151 COMPARISON OF CELL NUMBERS OF ZONA-INTACT AND ZONA-FREE PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS CULTURED IN VITRO

Abstract

Application of an artificial stimulus to activate oocytes and induce development is essential for the successful animal cloning by nuclear transfer (Zhu et al. 2002 Biol. Reprod. 66, 635-641). The embryo’s developmental competence could be further improved with optimal in vitro culture conditions (Du et al. 2007 Theriogenology 68, 1104-1110). Cell number determination is a commonly used and simple criterion to assess developmental quality of pre-implantation stage mammalian embryos (Lagutina et al. 2007 Theriogenology 67, 90-98). Our aim of the study was to investigate porcine embryos activated and cultured in different ways using total cell numbers as the only quality measure. After 43-44 h of in vitro maturation and cumulus cell removal, zona-intact (PAZI) or zona-free oocytes (PAZF; after pronase treatment) were subjected to parthenogenetic activation (Day 0) with a single 80-μs DC pulse of 1.26 kV cm^-1 or 0.86 kV cm^-1 (Kragh et al. 2005 Theriogenology 64, 1536-1545), followed by a 4-h treatment with 5 μg mL^-1 of cytochalasin B and 10 μg mL^-1 of cycloheximide. Subsequently, the well of the well system (Vajta in vitro 2000 Mol. Reprod. Dev. 55, 256-264) was used for culture of all PAZF and half of the PAZI embryos (PAZF-WOW and PAZI-WOW groups, respectively), whereas the remaining PAZI embryos were cultured in groups of 25-30 (PAZI group). All cultures were performed in porcine zygote medium 3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119). On Day 6, all these in vitro cultured embryos were fixed and stained with Hoechst 33342 and cell numbers were counted on the microscopic pictures taken using fluorescent light. Data analysis was performed using ANOVA. Four replicates were performed with a total of 462 PAZF-WOW, 484 PAZI-WOW, and 467 PAZI group embryos. Embryos of each group were then divided into 5 groups based on their cell number (<5 cells, 5-8 cells, 9-16 cells, 17-32 cells, >32 cells). Percentages of embryos in each group are shown in Table 1. The average cell numbers of zona-intact embryos from PAZI-WOW and the PAZI group were similar to each other (P > 0.05), whereas the cell numbers of PAZF-WOW embryos were significantly different from both PAZI-WOW and PAZI group embryos (P < 0.05), with more embryos having higher cell numbers.The results demonstrate that zona-free parthenogenetically activated embryos cultured in WOW have higher cell numbers than embryos with intact zona pellucida. Accordingly, the presence of zona pellucida may compromise embryo development under certain in vitro culture situations.

Table 1.  Distribution (in %) of parthenogenetically activated porcine embryos according to their cell number on Day 6 after activation