74 EXAMINATION OF ABNORMAL CELL DIVISION AND CHROMOSOME ABERRATION IN PIG PARTHENOTES AND CLONED EMBRYOS

Abstract

Blastomere fragmentation is commonly observed in pig embryos and is associated with reduced blastocyst and pregnancy rates. This study examined the effect of the frequency of abnormal cell division and chromosome aberration on the embryonic developmental ability of pig parthenotes and nuclear transferred (NT) embryos. Pig immature oocytes cultured in TCM-199 supplemented with 10% pig follicular fluid, 0.2 mM pyruvate, 10 ng/mL epidermal growth factor (EGF), 5 µg/mL Folltropin V, 1 µg/mL estradiol-17², and 25 µg/mL gentamycin for 44 h. Cumulus cells from matured oocytes were removed by vortexing for 1 min in TL-HEPES medium containing 0.1% hyarunonidase. Denuded oocytes were enucleated using 20 um micropipette in TCM-HEPES medium containing 7.5 µg/mL cytochalasin B (CB) and 10% fetal bovine serum, and were reconstructed with fetal fibroblasts by electrofusion (two DC pulses of 2.0 kV/cm for 30 µs). For production of parthenotes and reconstructed embryos, denuded oocytes were activated by a DC pulse of 1.0 kV/cm for 30 µs and then cultured for 4 h in NCSU23 with 10 µg/mL CB and 0.4% bovine serum albumin for inhibition of polar body extrusion. Subsequently, these oocytes were cultured in 50 µL of NCSU23 containing 0.4% BSA for 7 days at 39°C in a humidified atmosphere of 5% CO₂ in air. The frequency of chromosome aberrations was evaluated using fluorescent in situ hybridization technique with a porcine chromosome-1 submetacentric specific probe. Data were analyzed by Student's t-test and ANOVA using SAS software as appropriate (SAS Institute, Inc., Cary, NC, USA). Parthenotes and NT embryos showed similiar cleavage rates (61.4 and 62.9%), but the blastocyst rate of parthenotes (18.4%) was significantly higher (P < 0.05) than that of NT embryos (10.4%). The frequency of chromosome aberration in NT embryos (39.8%) at the 4-cell stage on Day 3 of culture was significantly higher (P < 0.05) than that of parthenotes (21.9%). The percentage of fragmentation was significantly higher (P < 0.05) in NT embryos (51.7%) than in parthenotes (27.1%). Furthermore, the developmental rates of non-fragmented parthenotes (40.0%) and NT (22.9%) embryos to the blastocyst stage were significantly higher (P < 0.05) than those of fragmented parthenote and NT embryos (17.3 and 5.9% respectively). The total cell number of non-fragmented parthenote and NT embryos (34.4 ± 10.0 and 29.7 ± 7.5) were significantly higher (P < 0.05) than those of fragmented parthnote and NT embryos (22.3 ± 9.6 and 18.4 ± 6.2 respectively). Therefore, these results indicate that chromosomal abnormality and embryonic fragmentation could be associated with reduced developmental ability in pig NT embryos.

This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.