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Optimization of parthenogenetic activation protocol in porcine
Author(s) -
Lee JangWon,
Tian X. Cindy,
Yang Xiangzhong
Publication year - 2004
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20043
Subject(s) - blastocyst , cytochalasin b , parthenogenesis , cycloheximide , andrology , biology , oocyte , in vitro , microbiology and biotechnology , embryo , embryogenesis , biochemistry , medicine , protein biosynthesis
The effects of the electrical field strengths, number of pulses, and post‐activation media on chromatin conformation and parthenogenetic development were studied to optimize the activation protocol for porcine nuclear transfer. In experiment 1, electrical field strengths were examined. Oocytes were subjected to square direct current pulses at output voltages of 1.2, 1.7, 2.2, and 2.7 kV/cm for 1 × 30 μsec. The voltage resulting from experiment 1 was 2.2 kV/cm, in which 50.0% of activated oocytes developed to blastocysts in vitro. In experiment 2, the influence of 1, 2, and 3 pulses on blastocyst development was tested using field strengths and post‐activation medium described in experiment 1. Oocytes activated by a single 30 μsec pulse of 2.2 kV/cm DC yielded a higher blastocyst rate (56.3%) than oocytes activated by 2 or 3 pulses (<42.5%). In experiment 3 and 4, we investigated the effects of cytochalasin B (CB), cycloheximide (CH), and CB + CH on nuclear development stages and parthenogenetic development following a single 30 μsec pulse of 2.2 kV/cm DC. The percentage of activated oocytes was not different among CB (93.3%), CB + CH (98.3%), control (80.0%), and CH (80.0%) groups 12 hr after activation. Treatment with CB (57.5%) or CB + CH (53.8%) enhanced the blastocyst rate compared with other groups, CH (23.8%) treated‐ and control group (18.8%). The results demonstrated that a single 30 μsec pulse of 2.2 kV/cm DC followed by culturing in post‐activation medium with CB for 5 hr were effective parameters for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes which suggests that a single calcium rise is sufficient to activate pig oocytes and to achieve high rate of blastocyst development. Mol. Reprod. Dev. 68: 51–57, 2004. © 2004 Wiley‐Liss, Inc.