this experiment has confirmed the increased chances of early-cleaving embryos to reach the blastocyst stage. The higher percentages of apoptosis in late cleaving embryos at Day 7 and in all blastocysts at day 8 as determined by TUNEL staining could be an indication of lower blastocyst quality.
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