49IN VITRO DEVELOPMENT OF PORCINE CLONED EMBRYOS FOLLOWING DIFFERENT ACTIVATION TREATMENTS

Abstract

The present study compared the development of cloned porcine embryos following different activation treatments. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 22h in NCSU#23 medium supplemented with 10% porcine follicular fluid, 0.57mM cysteine, 0.5μgmL−1 LH, 0.5μgmL−1 FSH and 10ngmL−1 EGF. The COCs were further cultured for an additional 22h in the same medium at 39°C in an atmosphere of 5% CO2 in air, without hormonal supplements. Primary cultures of fibroblasts from a female fetus on Day 40 of gestation were established in DMEM +15% FCS. For nuclear donation, cells at the 5th-6th passage were cultured in DMEM+0.5% FCS for 5 days in order to arrest the cells in G0/G1. Following enucleation, oocytes were reconstructed by transfer of donor cells and fusion by means of three DC pulses (1.4kVcm−1, 30μs) delivered by a BTX 200, in 0.28M mannitol containing 0.01mM CaCl2 and 0.01mMMgCl2. Eggs were then divided into three treatment groups; control (without further treatment, Group 1), eggs cultured in 10μgmL−1 cycloheximide (CHX) for 5h (Group 2), and eggs cultured in 1.9mM6-dimethylaminopurine (6-DMAP) for 5h (Group 3). The eggs were then cultured in sets of 30in 60μL drops of NCSU#23 supplemented with 4μgmL BSA (essentially fatty acid free) until Day 7 at 39°C in a humidified atmosphere of 5% CO2. On Day 4 the culture were fed by adding 20μL NCSU#23 supplemented with 10% FBS. All experiments were performed as 4 replicates and statistical analysis was performed by one-way ANOVA (P<0.05). Blastocyst development rates were significantly higher (P<0.05) in Group 3 embryos compared to Group 1 controls (27.6±2.7% v. 20.1±4.1%, respectively), but rates did not differ in Group 2 (23.8±5.7%) compared to control. Total cell number in Group 3 blastocysts was, however, significantly higher (P<0.05) than in Groups 1 and 2 (44.6±2.4 v. 19.9±1.9 and 21.9±2.1, respectively). These results suggest that 6-DMAP is more efficient than cycloheximide in the activation of electrically fused NT oocytes during in vitro production of cloned porcine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-A Pharm. Co. LTD.]