98 EFFECT OF ZONA PELLUCIDA ON PORCINE PARTHENOGENETICALLY ACTIVATED EMBRYOS

Abstract

The need for zona pellucida (ZP) during pre-implantation embryo development is still debated. In porcine parthenogenetically activated (PA) embryos, we have previously shown a different distribution in cell numbers on Day 6 blastocysts cultured with or without ZP (Li et al. 2010 Reprod. Fertil. Dev. 22, 234). In the present study, we expanded this study to include also the timing of early development and the resulting quality and robustness (for vitrification) of porcine PA embryos. Parthenogenetic activation was made first by an electric pulse (1.26 kV cm^–1, 80 μs) and then by incubation with 5 μg mL^–1 cytochalasin B and 10 μg mL^–1 cycloheximide in PZM-3 medium for 4 h. ZP was removed by 3.3 mg mL^–1 pronase. Both zona-intact (PAZI) and zona-free (PAZF) embryos were cultured individually for 6 days either in time-lapse incubator (Embryoscope D, Unisense A/S, Aarhus, Denmark) for 15-min observations (Exp. 1; 60 oocytes, 2 replicates) or in standard a incubator for blastocyst quality studies on Day 6 (Exp. 2; 524 oocytes, 11 replicates) or for cryo-tolerance studies with vitrification using Cryotop on Day 4, followed by warming and 2 days further culture (Exp. 3; 449 oocytes, 4 replicates). The timing of morulae was recorded when they completed compaction. Good blastocysts were defined when they expanded to 1.5 times larger than oocytes and formed regular blastocoel cavity with uniform colour and distribution of cells. Timing data were analysed by Student's t-test, while development rates and survival rates were analysed by chi-squared test. Exp. 1: after activation, 42 blastocysts formed on Day 6, during which the timing of development was monitored (Table 1). PAZF embryos developed faster than PAZI, especially during the first 3 cell cycles. Exp. 2: after activation, 212 and 197 blastocysts formed on Day 6 with or without ZP, respectively. Both rates of total blastocysts and good blastocysts of PAZI embryos were significantly higher than those of PAZF embryos (80.1 ± 2.7% vs 69.9 ± 1.1%, 61.9 ± 3.3% vs 49.5 ± 2.5%, respectively), but no difference was found in all blastocyst's cell numbers between PAZI and PAZF (48.2 ± 2.3 and 47.9 ± 3.2, respectively). Exp. 3: after activation, 107 PAZI and 44 PAZF embryos were vitrified on Day 4. More PAZI than PAZF embryos survived (60.8 ± 8.3% vs 30.4 ± 11.9%; P < 0.05). In conclusion, removal of ZP can increase the speed of development of porcine PA embryos, especially at the timing of embryonic genome activation (5-cell stage). Furthermore, the zona pellucida can benefit the blastocyst formation and cryo-tolerance for PA embryos, perhaps by creating a more stable microenvironement.

Table 1.  Timing of developmental stages of porcine PA embryos with (PAZI) or without (PAZF) zona pellucida*